The effect of Boric Acid and Calcium Fructoborate on T Helper Cell Differentiation by Influencing Foxp3 and Ror-γt in Rheumatoid Arthritis and Systemic Lupus Erythematosus

The effect of Boric Acid and Calcium Fructoborate on T Helper Cell Differentiation by Influencing Foxp3 and Ror-γt in Rheumatoid Arthritis and Systemic Lupus Erythematosus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The effect of Boric Acid and Calcium Fructoborate on T Helper Cell Differentiation by Influencing Foxp3 and Ror-γt in Rheumatoid Arthritis and Systemic Lupus Erythematosus Rehime Yapar, Özgül Soysal Gündüz, Feyzan Özdal Kurt, Mehmet Korkmaz This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4706771/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Oct, 2024 Read the published version in Biological Trace Element Research → Version 1 posted 15 You are reading this latest preprint version Abstract Many animal and human studies indicate that Boric Acid and Calcium Fructoborate have effects on helper T cells in immunity. The aim of our study is to evaluate effects of Boric Acid and Calcium Fructoborate on Treg ( CD4 + Foxp3 + ) and Th17 ( CD4 + Ror-γt + ) cell populations and related cytokine levels in mononuclear cells isolated from peripheral blood samples of Rheumatoid Arthritis and Systemic Lupus Erythematosus patients. Newly diagnosed Rheumatoid Arthritis (n = 10) patients, Systemic Lupus Erythematosus (n = 5) pateints and healthy individuals (n = 9) were included in this study. Consent forms were obtained from all individuals participating the study, blood samples were taken, peripheral blood mononuclear cells were isolated. Isolated cells were exposed to low dose and high dose Boric Acid and Calcium Fructoborate in cell culture. Treg and Th17 cell populations were analyzed by flow cytometry after 48 hours of exposure. IL-2, IL-6, IL-17, IL-23, TNF-α and TGF-β levels in the culture medium were tested by ELISA method. At the end of the study, in healthy controls high dose BA improved the Treg/Th17 population but couldn’t display similar effect on RA and SLE group. However, both Boric Acid and Calcium Fructoborate at different doses showed an increasing effect on Ror-γt in RA and SLE group. Different doses of BA and CaF treatment found to have a variable effect on cytokine. Both BA and CaF in low doses decreased TNF-α levels in RA group shows that these boron compounds could contribute positively to the treatment of autoimmune diseases. Th17 Treg Foxp3 Ror-γt Autoimmunity TNF-α Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune, inflammatory disease caused by the accumulation of immune complex, especially in the joints, formed by the combination of modified proteins in the body and autoantibodies developed against them [ 1 ]. In Systemic Lupus Erythematosus (SLE), another rheumatic autoimmune disease, autoantibodies formed against nuclear compartments generate immunocomplex and cause tissue inflammation and multiorgan damage [ 2 , 3 ]. The worldwide prevalence of RA were estimated to be 460 per 100,000 persons according to an article published in 2020 and for SLE the number were 43.7 per 100,000 persons according to an article published in 2022 [ 4 , 5 ]. This means that millions of people are affected by RA and SLE globally in every year. Till now no cure for these diseases and the etiology also is not clear. Although the etiology of both RA and SLE are not clearly elucidated, recent studies demonstrated the role of different types of helper T cells in their pathogenesis. Immunosuppressor T helper cells expressing Forkhead box protein 3 ( Foxp3 ) transcription factor usually called Regulator T (Treg) cells and inflammatory T helper cells expressing retinoid-related orphan receptor gamma t ( Ror-γt ) transcription factor called Th17 cells are essential CD4 + T cell subsets that play important role in pathogenesis of RA and SLE [ 6 – 9 ]. Expression of transcription factor Foxp3 is essential for the suppresser role of Treg cell in peripheral and it is one of the most specific markers for identify Treg cells [ 10 ]. High and stabile expression of Foxp3 manage the transcriptional and functional landscape of Treg cells. Mutation of Foxp3 had been found related to autoimmunity in human and mouse [ 11 , 12 ]. Treg suppression conduct in four mechanisms directly or indirectly: Treg secreted inhibitory cytokines (IL-10, IL-35, TGF-β) display suppression; Treg induce target cell cytolysis; Treg disrupt target metabolism; Treg regulate dendritic cells maturation or function to suppress inflammation [ 13 – 15 ]. Due to those positive effects of Treg cells several approaches including IL-2 therapy and Treg cell expansion therapies were investigated in autoimmune disease such as RA [ 16 ]. Treg cells from RA patients that expanded with rapamycin were found to display stable Foxp3 expression and suppressed synovial conventional T cell proliferation more effectively compared to their ex vivo counterparts [ 17 ]. Rituximab treatment in the early phase of B-cell depletion increased mRNA levels of Foxp3 in patients with active SLE. During follow-up, patients in clinical remission revealed persistently elevated Foxp3 mRNA levels, whereas patients with active disease have lower expression of this marker [ 18 ]. Th17 cells contribute to the tissue inflammation and pathogenesis of diverse autoimmune diseases, including psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD) and multiple sclerosis [ 19 ]. Th17 cells inflame inflammation in the RA joint by secreting several inflammatory cytokines such as IL-17, GM-CSF. And together with fibroblast like synoviocytes Th17 cells produce TNF-α and IL-1β. GM-CSF production was found to responsible for the chronic inflammation induced by Th17 in RA [ 20 , 9 ]. Paradowska-Gorycka A. and his colleges investigated the Th17/Treg-related transcriptional factor expression and cytokine profiles of RA patients and revealed that in isolated Peripheral Blood Mononuclear Cell (PBMC)s Treg(CD4 + CD25 high CD127 − )/Th17(CD4 + CCR6 + CXCR3 − ) ratio were lower in RA patients compared to the Healthy Controls [ 21 ]. Yao M.X. et al. found Th17(CD4 + IL-17A + )/Treg(CD4 + Foxp3 + ) ratio were higher in collagen induced arthritis Rat model [ 22 ]. Oxidative stress and metabolic abnormalities have been found to related to the imbalance of Treg and Th17 in SLE [ 23 , 24 ]. In a SLE mice model Cucurbitacin IIb, an substrate extracted from a traditional Chinese medicine, found to be improved kidney injury by balancing the Treg(CD4 + Foxp3 + ) /Th17(CD4 + IL-17A + ) percentage in the meanwhile upregulated Foxp3 expression and downregulated Ror-γt expression in splenic lymphocytes [ 25 ]. Latest studies signify that attempts to reduce pathogenic Th17 cell population and increase Treg cell stability may provide promising therapeutic approaches in both SLE and RA treatment. Boron and its compounds have been found to have anti-inflammatory, immunomodulatory effects [ 26 , 27 ]. Boric Acid (BA) is one of the most important organoboron compound that found physiologically in biologic systems [ 28 ]. Calcium Fructoborate (CaF) is a sugar-borate compound naturally found in fruits and vegetables [ 29 ]. Both BA and CaF have been studied by researchers in terms of their effects on inflammation. Jin ErHui et al. demonstrated that low dose BA could induce splenic lymphocyte proliferation and reduce CD4 + /CD8 + cell ratio [ 30 ]. Routray I. and Shakir A. also found that sodium tetraborate increase CD4 + T helper cell population in a dose-depended manner [ 31 ]. Bortezomib has been studied in SLE patients’ PBMCs and it revealed that 4 ug/ml bortezomib decreased CD3 + CD4 + and CD3 + CD8 + cell proliferation [ 32 ]. In another study, CaF had given to the RA and Osteoarthritis patients as an adjuvant with entanercept. Patients took 220 mg/day CaF for 60 days and ESR, CRP, IL-1α, IL-6 ve TNF-α levels were found significantly decreased [ 33 ]. All these evidence demonstrate that boron compound could affect T cells and cytokines released from them. To sum up treatments and the understanding of pathogenesis of RA and SLE need an improvement. Boron compounds have promising potential in the treatment of autoimmune diseases. In our study we aimed to investigate the effects of BA and CaF on the Treg and Th17 cell population in PBMCs isolated form RA and SLE patients in terms of Foxp3 and Ror-γt expression. Which is very important to elucidate the boron compounds’ effects on autoimmunity and for the searching of new therapeutic approaches towards these diseases. Certain cytokines related to the differentiation of Navie T cells towards Treg or Th17 such as IL-6, IL-2, TGF-β and IL-23 along with proinflammatory cytokines IL-17 and TNF-α were also measured in this study. Materials and Methods Materials BA and CaF were supplied from BOREN (National Boron Research Institution-TURKEY). Antibodies: anti-CD4 PE, anti-Foxp3 FITC and anti-Ror-γt APC (Biolegend) were used for flow cytometry test. ELISA kits for IL-2, IL-17, IL-23, and TGF-β cytokines were purchased from Sunred Biotechnolog Company, China. ELISA kits for IL-6 and TNF-α cytokines were purchased from ELK Biotechnolog Company, China. RPMI-1640 (NutriCulture), Fetal Bovine Serum Heat Inactivated (Biosera), Penicilin-Streptomycin (Gibco by Life Technology), Cell Viability Detection Kit-8 (Eco-Tech Biotechnology), Dulbecco’s Phosphate Bufferd Saline 10X (Biosera), Dimethyl Sulfoxide Cell Culture Grade (AppliChem), Lymphocytes Separation Media (Capricorn) were used in cell culture. Patients and Study Design Those who were over the age of 18 and diagnosed with RA in accordance with the ACR/EULAR 2019 RA classification were included in our study and determined as the RA Group (n = 10). Those who were over the age of 18 and diagnosed with SLE in accordance with the ACR/EULAR 2019 SLE classification were included in our study and determined as the SLE Group (n = 5). For RA and SLE patients, those who use biological agents (such as TNFα and interleukin inhibitors), pregnant women, those with active infections and those with other autoimmune diseases were not included in the study. For healthy volunteers, individuals over the age of 18 who did not have any autoimmune disease history, did not have any recent surgery, were not pregnant and did not have active infection were selected as the Control Group (n = 9). Informed consents were taken from all participants according to the principles of the Declaration of Helsinki. The local scientific ethics committee of Manisa Celal Bayar University, Institute of Medicine approved the study protocol. Approval document number: 20.478.486. A maximum of 10 ml of peripheral blood samples were collected from all participants for further isolation of PBMCs. Blood samples were collected in sterile environment in blood tubes containing heparin. Clinical data describing study subject were given in Table 1 . Isolation of PBMCs and Cell Culture For PBMC isolation 3.5 ml of lymphocyte isolation solution was transferred to a sterile 15 ml falcon tube. Fresh blood was slowly dropped over the solution and two layers were formed with the isolation solution on the bottom and the blood sample on the top. Four separate gradient layers were obtained by centrifuging the Falcon tube at 1800 rpm for 30 min at room temperature. After centrifugation PBMC containing white blood cells were in the second layer of the whole gradient. The upper layer containing the serum is removed with a sterile pipette. The second layer is the floor with PBMCs and the PBMCs were transferred to a separate falcon tube with a sterile pipette. For washing sterile 1X PBS was added to fill the falcon and centrifuged at 1800 rpm for 10 minutes and the wash step repeated three times. After washing step, the pellets were dissolved with 1 ml RPMI-1640 cell culture medium containing 10% Fetal Bovine Serum (FBS) 1% penicillin-streptomycin and cell counting was performed. This procedure was applied to the blood samples of all volunteers participating in the study. Average 15x 10 6 − 30x 10 6 cells were isolated from each participant. Cell Proliferation and Cytotoxicity Analysis T cells are suspension cells thus WST-8 test was performed to test the proliferative and/or cytotoxic effects of BA and CaF on isolated PBMCs. Because supernatant doesn’t need to be removed before reading the formazone in microplate reader in the WST-8 test, PBMCs obtained from volunteers were cultured into 96 well plates and cell proliferation or inhibition effects were determined after 24, 48 and 72 hours. Cells were cultured in density of 10000 cell/well in 180 ul culture medium. BA and CaF substances prepared by dissolving 10-100-250-500-1000-2500-5000 µM doses with distilled water were applied 10 µl/well after the cultured cells were rested and kept overnight in an incubator containing 5% CO 2 at 37°C. 10 µl of WST-8 solution was added to the plates at the 24th, 48th and 72nd hours and kept in the incubator for 3 hours, wrapped with aluminum foil. Dihydrogenase in living cells stains WST-8 in orange color and the amount of formazone formed from this color was read in a microplate reader (BioTek) device at 450 nm wavelength in accordance with the manufacturer's instructions The experiments were designed in triplicate. After the results were saved in Excel, the IC50 value of the items was determined according to the %Radical Scavenging Activity. %Radical Scavenging Activity were calculated with the formula: $$\:\text{%}\text{R}\text{a}\text{d}\text{i}\text{c}\text{a}\text{l}\:\text{S}\text{c}\text{a}\text{r}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}\:\text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}=\frac{\text{A}\text{b}\text{s}\text{r}\text{o}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\:-\:\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{S}\text{a}\text{m}\text{p}\text{l}\text{e}}{\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}\:\text{o}\text{f}\:\text{C}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}}\times\:100$$ Flow Cytometry Analysis Flow cytometry method was used to determine the populations of helper T cell subsets. Cells (3x 10 5 cells /ml/well) were cultured in 24 well plates. After overnight incubation, no substance was applied to one well as a control group. A low dose of 100 µM BA and a high dose of 1000 µM BA were administered to the BA group. A low dose of 250 µM CaF and a high dose of 2500 µM CaF were administered to the CaF group. HC (Healthy Control) Group : PBMCs isolated from healthy controls with no treatment; HC + 100 BA Group : PBMCs isolated from healthy controls and treated with 100 uM BA; HC + 1000 BA Group : PBMCs isolated from healthy controls and treated with 1000 uM BA; HC + 250 CaF Group : PBMCs isolated from healthy controls and treated with 250 uM CaF; HC + 2500 CaF Group : PBMCs isolated from healthy controls and treated with 2500 uM CaF. RA Group : PBMCs isolated from RA patients without treatment; RA + 100 BA Group : PBMCs isolated from RA patients and treated with 100 uM BA; RA + 1000 BA Group : PBMCs isolated from RA patients and treated with 1000 uM BA; RA + 250 CaF Group : PBMCs isolated from RA patients and treated with 250 uM CaF; RA + 2500 CaF Group : PBMCs isolated from RA patients and treated with 2500 uM CaF. SLE Group : PBMCs isolated from SLE patients without treatment; SLE + 100 BA Group : PBMCs isolated from SLE patients and treated with 100 uM BA; SLE + 1000 BA Group : PBMCs isolated from SLE patients and treated with 1000 uM BA; SLE + 250 CaF Group : PBMCs isolated from SLE patients and treated with 250 uM CaF; SLE + 2500 CaF Group : PBMCs isolated from SLE patients and treated with 2500 uM CaF. Since the helper T cell surface marker CD4 and transcription factors Ror-γt and Foxp3 will be stained, a combination of surface staining and intracellular staining was used [ 34 ]. Anti-CD4 PE, anti-Foxp3 FITC and anti-Ror-γt APC (Biolegend) antibodies were used for analysis. Cells that were cultured on 24 well plates and treated with different doses of substance were collected by washing them into 1.5 ml Eppendorf after 48hs. Since PBMCs were suspension cells, no removal from the surface was measured. Samples were analyzed by LSR FORTESSA flow cytometry and graphics were measured by flowjo program. Cytokine Analysis by ELISA Test Cytokines secreted in the culture medium were detected using ELISA method. BA and CaF treated or untreated culture medium of PBMCs were collected in sterile 1,5 ml Eppendorf and stored in -20 o C until the measurement of ELISA test. Standard solutions were prepared for each cytokine according to the manufacturer’s constructions. Standards, blank and sample wells were determined and after staining and washing steps stop solution were added. At the end of the procedure plates were immediately read with microplate reader in 450 nm wavelength. Standard curves were obtained, and cytokine levels were determined. Table 1 Study Subject Clinical Data. Groups Characters Healthy Donors (n = 9) SLE Patients (n = 5) RA Patients (n = 10) Age 26–53, 33 (Median) 20–55, 25 (Median) 36–64, 52 (Median) Gender Female (5) Male (4) Female (5) Female (4) Male (6) DAS28 --- --- 5,33 ± 0,84 SADAI --- 4–16 --- CRP (mg/dl) < 0,3 1,3 ± 1,4 1,2 ± 1,1 ESR (mm/hour) 18 ± 3 34 ± 20 44 ± 23 RF (IU/ml) Negative Negative Negative (2) Positive (8) Anti-CCP (U/ml) < 20 200 (5) Anti-dsDNA Negative Negative Positive (4) Comorbidities --- Varix (1) Hypertension (2) Statistical Analysis Data obtained after cell culture were statistically evaluated using Mixed ANOVA test. Analyzed data were given in means and standard error. P values under 0,05, 0,01 or 0,001 were determined as statistically significant. P > 0,05 were evaluated as statistically insignificant. Results Proliferative/Cytotoxic Effects of BA and CaF on PBMC Cells Isolated from Healty Donors, RA and SLE patients The different doses of BA and CaF were applied to the PBMCs isolated from HC, SLE and RA patients. After 24h, 48h, 72h incubation with BA in different doses, PBMCs in all HC, SLE and RA groups did not show any significant proliferative or cytotoxic effects (Fig. 1 A, 1 B). After 24h, 48h, 72h incubation with CaF, PBMCs in all HC, SLE and RA groups didn’t show any significant proliferative or cytotoxic effect in doses 10- 100- 250- 500- 1000 µM. However, higher doses of CaF including 2500–5000 µM showed proliferative effects in all HC, SLE and RA groups (p < 0,001). According to these results for further analysis 100 µM and 1000 µM were chosen for BA treatment and 250 µM and 2500 µM were chosen for CaF treatment. Effects of BA and CaF on Treg (CD4 + Foxp3 + ) and Th17 (CD4 + Ror-γt + ) cell population in isolated PBMCs The Low (100 µM BA and 250 µM CaF) and the high doses (1000 µM BA and 2500 µM CaF) of BA and CaF were applied to the isolated PBMCs then Treg (CD4 + Foxp3 + ) and Th17 (CD4 + Ror-γt + ) cell populations were tested in flow cytometry ( Fig. 2 ). Treg cell population in HC group, SLE group and RA group were 4,71% (0,58), 4,78% (0,79) and 10,59% (0,56) respectively. SLE group was similar Treg population compared to the HC group in nontreated controls. RA group found to have higher Treg population without any treatment compared to the HC group and SLE group. In HC Group, both low and high dose BA administration caused an increase in the CD4 + Foxp3 + Treg population compared to the substance-free control, while both doses of CaF administration caused a decrease in Treg population (Fig. 2 A). However, the effects of both BA and CaF application were insignificant (HC + 100 BA Group p = 0,999, HC + 1000 BA Group p = 0,660, HC + 250 CaF Group p = 0,999 and HC + 2500 CaF Group p = 0,990, p > 0,05). In SLE Group, low dose BA administration decreased the Treg population while high dose BA administration increased its population, but these results were not statistically significant (SLE + 100 BA Group p = 0,644, SLE + 1000 BA Group p = 0,642, p > 0.05). In SLE + 2500 CaF group CD4 + Foxp3 + Treg population were decreased, whereas in SLE + 250 CaF group Treg population were increased compared to the nontreated control (Fig. 2 B), but these results also were statistically insignificant (p = 0.786 and p = 0.686, respectively p > 0.05). In the RA group, high-dose (1000 µM) BA administration, as well as both low-dose (250 µM) and high-dose (2500 µM) CaF administration, tend to decrease the Treg population compared to the substance-free control. Only low-dose BA application increased the Treg population (Fig. 2 C). However, these values were found to be statistically significant only in RA + 2500 CaF group (p = 0.001, p < 0,01). In nontreated controls Th17 cell rate were 16.99% (1.94) in the HC Group, 15.36% (2.60) in the SLE Group and 20.36% (1.84) in the RA Group. When both doses of BA and high dose of CaF applied groups were compared with the nontreated control group, it was observed that there was a decrease in Th17 cell population in the PBMCs isolated from healthy donors (Fig. 3 A). But this effect was found to be statistically insignificant (HC + 100 BA p = 0.999, HC + 1000 BA p = 0.144, HC + 2500 CaF p = 0.786, p > 0.05). In HC + 250 CaF group Th17 population tend to elevate but this effect also statistically insignificant (p = 0.999, p > 0.05). In the SLE, it was observed that both doses of BA and both doses of CaF increased the Th17 cell population compared to the drug-free control (Fig. 3 B). This effect was found to be statistically significant except SLE + 1000 BA group (SLE + 100 BA p = 0.042, p < 0.05. SLE + 250 CaF p = 0.001, SLE + 2500 CaF p = 0.007, p < 0.01. RA + 1000 BA p = 0.015, RA + 250 CaF p = 0.018, p < 0.05). Similar to the SLE group, in the RA group, both doses of BA and both doses of CaF were observed to increase the Th17 cell population when compared to the drug-free control (Fig. 3 C). And this effect was found statistically significant in high dose BA and low dose CaF application (RA + 100 BA p = 0.999, RA + 2500 CaF p = 0.999, p > 0.05. RA + 1000 BA p = 0.015, RA + 250 CaF p = 0.018, p < 0.05). According to the above results Th17/Treg ratio were calculated (Fig. 4 ). Both Th17 and Treg percentages were higher in the RA group than in the control and SLE groups. The Th17/Treg ratio in three groups were HC Group 3.65% (0,36), SLE Group 3,69% (0,49) and RA Group 2,01% (0,35). Only in HC + 1000 BA group Th17/Treg percentage were significantly decreased. However not similar effects were observed in SLE and RA groups. Effects of BA and CaF on IL-2, IL-6, IL-17, IL-23, TNF-α and TGF-β Cytokine Levels Cytokine (IL-2, IL-6, IL-17, IL-23, TNF-α and TGF-β) levels of cultured PBMCs were analyzed by ELISA test. Effects of different doses of BA on cytokine levels were given in Table 2 . TNF-α levels were increased dose-depended pattern with BA application in HC group and SLE group compared to the control. And these effects were statistically significant in high dose BA groups. However, in RA group low dose BA decreased TNF-α level significantly compared to the control (p < 0,034). Both dose of BA treatment significantly affected the IL-17 level in HC group. IL-17 level was decreased significantly with both dose of BA applications in SLE and RA group. IL-23 level was increased with low dose BA treatment in all groups. And statistically significant in HC group and RA group. In SLE group both doses of BA administration didn’t significantly change the IL-23 level. Different doses of BA application decreased the IL-2 level in HC group and low dose BA decreased more effectively. Low and high dose of BA treatment tend to increase IL-2 level in SLE group but this effect was not statistically significant. BA administration doesn’t effectively change the IL-2 level in RA group. BA application increased the IL-6 level in HC group and this effect was statistically significant only in HC + 100 BA group. Both low and high doses of BA application showed similar effects in IL-6 levels of SLE and RA groups. Low dose BA administration decreased IL-6 level more effectively than high dose BA in both SLE and RA groups. In HC group, SLE group and RA group low dose BA treatment increased the TGF-β secreted by PBMCs in cell culture media. However, this effect was only statistically significant in SLE + 100 BA group. Effects of different doses of CaF on cytokine levels were given in Table 3 . Similar to BA low dose CaF decreased TNF-α level significantly compared to the nontreated control in RA group. In SLE and HC groups different dose of CaF treatment increased TNF-α level in dose-depended manner compared to the control. However, this effect was statistically insignificant. Low dose CaF significantly decreased the IL-17 levels in SLE group but doesn’t change it in HC and RA groups. However, high dose CaF significantly increased IL-17 levels in all groups compared to the nontreated control. IL-23 level was displayed similar patterns after low and high dose CaF applications in all groups which is increased with low dose CaF and decreased with high dose CaF. But these results were statistically significant in SLE + 250 CaF group, HC + 250 CaF group and HC + 2500 CaF group. IL-2 level were not significantly changed with both dose CaF applications in RA group. In SLE group IL-2 level were increased with low dose CaF application and doesn’t change with high dose compared to the nontreated control. In HC group both doses CaF application significantly decreased the IL-2 levels. IL-6 levels were not changed with both dose CaF administration in HC group. Low dose CaF application decreased IL-6 levels while high dose significantly increased its levels in RA group. In SLE group low dose CaF increased the IL-6 levels. However, IL-6 level were stayed steady with high dose CaF. Effects of two different dose of CaF on TGF-β levels in HC group and SLE group were showed similar patterns. In which TGF- β levels were increased dose-depended manner and its statistically significant in HC + 250 CaF group, HC + 2500 CaF group and SLE + 2500 CaF group. In RA group both dose CaF application decreased the TGF-β insignificantly. Table 2 Cytokine levels of all Groups after different doses of BA application. Characters Groups Control (Mean, St.E) 100 µM BA (Mean, St.E) 1000 µM BA (Mean, St.E) p-value (Control vs 100BA) p-value (Control vs 1000BA) p-value (100BA vs 1000BA) Comparison of Effects of BA Among Groups (P-value) TNF-α (pg/ml) HC 174,68 (70,44) 222,66 (55,85) 426,78 (89,86) 0,138 0,035* 0,067 0,295 (HC vs SLE 0,788 HC vs RA 0,999 SLE vs RA 0,371) SLE 203,88 (94,51) 313,16 (74,93) 636,95 (120,56) 0,634 0,006* 0,025* RA 324,79 (66,82) 158,05 (52,98) 219,58 (85,25) 0,034* 0,711 0,999 IL-17 (pg/ml) HC 278,74 (39,42) 272,17 (15,66) 268,13 (15,79) 0,996 0,999 0,999 0,078 (HC vs SLE 0,200 HC vs RA 0,138 SLE vs RA 0,999) SLE 465,52 (52,89) 276,81 (20,96) 298,87 (21,19) < 0,001* < 0,001* 0,295 RA 394,54 (37,40) 320,97 (14,82) 304,06 (14,98) 0,026* 0,003* 0,224 IL-23 (ng/ml) HC 6,29 (0,42) 8,73 (0,41) 8,67 (0,36) < 0,001* < 0,001* 0,999 0,242 (HC vs SLE 0,337 HC vs RA 0,726 SLE vs RA 0,999) SLE 6,75 (0,56) 7,60 (0,55) 6,43 (0,48) 0,273 0,999 0,008* RA 6,63 (0,40) 7,71 (0,39) 7,60 (0,34) 0,014* 0,023* 0,999 IL-2 (ng/l) HC 36,47 (3,27) 22,14 (1,20) 29,31 (0,78) 0,001* 0,093 < 0,001* 0,359 (HC vs SLE 0,472 HC vs RA 0,999 SLE vs RA 0,999) SLE 21,81 (4,39) 29,38 (1,61) 26,56 (1,05) 0,265 0,794 0,335 RA 28,03 (3,11) 28,77 (1,14) 27,56 (0,74) 0,999 0,999 0,967 IL-6 (pg/ml) HC 18,98 (1,20) 23,66 (1,55) 21,06 (1,40) 0,017* 0,684 0,679 0,030* (HC vs SLE 0,568 HC vs RA 0,026 SLE vs RA 0,926) SLE 25,38 (1,61) 21,08 (2,08) 23,62 (1,88) 0,142 0,999 0,999 RA 29,19 (1,14) 20,92 (1,47) 24,80 (1,33) < 0,001* 0,035* 0,191 TGF-β (pg/ml) HC 19,32 (0,75) 21,01 (1,26) 21,95 (1,06) 0,365 0,030* 0,999 0,019* (HC vs SLE 0,026 HC vs RA 0,098 SLE vs RA 0,947) SLE 20,74 (1,00) 24,92 (1,69) 28,61 (1,42) 0,022* < 0,001* 0,139 RA 23,14 (0,71) 24,60 (1,19) 22,35 (1,01) 0,470 0,999 0,248 HC = Healthy Control Group. SLE = Systemic Lupus Erythematosus Group. RA = Rheumatoid Arthritis Group. St.E = Standard Error. *= Statistically Significant. Table 3 Cytokine levels of all Groups after different doses CaF application. Characters Groups Control (Mean, St.E) 250 µM Caf (Mean, St.E) 2500 µM caf (Mean, St.E) p-value (Control vs 250caf) p-value (Control vs 2500caf) p-value (250caf vs 2500caf) Comparison of Effects of caf Among Groups (P-value) TNF-α (pg/ml) HC 174,68 (70,44) 248,53 (25,17) 502,18 (136,18) 0,996 0,073 0,251 0,375 (HC vs SLE 0,999 HC vs RA 0,970 SLE vs RA 0,589) SLE 203,88 (94,51) 413,34 (75,35) 416,27 (182,70) 0,144 0,509 0,999 RA 324,79 (66,82) 125,99 (53,28) 207,44 (129,19) 0,031* 0,999 0,999 IL-17 (pg/ml) HC 278,74 (39,42) 279,60 (36,87) 376,24 (24,53) 0,996 < 0,001* < 0,001* 0,014 (HC vs SLE 0,016 HC vs RA 0,126 SLE vs RA 0,576) SLE 465,52 (52,89) 375,84 (49,43) 605,16 (32,91) < 0,001* < 0,001* < 0,001* RA 394,54 (37,40) 386,68 (34,96) 447,12 (23,27) 0,999 0,023* 0,008* IL-23 (ng/ml) HC 6,29 (0,42) 9,77 (0,93) 4,61 (0,43) 0,007* 0,008* < 0,001* 0,171 (HC vs SLE 0,196 HC vs RA 0,866 SLE vs RA 0,895) SLE 6,75 (0,56) 11,53 (1,25) 6,41 (0,57) 0,006* 0,999 0,002* RA 6,63 (0,40) 8,87 (0,88) 7,04 (0,41) 0,087 0,999 0,165 IL-2 (ng/l) HC 36,47 (3,27) 21,48 (0,84) 18,04 (1,22) < 0,001* < 0,001* 0,037* 0,171 (HC vs SLE 0,999 HC vs RA 0,509 SLE vs RA 0,257) SLE 21,81 (4,39) 26,53 (1,12) 23,43 (1,64) 0,751 0,999 0,241 RA 28,03 (3,11) 28,38 (0,79) 27,83 (1,16) 0,999 0,999 0,999 IL-6 (pg/ml) HC 18,98 (1,20) 20,46 (5,56) 19,13 (2,78) 0,999 0,999 0,999 < 0,001 (HC vs SLE 0,023 HC vs RA < 0,001 SLE vs RA 0,004) SLE 25,38 (1,61) 41,25 (7,45) 25,12 (3,72) 0,084 0,999 0,221 RA 29,19 (1,14) 23,92 (5,27) 79,68 (2,63) 0,840 < 0,001* < 0,001* TGF-β (pg/ml) HC 19,32 (0,75) 22,51 (0,99) 24,71 (0,98) 0,043* < 0,001* 0,249 0,249 (HC vs SLE 0,428 HC vs RA 0,999 SLE vs RA 0,369) SLE 20,74 (1,00) 24,74 (1,33) 25,91 (1,31) 0,063 0,003* 0,999 RA 23,14 (0,71) 21,47 (0,94) 21,75 (0,93) 0,470 0,473 0,999 HC = Healthy Control Group. SLE = Systemic Lupus Erythematosus Group. RA = Rheumatoid Arthritis Group. St.E = Standard Error. *= Statistically Significant. Discussion First of all, we investigated the proliferative/cytotoxic effects of different dose of BA and CaF. Interestingly we found that high dose CaF induce proliferation of PBMCs isolated from study subjects especially after 1000 µM. Though this is not the focus of our study this maybe important for the stimulation of lymphocytes or other type of PBMCs in culture conditions. We think that it’s important to determine the cell type proliferated for further investigations. In our study, the effect of different doses of BA and CaF on cytokines released from PBMCs isolated from RA and SLE patients in culture was investigated. As a result, it was observed in our study that BA and CaF application increased TNF-α level in healthy individuals and SLE patients, but both doses of BA and CaF could display anti-inflammatory effects by reducing TNF-α levels in RA patients. Routray I. and Shakir A. added sodium tetraborate dihydrate equivalent to BA at 2-, 2.5- and 3-mM concentrations to the drinking water of mice and isolated the splenic cells after feeding the mice with this drinking water for 10 days. They found that after stimulation with LPS (lipopolysaccharide) in the isolated cells, the CD4 + T cell population and the levels of TNF-α, IL-6 and IL-1β increased in a dose-dependent manner [ 31 ]. Similar situation maybe the case in our study in HC group and SLE group. And they pointed out that this effect is achieved stabilizing the antigen-receptor complex on the lymphocyte by boric acid. it was observed that high-dose CaF increased IL-17 secretion in cultured PBMCs isolated from both SLE patients and RA patients, on the contrary, high and low-dose BA and low-dose CaF could have anti-inflammatory effects by reducing IL-17 secretion; It was observed that administration of different doses of BA was insufficient to suppress IL-23 level, an inflammatory cytokine plays important role in RA and SLE pathogenesis, CaF could display an anti-inflammatory effect by lowering IL-23 level at high doses in healthy individuals, but it caused an increase in IL-23 secretion in autoimmune conditions such as SLE and RA where the inflammatory balance was impaired; IL-23 is mainly secreted from dendritic cells and macrophages and plays important role in the differentiation of naive T cells into Th17 cells in autoimmune diseases [ 35 ]. However, naive T cells must first be stimulated by TGF-β, IL-6 and IL-21 to express the IL-23 receptor [ 36 ]. Therefore, whether the increasing effect of boron compounds used in this study on IL-23 level contribute to the inflammation or not couldn’t be clearly demonstrated by the results obtained. Meanwhile, the increase in Th17 (CD4 + Ror-γt + ) cells after treatment with different boron compounds that observed in our study may be related to the increase in IL-23. IL-2 is a cytokine secreted by T cells and is also called T cell growth factor because it causes clonal expansion of T cells. IL-2 is involved in the proliferation of T and B cells, expression of antibodies from B cells, expression of other cytokines, and Fas-mediated apoptosis [ 37 ]. In our study, it was observed that administration of different doses of BA and low-dose CaF may be effective in regulating autoimmunity by increasing IL-2 level in SLE patients. However, it was found that different doses of BA and CaF were insufficient to increase the level of IL-2, which has an autoimmunity suppressive effect, in RA patients. IL-6 has many biological activities and was first known as B cell stimulatory factor-2. IL-6 activates acute phase reactants and plays an important role in Th17 cell differentiation together with TGF-β [ 38 ]. It was observed that the administration of low and high doses of BA could have an anti-inflammatory effect on PBMCs isolated from patients with SLE and RA by reducing the level of inflammatory cytokine IL-6. It was found that the effect of CaF on IL-6 secretion from PBMCs isolated from SLE and RA patients is complex, and that different doses of CaF may affect IL-6 levels differently in different disease groups. It has been observed that different doses of BA and CaF could display anti-inflammatory effect by increasing TGF-β level in SLE group, affecting helper T cell subgroup differentiation, for example CD4 + Foxp3 + Treg cell differentiation. It was found that different doses of BA and CaF did not have a significant effect on TGF-β levels in the culture medium of PBMCs isolated from RA patients. It is known that the disorder of Treg/Th17 balance in autoimmune diseases is associated with the pathogenesis of the disease [ 21 , 23 ], and recently strategies for maintaining the Treg/Th17 balance have been tried to be exhibited with increasing investigations. Treg cells belonging to the helper T cell subgroup require the Foxp3 transcription factor to fulfill its function of maintaining immune tolerance [ 13 , 39 ]. Th17 cells belonging to the inflammatory helper T cell subgroup, on the other hand, require the Ror-γt transcription factor [ 19 ]. It has been reported in many studies that Treg and Th17 population and function are also important in the pathogenesis of RA and SLE [ 6 – 8 , 40 ]. Xie M. et al. found in their study that there is Treg/Th17 (CD4 + Foxp3 + /CD4 + IL-17A + ) imbalance in PBMCs isolated from RA and SLE patients. And they also showed that this imbalance is due to decreased Foxp3 expression, which is caused by targeting the 3'untranslated region of Foxp3 gene by mi-R34a, and mi-R34a in cells is found to be higher compared to healthy controls due to TNF-α and IL-6. They found that it leads to increased expression of Ror-γt [ 41 ]. Yao M.X. et al. have investigated the therapeutical effect of Wu-Teng-Gao, one of the Chinese traditional medicines, on collagen induced arthritis model in terms of Th17/Treg balance. An increase in Foxp3 expression and a decrease in Ror-γt expression were observed. In addition, a decrease in pro-inflammatory cytokines (IL-1, IL-6, TNF-α, IL-17) and an increase in anti-inflammatory cytokines (IL-10 and TGF-β) were observed, and it was found that bone damage was inhibited in histological staining [ 22 ]. In a study conducted in a lupus mouse model, the effect of Cucurbitacin IIb on Th17 and Treg cell percentages, Foxp3 and Ror-γt expression levels in cells isolated from the spleen of mice was investigated. As a result, it was observed that there was an increase in Treg cell population, decrease in Th17 cell population, increase in Foxp3 expression, decrease in Ror-γt expression, increase in IL-10 and TGF-β expression, and decrease in IL-17 and IL-6 levels in Cucurbitacin IIb applied groups compared to control [ 25 ]. In our study, CD4 + Foxp3 + Treg and CD4 + Ror-γt + Th17 cell populations in PBMCs isolated from RA and SLE patients were investigated and Th17/Treg cell percentage were calculated ( Fig. 4 ). Th17/Treg ratio in our study were found to be lower in RA group compared to the HC group. This maybe because CD4 + Ror-γt + cells were investigated as Th17 in our study and in many other studies investigating Treg and Th17, CD4 + IL-17A + Th17 cells were studied [ 42 , 22 ]. We aimed to investigate the effects of BA and CaF on CD4 + Foxp3 + cells and CD4 + Ror-γt + cells. It is known that Th17 cells have pTh17 and non-pTh17 subgroups [ 19 ]. It has been reported that STAT proteins along with Foxp3 and Ror-γt are also important in Treg and Th17 functions. It has been reported that STAT3 protein contributes to the pathogenicity of Th17 cells [ 43 ]. STAT5, on the other hand, contributes to the differentiation of naive T cells into Foxp3 -expressing immunosuppressive Treg cells [ 44 , 13 ]. While as a result different doses of BA decreased the Th17/Treg ratio in healthy individuals in our study and different doses of CaF increased the Th17/Treg ratio in the RA group. Similar to the RA group different doses of CaF caused an increase in Th17/Treg ratio in the SLE group. In terms of Ror-γt increasing effects boron compounds studied in our study, whether it’s contributed to or attenuate the course of inflammation can be elucidated by further investigations towards the cofactors of these signaling pathways mentioned above. When these results are evaluated together with the cytokine levels, low dose BA decreased the pro-inflammatory cytokine IL-17, TNF-α level, except IL-23, and tended to increase IL-6 and TGF-β level. Despite the increased expression of Ror-γt, the decrease in the level of pro-inflammatory cytokines may indicate that BA has an anti-inflammatory effect on non-pTh17 cells. All these results show that BA and CaF could affect Helper T cell differentiation in autoimmune diseases by affecting both Foxp3 and Ror-γt levels in CD4 + T cells. Changes in the Foxp3 and Ror-γt expression may related to the alteration in the IL-17 and TGF-β levels. In particular, the effect of BA and CaF on TNF-α level shows that it could contribute positively to the treatment of autoimmune diseases. However, the effect of BA and CaF on the cytokine network was found to be very complex. Therefore, the effect of BA and CaF on the course of inflammation can be elucidated by further investigation of molecules such as STAT, IL-1, IL-10 and GM-CSF in the downstream signaling pathways. Declarations This study was performed under the principles of the Declaration of Helsinki. The local scientific ethics committee of Manisa Celal Bayar University, Institute of Medicine approved the study protocol. Approval document number: 20.478.486. Informed consent was obtained from all participants included in the study. The authors have no financial or proprietary interests in any material discussed in this article. All the authors contributed to the study in different ways. M.K. is the supervisor of the study. Ö.SG. managed patient evaluation and patient data collection. F.ÖK. performed part of the cell culture analysis and contributed to the data analysis. R.Y. involved in the literature investigations, sample collection, laboratory analysis, data analysis and manuscript preparation. All authors evaluated and approved the final manuscript. Author Contribution All the authors contributed to the study in different ways. M.K. is the supervisor of the study. Ö.SG. managed patient evaluation and patient data collection. F.ÖK. performed part of the cell culture analysis and contributed to the data analysis. R.Y. involved in the literature investigations, sample collection, laboratory analysis, data analysis and manuscript preparation. All authors evaluated and approved the final manuscript. Acknowledgement We would like to thank the National Boron Research Institute, BOREN, for providing us Boric Acid and Calcium Fructoborate. 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Treg cell population decreased significantly in RA+ 2500 CaF group (**= p\u0026lt;0,01).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4706771/v1/baa0a23c5a7bb539d163a049.png"},{"id":62155116,"identity":"d3e95c1c-1c5c-410c-8ff1-6a549d52c44c","added_by":"auto","created_at":"2024-08-09 21:02:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":448005,"visible":true,"origin":"","legend":"\u003cp\u003eTh17 cell population in HC, SLE and RA patients PBMCs after low and high dose BA and CaF treatment. Th17 cell population increased in SLE+ 100 BA group, SLE+ 250 CaF group, SLE+ 2500 group, RA+ 1000 BA group and RA+ 250 CaF group (* p\u0026lt;0,05, ** p\u0026lt;0,01).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4706771/v1/00b7b3f56a627b463f3da650.png"},{"id":62153783,"identity":"16dd93f8-1ade-42c6-a254-beba0433d2d1","added_by":"auto","created_at":"2024-08-09 20:54:27","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":73586,"visible":true,"origin":"","legend":"\u003cp\u003eTh17/Treg cell percentage in HC, SLE and RA patients PBMCs after low and high dose BA and CaF treatment.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4706771/v1/6c1078dd1ee2b17d18af5253.jpeg"},{"id":67681995,"identity":"f716ef5f-063d-46dd-a7a8-b3d6f839a7a5","added_by":"auto","created_at":"2024-10-28 16:12:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1949018,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4706771/v1/1ff47b3d-3546-4241-b30d-ecbe0ba6fed6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of Boric Acid and Calcium Fructoborate on T Helper Cell Differentiation by Influencing Foxp3 and Ror-γt in Rheumatoid Arthritis and Systemic Lupus Erythematosus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA) is a chronic, systemic, autoimmune, inflammatory disease caused by the accumulation of immune complex, especially in the joints, formed by the combination of modified proteins in the body and autoantibodies developed against them [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In Systemic Lupus Erythematosus (SLE), another rheumatic autoimmune disease, autoantibodies formed against nuclear compartments generate immunocomplex and cause tissue inflammation and multiorgan damage [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The worldwide prevalence of RA were estimated to be 460 per 100,000 persons according to an article published in 2020 and for SLE the number were 43.7 per 100,000 persons according to an article published in 2022 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This means that millions of people are affected by RA and SLE globally in every year. Till now no cure for these diseases and the etiology also is not clear. Although the etiology of both RA and SLE are not clearly elucidated, recent studies demonstrated the role of different types of helper T cells in their pathogenesis. Immunosuppressor T helper cells expressing Forkhead box protein 3 (\u003cem\u003eFoxp3\u003c/em\u003e) transcription factor usually called Regulator T (Treg) cells and inflammatory T helper cells expressing retinoid-related orphan receptor gamma t (\u003cem\u003eRor-γt\u003c/em\u003e) transcription factor called Th17 cells are essential CD4\u003csup\u003e+\u003c/sup\u003e T cell subsets that play important role in pathogenesis of RA and SLE [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Expression of transcription factor Foxp3 is essential for the suppresser role of Treg cell in peripheral and it is one of the most specific markers for identify Treg cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. High and stabile expression of Foxp3 manage the transcriptional and functional landscape of Treg cells. Mutation of Foxp3 had been found related to autoimmunity in human and mouse [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Treg suppression conduct in four mechanisms directly or indirectly: Treg secreted inhibitory cytokines (IL-10, IL-35, TGF-β) display suppression; Treg induce target cell cytolysis; Treg disrupt target metabolism; Treg regulate dendritic cells maturation or function to suppress inflammation [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Due to those positive effects of Treg cells several approaches including IL-2 therapy and Treg cell expansion therapies were investigated in autoimmune disease such as RA [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Treg cells from RA patients that expanded with rapamycin were found to display stable Foxp3 expression and suppressed synovial conventional T cell proliferation more effectively compared to their ex vivo counterparts [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Rituximab treatment in the early phase of B-cell depletion increased mRNA levels of Foxp3 in patients with active SLE. During follow-up, patients in clinical remission revealed persistently elevated Foxp3 mRNA levels, whereas patients with active disease have lower expression of this marker [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Th17 cells contribute to the tissue inflammation and pathogenesis of diverse autoimmune diseases, including psoriasis, rheumatoid arthritis, inflammatory bowel disease (IBD) and multiple sclerosis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Th17 cells inflame inflammation in the RA joint by secreting several inflammatory cytokines such as IL-17, GM-CSF. And together with fibroblast like synoviocytes Th17 cells produce TNF-α and IL-1β. GM-CSF production was found to responsible for the chronic inflammation induced by Th17 in RA [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Paradowska-Gorycka A. and his colleges investigated the Th17/Treg-related transcriptional factor expression and cytokine profiles of RA patients and revealed that in isolated Peripheral Blood Mononuclear Cell (PBMC)s Treg(CD4\u003csup\u003e+\u003c/sup\u003eCD25\u003csup\u003ehigh\u003c/sup\u003eCD127\u003csup\u003e\u0026minus;\u003c/sup\u003e)/Th17(CD4\u003csup\u003e+\u003c/sup\u003eCCR6\u003csup\u003e+\u003c/sup\u003eCXCR3\u003csup\u003e\u0026minus;\u003c/sup\u003e) ratio were lower in RA patients compared to the Healthy Controls [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Yao M.X. et al. found Th17(CD4\u003csup\u003e+\u003c/sup\u003eIL-17A\u003csup\u003e+\u003c/sup\u003e)/Treg(CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e) ratio were higher in collagen induced arthritis Rat model [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Oxidative stress and metabolic abnormalities have been found to related to the imbalance of Treg and Th17 in SLE [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In a SLE mice model Cucurbitacin IIb, an substrate extracted from a traditional Chinese medicine, found to be improved kidney injury by balancing the Treg(CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e) /Th17(CD4\u003csup\u003e+\u003c/sup\u003eIL-17A\u003csup\u003e+\u003c/sup\u003e) percentage in the meanwhile upregulated Foxp3 expression and downregulated \u003cem\u003eRor-γt\u003c/em\u003e expression in splenic lymphocytes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Latest studies signify that attempts to reduce pathogenic Th17 cell population and increase Treg cell stability may provide promising therapeutic approaches in both SLE and RA treatment.\u003c/p\u003e \u003cp\u003eBoron and its compounds have been found to have anti-inflammatory, immunomodulatory effects [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Boric Acid (BA) is one of the most important organoboron compound that found physiologically in biologic systems [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Calcium Fructoborate (CaF) is a sugar-borate compound naturally found in fruits and vegetables [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Both BA and CaF have been studied by researchers in terms of their effects on inflammation. Jin ErHui et al. demonstrated that low dose BA could induce splenic lymphocyte proliferation and reduce CD4\u003csup\u003e+\u003c/sup\u003e/CD8\u003csup\u003e+\u003c/sup\u003e cell ratio [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Routray I. and Shakir A. also found that sodium tetraborate increase CD4\u003csup\u003e+\u003c/sup\u003e T helper cell population in a dose-depended manner [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Bortezomib has been studied in SLE patients\u0026rsquo; PBMCs and it revealed that 4 ug/ml bortezomib decreased CD3\u003csup\u003e+\u003c/sup\u003eCD4\u003csup\u003e+\u003c/sup\u003e and CD3\u003csup\u003e+\u003c/sup\u003eCD8\u003csup\u003e+\u003c/sup\u003e cell proliferation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In another study, CaF had given to the RA and Osteoarthritis patients as an adjuvant with entanercept. Patients took 220 mg/day CaF for 60 days and ESR, CRP, IL-1α, IL-6 ve TNF-α levels were found significantly decreased [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. All these evidence demonstrate that boron compound could affect T cells and cytokines released from them.\u003c/p\u003e \u003cp\u003eTo sum up treatments and the understanding of pathogenesis of RA and SLE need an improvement. Boron compounds have promising potential in the treatment of autoimmune diseases. In our study we aimed to investigate the effects of BA and CaF on the Treg and Th17 cell population in PBMCs isolated form RA and SLE patients in terms of Foxp3 and Ror-γt expression. Which is very important to elucidate the boron compounds\u0026rsquo; effects on autoimmunity and for the searching of new therapeutic approaches towards these diseases. Certain cytokines related to the differentiation of Navie T cells towards Treg or Th17 such as IL-6, IL-2, TGF-β and IL-23 along with proinflammatory cytokines IL-17 and TNF-α were also measured in this study.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eBA and CaF were supplied from BOREN (National Boron Research Institution-TURKEY). Antibodies: anti-CD4 PE, anti-Foxp3 FITC and anti-Ror-γt APC (Biolegend) were used for flow cytometry test. ELISA kits for IL-2, IL-17, IL-23, and TGF-β cytokines were purchased from Sunred Biotechnolog Company, China. ELISA kits for IL-6 and TNF-α cytokines were purchased from ELK Biotechnolog Company, China. RPMI-1640 (NutriCulture), Fetal Bovine Serum Heat Inactivated (Biosera), Penicilin-Streptomycin (Gibco by Life Technology), Cell Viability Detection Kit-8 (Eco-Tech Biotechnology), Dulbecco\u0026rsquo;s Phosphate Bufferd Saline 10X (Biosera), Dimethyl Sulfoxide Cell Culture Grade (AppliChem), Lymphocytes Separation Media (Capricorn) were used in cell culture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePatients and Study Design\u003c/h2\u003e \u003cp\u003eThose who were over the age of 18 and diagnosed with RA in accordance with the ACR/EULAR 2019 RA classification were included in our study and determined as the RA Group (n\u0026thinsp;=\u0026thinsp;10). Those who were over the age of 18 and diagnosed with SLE in accordance with the ACR/EULAR 2019 SLE classification were included in our study and determined as the SLE Group (n\u0026thinsp;=\u0026thinsp;5). For RA and SLE patients, those who use biological agents (such as TNFα and interleukin inhibitors), pregnant women, those with active infections and those with other autoimmune diseases were not included in the study. For healthy volunteers, individuals over the age of 18 who did not have any autoimmune disease history, did not have any recent surgery, were not pregnant and did not have active infection were selected as the Control Group (n\u0026thinsp;=\u0026thinsp;9). Informed consents were taken from all participants according to the principles of the Declaration of Helsinki. The local scientific ethics committee of Manisa Celal Bayar University, Institute of Medicine approved the study protocol. Approval document number: 20.478.486. A maximum of 10 ml of peripheral blood samples were collected from all participants for further isolation of PBMCs. Blood samples were collected in sterile environment in blood tubes containing heparin. Clinical data describing study subject were given in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIsolation of PBMCs and Cell Culture\u003c/h2\u003e \u003cp\u003eFor PBMC isolation 3.5 ml of lymphocyte isolation solution was transferred to a sterile 15 ml falcon tube. Fresh blood was slowly dropped over the solution and two layers were formed with the isolation solution on the bottom and the blood sample on the top. Four separate gradient layers were obtained by centrifuging the Falcon tube at 1800 rpm for 30 min at room temperature. After centrifugation PBMC containing white blood cells were in the second layer of the whole gradient. The upper layer containing the serum is removed with a sterile pipette. The second layer is the floor with PBMCs and the PBMCs were transferred to a separate falcon tube with a sterile pipette. For washing sterile 1X PBS was added to fill the falcon and centrifuged at 1800 rpm for 10 minutes and the wash step repeated three times. After washing step, the pellets were dissolved with 1 ml RPMI-1640 cell culture medium containing 10% Fetal Bovine Serum (FBS) 1% penicillin-streptomycin and cell counting was performed. This procedure was applied to the blood samples of all volunteers participating in the study. Average 15x 10\u003csup\u003e6\u003c/sup\u003e \u0026minus;\u0026thinsp;30x 10\u003csup\u003e6\u003c/sup\u003e cells were isolated from each participant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell Proliferation and Cytotoxicity Analysis\u003c/h2\u003e \u003cp\u003eT cells are suspension cells thus WST-8 test was performed to test the proliferative and/or cytotoxic effects of BA and CaF on isolated PBMCs. Because supernatant doesn\u0026rsquo;t need to be removed before reading the formazone in microplate reader in the WST-8 test, PBMCs obtained from volunteers were cultured into 96 well plates and cell proliferation or inhibition effects were determined after 24, 48 and 72 hours. Cells were cultured in density of 10000 cell/well in 180 ul culture medium. BA and CaF substances prepared by dissolving 10-100-250-500-1000-2500-5000 \u0026micro;M doses with distilled water were applied 10 \u0026micro;l/well after the cultured cells were rested and kept overnight in an incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. 10 \u0026micro;l of WST-8 solution was added to the plates at the 24th, 48th and 72nd hours and kept in the incubator for 3 hours, wrapped with aluminum foil. Dihydrogenase in living cells stains WST-8 in orange color and the amount of formazone formed from this color was read in a microplate reader (BioTek) device at 450 nm wavelength in accordance with the manufacturer's instructions The experiments were designed in triplicate. After the results were saved in Excel, the IC50 value of the items was determined according to the %Radical Scavenging Activity. %Radical Scavenging Activity were calculated with the formula:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:\\text{%}\\text{R}\\text{a}\\text{d}\\text{i}\\text{c}\\text{a}\\text{l}\\:\\text{S}\\text{c}\\text{a}\\text{r}\\text{v}\\text{e}\\text{n}\\text{g}\\text{i}\\text{n}\\text{g}\\:\\text{A}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}=\\frac{\\text{A}\\text{b}\\text{s}\\text{r}\\text{o}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}\\:-\\:\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{S}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}{\\text{A}\\text{b}\\text{s}\\text{o}\\text{r}\\text{b}\\text{a}\\text{n}\\text{c}\\text{e}\\:\\text{o}\\text{f}\\:\\text{C}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}}\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eFlow Cytometry Analysis\u003c/h2\u003e \u003cp\u003eFlow cytometry method was used to determine the populations of helper T cell subsets. Cells (3x 10\u003csup\u003e5\u003c/sup\u003e cells /ml/well) were cultured in 24 well plates. After overnight incubation, no substance was applied to one well as a control group. A low dose of 100 \u0026micro;M BA and a high dose of 1000 \u0026micro;M BA were administered to the BA group. A low dose of 250 \u0026micro;M CaF and a high dose of 2500 \u0026micro;M CaF were administered to the CaF group. \u003cb\u003eHC (Healthy Control) Group\u003c/b\u003e: PBMCs isolated from healthy controls with no treatment; \u003cb\u003eHC\u0026thinsp;+\u0026thinsp;100 BA Group\u003c/b\u003e: PBMCs isolated from healthy controls and treated with 100 uM BA; \u003cb\u003eHC\u0026thinsp;+\u0026thinsp;1000 BA Group\u003c/b\u003e: PBMCs isolated from healthy controls and treated with 1000 uM BA; \u003cb\u003eHC\u0026thinsp;+\u0026thinsp;250 CaF Group\u003c/b\u003e: PBMCs isolated from healthy controls and treated with 250 uM CaF; \u003cb\u003eHC\u0026thinsp;+\u0026thinsp;2500 CaF Group\u003c/b\u003e: PBMCs isolated from healthy controls and treated with 2500 uM CaF. \u003cb\u003eRA Group\u003c/b\u003e: PBMCs isolated from RA patients without treatment; \u003cb\u003eRA\u0026thinsp;+\u0026thinsp;100 BA Group\u003c/b\u003e: PBMCs isolated from RA patients and treated with 100 uM BA; \u003cb\u003eRA\u0026thinsp;+\u0026thinsp;1000 BA Group\u003c/b\u003e: PBMCs isolated from RA patients and treated with 1000 uM BA; \u003cb\u003eRA\u0026thinsp;+\u0026thinsp;250 CaF Group\u003c/b\u003e: PBMCs isolated from RA patients and treated with 250 uM CaF; \u003cb\u003eRA\u0026thinsp;+\u0026thinsp;2500 CaF Group\u003c/b\u003e: PBMCs isolated from RA patients and treated with 2500 uM CaF. \u003cb\u003eSLE Group\u003c/b\u003e: PBMCs isolated from SLE patients without treatment; \u003cb\u003eSLE\u0026thinsp;+\u0026thinsp;100 BA Group\u003c/b\u003e: PBMCs isolated from SLE patients and treated with 100 uM BA; \u003cb\u003eSLE\u0026thinsp;+\u0026thinsp;1000 BA Group\u003c/b\u003e: PBMCs isolated from SLE patients and treated with 1000 uM BA; \u003cb\u003eSLE\u0026thinsp;+\u0026thinsp;250 CaF Group\u003c/b\u003e: PBMCs isolated from SLE patients and treated with 250 uM CaF; \u003cb\u003eSLE\u0026thinsp;+\u0026thinsp;2500 CaF Group\u003c/b\u003e: PBMCs isolated from SLE patients and treated with 2500 uM CaF. Since the helper T cell surface marker CD4 and transcription factors Ror-γt and Foxp3 will be stained, a combination of surface staining and intracellular staining was used [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Anti-CD4 PE, anti-Foxp3 FITC and anti-Ror-γt APC (Biolegend) antibodies were used for analysis. Cells that were cultured on 24 well plates and treated with different doses of substance were collected by washing them into 1.5 ml Eppendorf after 48hs. Since PBMCs were suspension cells, no removal from the surface was measured. Samples were analyzed by LSR FORTESSA flow cytometry and graphics were measured by flowjo program.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCytokine Analysis by ELISA Test\u003c/h2\u003e \u003cp\u003eCytokines secreted in the culture medium were detected using ELISA method. BA and CaF treated or untreated culture medium of PBMCs were collected in sterile 1,5 ml Eppendorf and stored in -20 \u003csup\u003eo\u003c/sup\u003eC until the measurement of ELISA test. Standard solutions were prepared for each cytokine according to the manufacturer\u0026rsquo;s constructions. Standards, blank and sample wells were determined and after staining and washing steps stop solution were added. At the end of the procedure plates were immediately read with microplate reader in 450 nm wavelength. Standard curves were obtained, and cytokine levels were determined.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy Subject Clinical Data.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003cp\u003eCharacters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHealthy Donors\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSLE Patients (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRA Patients\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26\u0026ndash;53, 33 (Median)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20\u0026ndash;55, 25 (Median)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e36\u0026ndash;64, 52 (Median)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGender\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFemale (5)\u003c/p\u003e \u003cp\u003eMale (4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFemale (5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFemale (4)\u003c/p\u003e \u003cp\u003eMale (6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDAS28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5,33\u0026thinsp;\u0026plusmn;\u0026thinsp;0,84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSADAI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u0026ndash;16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCRP (mg/dl)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,3\u0026thinsp;\u0026plusmn;\u0026thinsp;1,4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1,2\u0026thinsp;\u0026plusmn;\u0026thinsp;1,1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eESR (mm/hour)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34\u0026thinsp;\u0026plusmn;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44\u0026thinsp;\u0026plusmn;\u0026thinsp;23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRF (IU/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative (2)\u003c/p\u003e \u003cp\u003ePositive (8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAnti-CCP (U/ml)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62\u0026thinsp;\u0026plusmn;\u0026thinsp;63\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;200 (5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAnti-dsDNA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive (4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eComorbidities\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e---\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVarix (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHypertension (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData obtained after cell culture were statistically evaluated using Mixed ANOVA test. Analyzed data were given in means and standard error. P values under 0,05, 0,01 or 0,001 were determined as statistically significant. P\u0026thinsp;\u0026gt;\u0026thinsp;0,05 were evaluated as statistically insignificant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eProliferative/Cytotoxic Effects of BA and CaF on PBMC Cells Isolated from Healty Donors, RA and SLE patients\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe different doses of BA and CaF were applied to the PBMCs isolated from HC, SLE and RA patients. After 24h, 48h, 72h incubation with BA in different doses, PBMCs in all HC, SLE and RA groups did not show any significant proliferative or cytotoxic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). After 24h, 48h, 72h incubation with CaF, PBMCs in all HC, SLE and RA groups didn\u0026rsquo;t show any significant proliferative or cytotoxic effect in doses 10- 100- 250- 500- 1000 \u0026micro;M. However, higher doses of CaF including 2500\u0026ndash;5000 \u0026micro;M showed proliferative effects in all HC, SLE and RA groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0,001). According to these results for further analysis 100 \u0026micro;M and 1000 \u0026micro;M were chosen for BA treatment and 250 \u0026micro;M and 2500 \u0026micro;M were chosen for CaF treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eEffects of BA and CaF on Treg (CD4\u003c/em\u003e \u003csup\u003e \u003cem\u003e+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eFoxp3\u003c/em\u003e \u003csup\u003e \u003cem\u003e+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003e) and Th17 (CD4\u003c/em\u003e \u003csup\u003e \u003cem\u003e+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003eRor-γt\u003c/em\u003e \u003csup\u003e \u003cem\u003e+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003e) cell population in isolated PBMCs\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe Low (100 \u0026micro;M BA and 250 \u0026micro;M CaF) and the high doses (1000 \u0026micro;M BA and 2500 \u0026micro;M CaF) of BA and CaF were applied to the isolated PBMCs then Treg (CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e) and Th17 (CD4\u003csup\u003e+\u003c/sup\u003eRor-γt\u003csup\u003e+\u003c/sup\u003e) cell populations were tested in flow cytometry \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Treg cell population in HC group, SLE group and RA group were 4,71% (0,58), 4,78% (0,79) and 10,59% (0,56) respectively. SLE group was similar Treg population compared to the HC group in nontreated controls. RA group found to have higher Treg population without any treatment compared to the HC group and SLE group.\u003c/p\u003e \u003cp\u003eIn HC Group, both low and high dose BA administration caused an increase in the CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg population compared to the substance-free control, while both doses of CaF administration caused a decrease in Treg population (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, the effects of both BA and CaF application were insignificant (HC\u0026thinsp;+\u0026thinsp;100 BA Group p\u0026thinsp;=\u0026thinsp;0,999, HC\u0026thinsp;+\u0026thinsp;1000 BA Group p\u0026thinsp;=\u0026thinsp;0,660, HC\u0026thinsp;+\u0026thinsp;250 CaF Group p\u0026thinsp;=\u0026thinsp;0,999 and HC\u0026thinsp;+\u0026thinsp;2500 CaF Group p\u0026thinsp;=\u0026thinsp;0,990, p\u0026thinsp;\u0026gt;\u0026thinsp;0,05). In SLE Group, low dose BA administration decreased the Treg population while high dose BA administration increased its population, but these results were not statistically significant (SLE\u0026thinsp;+\u0026thinsp;100 BA Group p\u0026thinsp;=\u0026thinsp;0,644, SLE\u0026thinsp;+\u0026thinsp;1000 BA Group p\u0026thinsp;=\u0026thinsp;0,642, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In SLE\u0026thinsp;+\u0026thinsp;2500 CaF group CD4\u0026thinsp;+\u0026thinsp;Foxp3\u0026thinsp;+\u0026thinsp;Treg population were decreased, whereas in SLE\u0026thinsp;+\u0026thinsp;250 CaF group Treg population were increased compared to the nontreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), but these results also were statistically insignificant (p\u0026thinsp;=\u0026thinsp;0.786 and p\u0026thinsp;=\u0026thinsp;0.686, respectively p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the RA group, high-dose (1000 \u0026micro;M) BA administration, as well as both low-dose (250 \u0026micro;M) and high-dose (2500 \u0026micro;M) CaF administration, tend to decrease the Treg population compared to the substance-free control. Only low-dose BA application increased the Treg population (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, these values were found to be statistically significant only in RA\u0026thinsp;+\u0026thinsp;2500 CaF group (p\u0026thinsp;=\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0,01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn nontreated controls Th17 cell rate were 16.99% (1.94) in the HC Group, 15.36% (2.60) in the SLE Group and 20.36% (1.84) in the RA Group. When both doses of BA and high dose of CaF applied groups were compared with the nontreated control group, it was observed that there was a decrease in Th17 cell population in the PBMCs isolated from healthy donors (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). But this effect was found to be statistically insignificant (HC\u0026thinsp;+\u0026thinsp;100 BA p\u0026thinsp;=\u0026thinsp;0.999, HC\u0026thinsp;+\u0026thinsp;1000 BA p\u0026thinsp;=\u0026thinsp;0.144, HC\u0026thinsp;+\u0026thinsp;2500 CaF p\u0026thinsp;=\u0026thinsp;0.786, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In HC\u0026thinsp;+\u0026thinsp;250 CaF group Th17 population tend to elevate but this effect also statistically insignificant (p\u0026thinsp;=\u0026thinsp;0.999, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In the SLE, it was observed that both doses of BA and both doses of CaF increased the Th17 cell population compared to the drug-free control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This effect was found to be statistically significant except SLE\u0026thinsp;+\u0026thinsp;1000 BA group (SLE\u0026thinsp;+\u0026thinsp;100 BA p\u0026thinsp;=\u0026thinsp;0.042, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. SLE\u0026thinsp;+\u0026thinsp;250 CaF p\u0026thinsp;=\u0026thinsp;0.001, SLE\u0026thinsp;+\u0026thinsp;2500 CaF p\u0026thinsp;=\u0026thinsp;0.007, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01. RA\u0026thinsp;+\u0026thinsp;1000 BA p\u0026thinsp;=\u0026thinsp;0.015, RA\u0026thinsp;+\u0026thinsp;250 CaF p\u0026thinsp;=\u0026thinsp;0.018, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similar to the SLE group, in the RA group, both doses of BA and both doses of CaF were observed to increase the Th17 cell population when compared to the drug-free control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). And this effect was found statistically significant in high dose BA and low dose CaF application (RA\u0026thinsp;+\u0026thinsp;100 BA p\u0026thinsp;=\u0026thinsp;0.999, RA\u0026thinsp;+\u0026thinsp;2500 CaF p\u0026thinsp;=\u0026thinsp;0.999, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05. RA\u0026thinsp;+\u0026thinsp;1000 BA p\u0026thinsp;=\u0026thinsp;0.015, RA\u0026thinsp;+\u0026thinsp;250 CaF p\u0026thinsp;=\u0026thinsp;0.018, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the above results Th17/Treg ratio were calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Both Th17 and Treg percentages were higher in the RA group than in the control and SLE groups. The Th17/Treg ratio in three groups were HC Group 3.65% (0,36), SLE Group 3,69% (0,49) and RA Group 2,01% (0,35). Only in HC\u0026thinsp;+\u0026thinsp;1000 BA group Th17/Treg percentage were significantly decreased. However not similar effects were observed in SLE and RA groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEffects of BA and CaF on IL-2, IL-6, IL-17, IL-23, TNF-α and TGF-β Cytokine Levels\u003c/h2\u003e \u003cp\u003eCytokine (IL-2, IL-6, IL-17, IL-23, TNF-α and TGF-β) levels of cultured PBMCs were analyzed by ELISA test. Effects of different doses of BA on cytokine levels were given in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. TNF-α levels were increased dose-depended pattern with BA application in HC group and SLE group compared to the control. And these effects were statistically significant in high dose BA groups. However, in RA group low dose BA decreased TNF-α level significantly compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0,034). Both dose of BA treatment significantly affected the IL-17 level in HC group. IL-17 level was decreased significantly with both dose of BA applications in SLE and RA group. IL-23 level was increased with low dose BA treatment in all groups. And statistically significant in HC group and RA group. In SLE group both doses of BA administration didn\u0026rsquo;t significantly change the IL-23 level. Different doses of BA application decreased the IL-2 level in HC group and low dose BA decreased more effectively. Low and high dose of BA treatment tend to increase IL-2 level in SLE group but this effect was not statistically significant. BA administration doesn\u0026rsquo;t effectively change the IL-2 level in RA group. BA application increased the IL-6 level in HC group and this effect was statistically significant only in HC\u0026thinsp;+\u0026thinsp;100 BA group. Both low and high doses of BA application showed similar effects in IL-6 levels of SLE and RA groups. Low dose BA administration decreased IL-6 level more effectively than high dose BA in both SLE and RA groups. In HC group, SLE group and RA group low dose BA treatment increased the TGF-β secreted by PBMCs in cell culture media. However, this effect was only statistically significant in SLE\u0026thinsp;+\u0026thinsp;100 BA group.\u003c/p\u003e \u003cp\u003eEffects of different doses of CaF on cytokine levels were given in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Similar to BA low dose CaF decreased TNF-α level significantly compared to the nontreated control in RA group. In SLE and HC groups different dose of CaF treatment increased TNF-α level in dose-depended manner compared to the control. However, this effect was statistically insignificant. Low dose CaF significantly decreased the IL-17 levels in SLE group but doesn\u0026rsquo;t change it in HC and RA groups. However, high dose CaF significantly increased IL-17 levels in all groups compared to the nontreated control. IL-23 level was displayed similar patterns after low and high dose CaF applications in all groups which is increased with low dose CaF and decreased with high dose CaF. But these results were statistically significant in SLE\u0026thinsp;+\u0026thinsp;250 CaF group, HC\u0026thinsp;+\u0026thinsp;250 CaF group and HC\u0026thinsp;+\u0026thinsp;2500 CaF group. IL-2 level were not significantly changed with both dose CaF applications in RA group. In SLE group IL-2 level were increased with low dose CaF application and doesn\u0026rsquo;t change with high dose compared to the nontreated control. In HC group both doses CaF application significantly decreased the IL-2 levels. IL-6 levels were not changed with both dose CaF administration in HC group. Low dose CaF application decreased IL-6 levels while high dose significantly increased its levels in RA group. In SLE group low dose CaF increased the IL-6 levels. However, IL-6 level were stayed steady with high dose CaF. Effects of two different dose of CaF on TGF-β levels in HC group and SLE group were showed similar patterns. In which TGF- β levels were increased dose-depended manner and its statistically significant in HC\u0026thinsp;+\u0026thinsp;250 CaF group, HC\u0026thinsp;+\u0026thinsp;2500 CaF group and SLE\u0026thinsp;+\u0026thinsp;2500 CaF group. In RA group both dose CaF application decreased the TGF-β insignificantly.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCytokine levels of all Groups after different doses of BA application.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCharacters\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eGroups\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(Mean, St.E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100 \u0026micro;M BA\u003c/p\u003e \u003cp\u003e(Mean, St.E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1000 \u0026micro;M BA\u003c/p\u003e \u003cp\u003e(Mean, St.E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003e(Control vs 100BA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003e(Control vs 1000BA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003e(100BA vs 1000BA)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eComparison of Effects of BA Among Groups\u003c/p\u003e \u003cp\u003e(P-value)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eTNF-α (pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e174,68 (70,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e222,66 (55,85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e426,78 (89,86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,035*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,295\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,788\u003c/p\u003e \u003cp\u003eHC vs RA 0,999\u003c/p\u003e \u003cp\u003eSLE vs RA 0,371)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e203,88 (94,51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e313,16 (74,93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e636,95 (120,56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,634\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,006*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,025*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e324,79 (66,82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e158,05 (52,98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e219,58 (85,25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,034*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,711\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-17\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e278,74 (39,42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e272,17 (15,66)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e268,13 (15,79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,078\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,200\u003c/p\u003e \u003cp\u003eHC vs RA 0,138\u003c/p\u003e \u003cp\u003eSLE vs RA 0,999)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e465,52 (52,89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e276,81 (20,96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e298,87 (21,19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,295\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e394,54 (37,40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e320,97 (14,82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e304,06 (14,98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,026*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,003*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,224\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-23 (ng/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,29 (0,42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8,73 (0,41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8,67 (0,36)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,242\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,337\u003c/p\u003e \u003cp\u003eHC vs RA 0,726\u003c/p\u003e \u003cp\u003eSLE vs RA 0,999)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,75 (0,56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7,60 (0,55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6,43 (0,48)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,008*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,63 (0,40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7,71 (0,39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7,60 (0,34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,014*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,023*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-2 (ng/l)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36,47 (3,27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22,14 (1,20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29,31 (0,78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,093\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,359\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,472\u003c/p\u003e \u003cp\u003eHC vs RA 0,999\u003c/p\u003e \u003cp\u003eSLE vs RA 0,999)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21,81 (4,39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29,38 (1,61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26,56 (1,05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,335\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28,03 (3,11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28,77 (1,14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27,56 (0,74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,967\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-6 (pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18,98 (1,20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23,66 (1,55)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21,06 (1,40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,017*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,684\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,030*\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,568\u003c/p\u003e \u003cp\u003eHC vs RA 0,026\u003c/p\u003e \u003cp\u003eSLE vs RA 0,926)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25,38 (1,61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21,08 (2,08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23,62 (1,88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29,19 (1,14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20,92 (1,47)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24,80 (1,33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,035*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,191\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eTGF-β (pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19,32 (0,75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21,01 (1,26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21,95 (1,06)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,365\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,030*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,019*\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,026\u003c/p\u003e \u003cp\u003eHC vs RA 0,098\u003c/p\u003e \u003cp\u003eSLE vs RA 0,947)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20,74 (1,00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24,92 (1,69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e28,61 (1,42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,022*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,139\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23,14 (0,71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24,60 (1,19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22,35 (1,01)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHC\u0026thinsp;=\u0026thinsp;Healthy Control Group. SLE\u0026thinsp;=\u0026thinsp;Systemic Lupus Erythematosus Group. RA\u0026thinsp;=\u0026thinsp;Rheumatoid Arthritis Group. St.E\u0026thinsp;=\u0026thinsp;Standard Error. *= Statistically Significant.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCytokine levels of all Groups after different doses CaF application.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCharacters\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003eGroups\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003cp\u003e(Mean, St.E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e250 \u0026micro;M Caf\u003c/p\u003e \u003cp\u003e(Mean, St.E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2500 \u0026micro;M caf\u003c/p\u003e \u003cp\u003e(Mean, St.E)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003e(Control vs 250caf)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003e(Control vs 2500caf)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003cp\u003e(250caf vs 2500caf)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eComparison of Effects of caf Among Groups\u003c/p\u003e \u003cp\u003e(P-value)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eTNF-α (pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e174,68 (70,44)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e248,53 (25,17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e502,18 (136,18)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,073\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,375\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,999\u003c/p\u003e \u003cp\u003eHC vs RA 0,970\u003c/p\u003e \u003cp\u003eSLE vs RA 0,589)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e203,88 (94,51)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e413,34 (75,35)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e416,27 (182,70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,509\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e324,79 (66,82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e125,99 (53,28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e207,44 (129,19)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,031*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-17\u003c/em\u003e\u003c/p\u003e \u003cp\u003e\u003cem\u003e(pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e278,74 (39,42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e279,60 (36,87)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e376,24 (24,53)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,996\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,014\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,016\u003c/p\u003e \u003cp\u003eHC vs RA 0,126\u003c/p\u003e \u003cp\u003eSLE vs RA 0,576)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e465,52 (52,89)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e375,84 (49,43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e605,16 (32,91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e394,54 (37,40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e386,68 (34,96)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e447,12 (23,27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,023*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,008*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-23 (ng/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,29 (0,42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9,77 (0,93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4,61 (0,43)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,007*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,008*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,171\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,196\u003c/p\u003e \u003cp\u003eHC vs RA 0,866\u003c/p\u003e \u003cp\u003eSLE vs RA 0,895)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,75 (0,56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11,53 (1,25)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6,41 (0,57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,006*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,002*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,63 (0,40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8,87 (0,88)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7,04 (0,41)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,087\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,165\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-2 (ng/l)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e36,47 (3,27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21,48 (0,84)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e18,04 (1,22)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,037*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,171\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,999\u003c/p\u003e \u003cp\u003eHC vs RA 0,509\u003c/p\u003e \u003cp\u003eSLE vs RA 0,257)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21,81 (4,39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26,53 (1,12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e23,43 (1,64)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,241\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28,03 (3,11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28,38 (0,79)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e27,83 (1,16)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eIL-6 (pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18,98 (1,20)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20,46 (5,56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19,13 (2,78)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,023\u003c/p\u003e \u003cp\u003eHC vs RA\u0026thinsp;\u0026lt;\u0026thinsp;0,001\u003c/p\u003e \u003cp\u003eSLE vs RA 0,004)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25,38 (1,61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41,25 (7,45)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25,12 (3,72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,221\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29,19 (1,14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23,92 (5,27)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e79,68 (2,63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eTGF-β (pg/ml)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eHC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19,32 (0,75)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22,51 (0,99)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24,71 (0,98)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,043*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0,001*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0,249\u003c/p\u003e \u003cp\u003e(HC vs SLE 0,428\u003c/p\u003e \u003cp\u003eHC vs RA 0,999\u003c/p\u003e \u003cp\u003eSLE vs RA 0,369)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eSLE\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20,74 (1,00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24,74 (1,33)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25,91 (1,31)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,003*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eRA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23,14 (0,71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21,47 (0,94)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21,75 (0,93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0,470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0,473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0,999\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHC\u0026thinsp;=\u0026thinsp;Healthy Control Group. SLE\u0026thinsp;=\u0026thinsp;Systemic Lupus Erythematosus Group. RA\u0026thinsp;=\u0026thinsp;Rheumatoid Arthritis Group. St.E\u0026thinsp;=\u0026thinsp;Standard Error. *= Statistically Significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eFirst of all, we investigated the proliferative/cytotoxic effects of different dose of BA and CaF. Interestingly we found that high dose CaF induce proliferation of PBMCs isolated from study subjects especially after 1000 \u0026micro;M. Though this is not the focus of our study this maybe important for the stimulation of lymphocytes or other type of PBMCs in culture conditions. We think that it\u0026rsquo;s important to determine the cell type proliferated for further investigations. In our study, the effect of different doses of BA and CaF on cytokines released from PBMCs isolated from RA and SLE patients in culture was investigated. As a result, it was observed in our study that BA and CaF application increased TNF-α level in healthy individuals and SLE patients, but both doses of BA and CaF could display anti-inflammatory effects by reducing TNF-α levels in RA patients. Routray I. and Shakir A. added sodium tetraborate dihydrate equivalent to BA at 2-, 2.5- and 3-mM concentrations to the drinking water of mice and isolated the splenic cells after feeding the mice with this drinking water for 10 days. They found that after stimulation with LPS (lipopolysaccharide) in the isolated cells, the CD4\u003csup\u003e+\u003c/sup\u003e T cell population and the levels of TNF-α, IL-6 and IL-1β increased in a dose-dependent manner [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Similar situation maybe the case in our study in HC group and SLE group. And they pointed out that this effect is achieved stabilizing the antigen-receptor complex on the lymphocyte by boric acid. it was observed that high-dose CaF increased IL-17 secretion in cultured PBMCs isolated from both SLE patients and RA patients, on the contrary, high and low-dose BA and low-dose CaF could have anti-inflammatory effects by reducing IL-17 secretion; It was observed that administration of different doses of BA was insufficient to suppress IL-23 level, an inflammatory cytokine plays important role in RA and SLE pathogenesis, CaF could display an anti-inflammatory effect by lowering IL-23 level at high doses in healthy individuals, but it caused an increase in IL-23 secretion in autoimmune conditions such as SLE and RA where the inflammatory balance was impaired; IL-23 is mainly secreted from dendritic cells and macrophages and plays important role in the differentiation of naive T cells into Th17 cells in autoimmune diseases [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, naive T cells must first be stimulated by TGF-β, IL-6 and IL-21 to express the IL-23 receptor [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, whether the increasing effect of boron compounds used in this study on IL-23 level contribute to the inflammation or not couldn\u0026rsquo;t be clearly demonstrated by the results obtained. Meanwhile, the increase in Th17 (CD4\u003csup\u003e+\u003c/sup\u003eRor-γt\u003csup\u003e+\u003c/sup\u003e) cells after treatment with different boron compounds that observed in our study may be related to the increase in IL-23. IL-2 is a cytokine secreted by T cells and is also called T cell growth factor because it causes clonal expansion of T cells. IL-2 is involved in the proliferation of T and B cells, expression of antibodies from B cells, expression of other cytokines, and Fas-mediated apoptosis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study, it was observed that administration of different doses of BA and low-dose CaF may be effective in regulating autoimmunity by increasing IL-2 level in SLE patients. However, it was found that different doses of BA and CaF were insufficient to increase the level of IL-2, which has an autoimmunity suppressive effect, in RA patients. IL-6 has many biological activities and was first known as B cell stimulatory factor-2. IL-6 activates acute phase reactants and plays an important role in Th17 cell differentiation together with TGF-β [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. It was observed that the administration of low and high doses of BA could have an anti-inflammatory effect on PBMCs isolated from patients with SLE and RA by reducing the level of inflammatory cytokine IL-6. It was found that the effect of CaF on IL-6 secretion from PBMCs isolated from SLE and RA patients is complex, and that different doses of CaF may affect IL-6 levels differently in different disease groups. It has been observed that different doses of BA and CaF could display anti-inflammatory effect by increasing TGF-β level in SLE group, affecting helper T cell subgroup differentiation, for example CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg cell differentiation. It was found that different doses of BA and CaF did not have a significant effect on TGF-β levels in the culture medium of PBMCs isolated from RA patients.\u003c/p\u003e \u003cp\u003eIt is known that the disorder of Treg/Th17 balance in autoimmune diseases is associated with the pathogenesis of the disease [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and recently strategies for maintaining the Treg/Th17 balance have been tried to be exhibited with increasing investigations. Treg cells belonging to the helper T cell subgroup require the \u003cem\u003eFoxp3\u003c/em\u003e transcription factor to fulfill its function of maintaining immune tolerance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Th17 cells belonging to the inflammatory helper T cell subgroup, on the other hand, require the \u003cem\u003eRor-γt\u003c/em\u003e transcription factor [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It has been reported in many studies that Treg and Th17 population and function are also important in the pathogenesis of RA and SLE [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Xie M. et al. found in their study that there is Treg/Th17 (CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e/CD4\u003csup\u003e+\u003c/sup\u003eIL-17A\u003csup\u003e+\u003c/sup\u003e) imbalance in PBMCs isolated from RA and SLE patients. And they also showed that this imbalance is due to decreased \u003cem\u003eFoxp3\u003c/em\u003e expression, which is caused by targeting the 3'untranslated region of \u003cem\u003eFoxp3\u003c/em\u003e gene by mi-R34a, and mi-R34a in cells is found to be higher compared to healthy controls due to TNF-α and IL-6. They found that it leads to increased expression of \u003cem\u003eRor-γt\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Yao M.X. et al. have investigated the therapeutical effect of Wu-Teng-Gao, one of the Chinese traditional medicines, on collagen induced arthritis model in terms of Th17/Treg balance. An increase in \u003cem\u003eFoxp3\u003c/em\u003e expression and a decrease in \u003cem\u003eRor-γt\u003c/em\u003e expression were observed. In addition, a decrease in pro-inflammatory cytokines (IL-1, IL-6, TNF-α, IL-17) and an increase in anti-inflammatory cytokines (IL-10 and TGF-β) were observed, and it was found that bone damage was inhibited in histological staining [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In a study conducted in a lupus mouse model, the effect of Cucurbitacin IIb on Th17 and Treg cell percentages, \u003cem\u003eFoxp3\u003c/em\u003e and \u003cem\u003eRor-γt\u003c/em\u003e expression levels in cells isolated from the spleen of mice was investigated. As a result, it was observed that there was an increase in Treg cell population, decrease in Th17 cell population, increase in \u003cem\u003eFoxp3\u003c/em\u003e expression, decrease in \u003cem\u003eRor-γt\u003c/em\u003e expression, increase in IL-10 and TGF-β expression, and decrease in IL-17 and IL-6 levels in Cucurbitacin IIb applied groups compared to control [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our study, CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e Treg and CD4\u003csup\u003e+\u003c/sup\u003eRor-γt\u003csup\u003e+\u003c/sup\u003e Th17 cell populations in PBMCs isolated from RA and SLE patients were investigated and Th17/Treg cell percentage were calculated \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Th17/Treg ratio in our study were found to be lower in RA group compared to the HC group. This maybe because CD4\u003csup\u003e+\u003c/sup\u003eRor-γt\u003csup\u003e+\u003c/sup\u003e cells were investigated as Th17 in our study and in many other studies investigating Treg and Th17, CD4\u003csup\u003e+\u003c/sup\u003eIL-17A\u003csup\u003e+\u003c/sup\u003e Th17 cells were studied [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We aimed to investigate the effects of BA and CaF on CD4\u003csup\u003e+\u003c/sup\u003eFoxp3\u003csup\u003e+\u003c/sup\u003e cells and CD4\u003csup\u003e+\u003c/sup\u003e Ror-γt\u003csup\u003e+\u003c/sup\u003e cells. It is known that Th17 cells have pTh17 and non-pTh17 subgroups [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. It has been reported that STAT proteins along with \u003cem\u003eFoxp3\u003c/em\u003e and \u003cem\u003eRor-γt\u003c/em\u003e are also important in Treg and Th17 functions. It has been reported that STAT3 protein contributes to the pathogenicity of Th17 cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. STAT5, on the other hand, contributes to the differentiation of naive T cells into \u003cem\u003eFoxp3\u003c/em\u003e-expressing immunosuppressive Treg cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While as a result different doses of BA decreased the Th17/Treg ratio in healthy individuals in our study and different doses of CaF increased the Th17/Treg ratio in the RA group. Similar to the RA group different doses of CaF caused an increase in Th17/Treg ratio in the SLE group. In terms of Ror-γt increasing effects boron compounds studied in our study, whether it\u0026rsquo;s contributed to or attenuate the course of inflammation can be elucidated by further investigations towards the cofactors of these signaling pathways mentioned above. When these results are evaluated together with the cytokine levels, low dose BA decreased the pro-inflammatory cytokine IL-17, TNF-α level, except IL-23, and tended to increase IL-6 and TGF-β level. Despite the increased expression of Ror-γt, the decrease in the level of pro-inflammatory cytokines may indicate that BA has an anti-inflammatory effect on non-pTh17 cells. All these results show that BA and CaF could affect Helper T cell differentiation in autoimmune diseases by affecting both \u003cem\u003eFoxp3\u003c/em\u003e and \u003cem\u003eRor-γt\u003c/em\u003e levels in CD4\u0026thinsp;\u003cem\u003e+\u003c/em\u003e\u0026thinsp;T cells. Changes in the \u003cem\u003eFoxp3\u003c/em\u003e and \u003cem\u003eRor-γt\u003c/em\u003e expression may related to the alteration in the IL-17 and TGF-β levels. In particular, the effect of BA and CaF on TNF-α level shows that it could contribute positively to the treatment of autoimmune diseases. However, the effect of BA and CaF on the cytokine network was found to be very complex. Therefore, the effect of BA and CaF on the course of inflammation can be elucidated by further investigation of molecules such as STAT, IL-1, IL-10 and GM-CSF in the downstream signaling pathways.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e This study was performed under the principles of the Declaration of Helsinki. The local scientific ethics committee of Manisa Celal Bayar University, Institute of Medicine approved the study protocol. Approval document number: 20.478.486. Informed consent was obtained from all participants included in the study. The authors have no financial or proprietary interests in any material discussed in this article. All the authors contributed to the study in different ways. M.K. is the supervisor of the study. \u0026Ouml;.SG. managed patient evaluation and patient data collection. F.\u0026Ouml;K. performed part of the cell culture analysis and contributed to the data analysis. R.Y. involved in the literature investigations, sample collection, laboratory analysis, data analysis and manuscript preparation. All authors evaluated and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll the authors contributed to the study in different ways. M.K. is the supervisor of the study. \u0026Ouml;.SG. managed patient evaluation and patient data collection. F.\u0026Ouml;K. performed part of the cell culture analysis and contributed to the data analysis. R.Y. involved in the literature investigations, sample collection, laboratory analysis, data analysis and manuscript preparation. All authors evaluated and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe would like to thank the National Boron Research Institute, BOREN, for providing us Boric Acid and Calcium Fructoborate. This study was founded by the Scientific Research Projects Coordination Unit of Manisa Celal Bayar University, Manisa, Turkey.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLee DM, Weinblatt ME (2001) Rheumatoid arthritis. 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Cell Mol Immunol 15(5):458\u0026ndash;469. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41423-018-0004-4\u003c/span\u003e\u003cspan address=\"10.1038/s41423-018-0004-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Th17, Treg, Foxp3, Ror-γt, Autoimmunity, TNF-α","lastPublishedDoi":"10.21203/rs.3.rs-4706771/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4706771/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMany animal and human studies indicate that Boric Acid and Calcium Fructoborate have effects on helper T cells in immunity. The aim of our study is to evaluate effects of Boric Acid and Calcium Fructoborate on Treg (\u003cem\u003eCD4\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eFoxp3\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e) and Th17 (\u003cem\u003eCD4\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eRor-γt\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e) cell populations and related cytokine levels in mononuclear cells isolated from peripheral blood samples of Rheumatoid Arthritis and Systemic Lupus Erythematosus patients. Newly diagnosed Rheumatoid Arthritis (n\u0026thinsp;=\u0026thinsp;10) patients, Systemic Lupus Erythematosus (n\u0026thinsp;=\u0026thinsp;5) pateints and healthy individuals (n\u0026thinsp;=\u0026thinsp;9) were included in this study. Consent forms were obtained from all individuals participating the study, blood samples were taken, peripheral blood mononuclear cells were isolated. Isolated cells were exposed to low dose and high dose Boric Acid and Calcium Fructoborate in cell culture. Treg and Th17 cell populations were analyzed by flow cytometry after 48 hours of exposure. IL-2, IL-6, IL-17, IL-23, TNF-α and TGF-β levels in the culture medium were tested by ELISA method. At the end of the study, in healthy controls high dose BA improved the Treg/Th17 population but couldn\u0026rsquo;t display similar effect on RA and SLE group. However, both Boric Acid and Calcium Fructoborate at different doses showed an increasing effect on Ror-γt in RA and SLE group. Different doses of BA and CaF treatment found to have a variable effect on cytokine. Both BA and CaF in low doses decreased TNF-α levels in RA group shows that these boron compounds could contribute positively to the treatment of autoimmune diseases.\u003c/p\u003e","manuscriptTitle":"The effect of Boric Acid and Calcium Fructoborate on T Helper Cell Differentiation by Influencing Foxp3 and Ror-γt in Rheumatoid Arthritis and Systemic Lupus Erythematosus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-09 20:54:22","doi":"10.21203/rs.3.rs-4706771/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-05T01:38:45+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-04T20:11:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-04T09:13:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-01T20:40:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-27T12:14:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93464159731535349843659988115687404883","date":"2024-08-25T18:03:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170099481797012770571271758492894329671","date":"2024-08-24T09:55:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150187079664835635562116002538108055524","date":"2024-08-24T09:55:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"110371894730407849504090082114898997760","date":"2024-07-24T11:57:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T07:03:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229704039243320199060528403875699317534","date":"2024-07-11T02:59:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-10T22:15:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-08T23:34:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-08T22:57:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Trace Element Research","date":"2024-07-08T15:45:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"beab7419-6ff0-4c3a-b7c1-348e1ee21bed","owner":[],"postedDate":"August 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:05:09+00:00","versionOfRecord":{"articleIdentity":"rs-4706771","link":"https://doi.org/10.1007/s12011-024-04425-9","journal":{"identity":"biological-trace-element-research","isVorOnly":false,"title":"Biological Trace Element Research"},"publishedOn":"2024-10-24 15:58:04","publishedOnDateReadable":"October 24th, 2024"},"versionCreatedAt":"2024-08-09 20:54:22","video":"","vorDoi":"10.1007/s12011-024-04425-9","vorDoiUrl":"https://doi.org/10.1007/s12011-024-04425-9","workflowStages":[]},"version":"v1","identity":"rs-4706771","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4706771","identity":"rs-4706771","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}