{"paper_id":"4e052c2c-864b-4b19-9f89-cd1a77fbcd29","body_text":"Creative Commons licenses: This is an Open Access article distributed under the terms of the Creative Commons  \nAttribution-NonCommercial-ShareAlike 4.0 International (CC BY -NC -SA 4.0). License (http://creativecommons.org/licenses/by-nc-sa/4.0/).\nExperimental research \nCorresponding author:\nJianfang Gong PhD \nDepartment of Traditional \nChinese Medicine\nShanxi Bethune Hospital\nShanxi Academy of Medical \nSciences\nTongji Shanxi Hospital\nThird Hospital of Shanxi \nMedical University\nTaiyuan, 030032, China;\nTongji Hospital\nTongji Medical College\nHuazhong University  \nof Science and Technology\nWuhan, 430030, China\nE-mail: jianfanggong@ \n163.com\n1 Department of Traditional Chinese Medicine, Shanxi Bethune Hospital, Shanxi \nAcademy of Medical Sciences,Tongji Shanxi Hospital, Third Hospital of Shanxi \nMedical University, Taiyuan, 030032, China; Tongji Hospital, Tongji Medical College, \nHuazhong University of Science and Technology, Wuhan, 430030, China \n2 Department of Radiology, Shanxi Bethune Hospital,Shanxi Academy of Medical \nSciences, Tongji Shanxi Hospital, Third Hospital of Shanxi Medical University, \nTaiyuan, 030032, China; Tongji Hospital, Tongji Medical College, Huazhong University \nof Science and Technology, Wuhan, 430030, China\n3 Department of Obstetrics and Gynecology, Shanxi Bethune Hospital, Shanxi \nAcademy of Medical Sciences, Tongji Shanxi Hospital, Third Hospital of Shanxi \nMedical University, Taiyuan, 030032, China; Tongji Hospital, Tongji Medical College, \nHuazhong University of Science and Technology, Wuhan, 430030, China\nSubmitted: 31 May 2021; Accepted: 28 October 2021\nOnline publication: 7 November 2021\nArch Med Sci  \nDOI: https://doi.org/10.5114/aoms/143423\nCopyright © 2021 Termedia & Banach\nLiriodendrin alleviates sciatic endometriosis-associated \npain in rats via suppressing the inflammatory response \nand regulating the signaling pathway of Pl3K/Akt/mToR\nJianfang Gong1, Liangliang Xue2, Mengling Wei1, Wenli Han3, Shen Jing1\nAbstract  \nIntroduction:  LY294002 has been validated as a PI3K pan-inhibitor in the \npathogenesis of airway inflammation, which attenuated IL-25-induced asth -\nma-like AHR. Liriodendrin was proved to play essential roles in attenuating \nendometriosis-associated pain. This work aimed to gain a  mechanical in -\nsight into the therapeutic efficiency of liriodendrin administration in atten -\nuating endometriosis-associated pain. \nMaterial and methods:  The von Frey filament test and thermal hyperalgesia \ntest were carried out to evaluate the acute and daily efficiency of drug ad -\nministration. Western blot was used to analyze the expression of substance \nP , PI3K/AKT/mTOR, p-PI3K/p-AKT/p-mTOR, and CGRP. ELISA was performed to \nexamine interleukin (IL)-1, IL-2, IL-6, TNF- α and PGE2 levels. \nResults:  As the results showed, liriodendrin significantly attenuated pain \nin endometriosis rats and restored the up-regulation of p-PI3K/p-AKT/  \np-mTOR in the endometriosis rats. The increased IL-1, IL-2, IL-6, TNF- α, and \nPGE2 levels were remarkably restored by liriodendrin in endometriosis rats. \nMoreover, the activated expression of substance P in the ventral horn of the \nspinal cord of endometriosis rats was notably restored by liriodendrin in \naddition to CGRP. Furthermore, liriodendrin effectively restored the lipopoly -\nsaccharide (LPS)-induced up-regulation of p-PI3K/p-AKT/p-mTOR protein as \nwell as the LPS activated IL-6, TNF- α, and IL-1 β mRNA and protein expres -\nsion in 12Z cells. \nConclusions:  We found that compared with LY294200, liriodendrin exerted \na more evident therapeutic effect in the treatment of endometriosis-associ -\nated pain by suppressing the secretion of pro-inflammatory cytokines.\nKey words: liriodendrin, endometriosis, pain, inflammation, Pl3K, Akt, \nmToR.\n\nJianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing\n2 Arch Med Sci\nIntroduction\nThe presence of stromal or glandular cells on \nthe edge of the uterine cavity is a defining char -\nacteristic of endometriosis [1]. Chronic pain such \nas radicular pain, back pain, dyspareunia, and dys-\nmenorrhea affects about 5–10% of young women \n[2, 3]. Endometriosis affecting the sciatic nerve is \na very rare condition [4]. The current treatment op-\ntions include extracting the ectopic lesions and ad-\nministration of hormones and non-steroidal anti-  \ninflammatory drugs (NSAIDs) [5]. Nonetheless, the \ncurrent treatment options have limited efficacy \nand unwanted side effects [6].\nNormal cellular functions such as metabolism, \nsurvival and growth are regulated by two signal -\ning pathways: the phosphatidylinositol-3 kinases \n(PI3Ks) and the mammalian target of rapamycin \n(mTOR) [7, 8]. Both the pathways are intercon -\nnected by a  type of serine/threonine kinase-like \nAKT [9]. Protein kinase B, also known as AKT, plays \na  role in several cellular functions via PI3K and \nmTOR [10]. Stimuli from different types of recep -\ntors, such as antigen receptors, cytokine receptors, \ninsulin receptors, or the insulin-like growth factor \nI receptor, activate the PI3K and mTOR pathways. \nUpon activation, PI3K causes phosphorylation of \nthe phosphorylate phosphatidylinositol 4,5-bis -\nphosphate (PIP2), which produces a second mes -\nsenger, phosphatidylinositol-3,4,5-trisphosphate \n(PIP3). PIP3 further stimulates downstream path -\nways such as AKT. Elevated levels of the PI3K/Akt/\nmTOR pathway components were observed in the \neutopic endometrium of women with endometri -\nosis [11]. The multilevel signaling pathway involv-\ning PI3K, Akt and mTOR correlated well with pain \nand endometriosis [12].\nDeying Dai, an expert in Chinese medicine, \ninvented the prescription formula of Caulis Sar -\ngentodoxae prescription. The formula was found \nto be effective against EMs (90%), menstrual fe -\nver (90.63%) and dysmenorrhea (96.25%) [13]. \nExtensive research has been performed to assess \nits effects in ulcerative colitis in humans. Sargent-\nodoxa cuneata is very effective for treating several \ninflammation conditions affecting the appendix, \nstomach and rheumatic arthritis. However, the \nmechanism of action of the medicine remains \nelusive. Another example of traditional medi -\ncine called liriodendrin, a type of lignin, has been \nstudied for its therapeutic effects. This disaccha -\nride lignan named liriodendrin, (+)-syringaresinol \ndi-O-b-D-glucopyranoside, is the bioactive com -\nponent of Sargentodoxa cuneata  (Oliv.) Rehd. Et \nWils, and a plethora of biological effects against \ninflammation, pain, and arrhythmia have been \nobserved for the medicine [14–16]. This medicine \nhas shown efficacy against disorders such as ar -\nrhythmia, myocardial ischemia, inflammation and \noxidative stress [14, 17]. Moreover, according to \nprevious findings, liriodendrin administration re -\nstored the elevation of neuropeptides in the ven -\ntral horn of the spinal cord of ENDO rat models. \nUpon oral administration liriodendrin is trans -\nformed to syringaresinol, which is attributable to \nthe anti-inflammatory effect [17]. In various hor -\nmone-dependent conditions, the phytoestrogen \nsyringaresinol binds selectively to the estrogen \nreceptors [18]. \nThe proposed mechanism of action of lirio -\ndendrin involved reduced secretion of inflamma -\ntion-inducing cytokines such as interleukin (IL)-6, \ntumor necrosis factor α (TNF-α) and IL-1 β in the \ncolon tissues. A similar decrease in proinflamma -\ntory cytokines was observed in DSS-induced co -\nlon damage models. In the intestine, a  link was \nobserved between the nuclear factor κB (NF- κB) \ncascade and the AKT signaling at transcription \nand translation levels [19, 20]. Liriodendrin blocks \nprotein-1 and/or NF-κB in SW982 human synovial \nsarcoma cells [21].\nBased on the above-mentioned evidence, \nLY294002 may alleviate endometriosis-associated \npain via regulating PI3K signaling, and liriodendrin \nas an extract of Sargentodoxa cuneata may also \ninhibit PI3K signaling, and in addition, liriodendrin \nmay also suppress the inflammatory response \nwhich is also involved in endometriosis-associat -\ned pain. We aimed to validate our hypothesis that \nthe administration of liriodendrin could exhibit \na therapeutic effect in the treatment of endome -\ntriosis-associated pain, the efficacy of which is \nsupposed to be better than LY294002 in endome-\ntriosis. And the novelty of our study was to com -\npare the efficacy of liriodendrin and LY294002 in \nthe treatment of endometriosis.\nMaterial and methods\nAnimal and treatment \nFemale SD rats with ages ranging between \nfourteen and eighteen weeks and weights in the \nrange of 180–220 g were purchased from Charles \nRiver Laboratories Portage, MI. The rats were \nhoused under alternating cycles of dark (12 h) \nand light (12 h) at room temperature and humidi-\nty near 70%. The rats were allowed access to food \nand water. Animals were divided into four groups \nwith 8 rats in each group, i.e.: 1) SHAM group \n(group of animals received the sham operation), \n2) ENDO group (group of animals were subject -\ned to the surgical procedure and established as \nendometriosis rat model), 3) ENDO + LY294002 \ngroup (endometriosis rat models subjected to the \nsurgical procedure to create a rat model of endo -\nmetriosis and received i.p. injection of LY294002) \nand 4) ENDO + liriodendrin group (endometriosis \n\nLiriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response  \nand regulating the signaling pathway of Pl3K/Akt/mToR\nArch Med Sci 3\nrat models subjected to the surgical procedure to \ncreate a rat model of endometriosis and received \noral administration of liriodendrin). Models of sci-\natic nerve endometriosis were created using pre -\nviously reported procedures [22]. \nIrrespective of the estrous cycle of the female \nrats, the autologous uterine tissue was grafted \nnear the site of the sciatic nerve. Initially, the \nrats were put under anesthesia using isoflurane \nthen a 2 cm long incision was created near the \nlower abdomen which exposed the right uterine \nhorn. Later, a 5 mm length of uterus tissue was \ndissected and extracted from the horn region \nof the uterus. It was kept in a Petri dish which \nwas preloaded with a phosphate buffer solution \ncontaining 1% penicillin and streptomycin. Sub -\nsequently, the right sciatic nerve was revealed on \nthe rats positioned on the left side and an inci -\nsion of 1 cm long near the thigh was made. The \nconnective tissues near the desired area were \ncautiously separated. The longitudinal section of \nthe uterus was opened and tied around the nerve \nwith uterus endometrial tissue next to the nerve. \nIn the case of rats that received the Sham opera -\ntions, no implant was installed near the exposed \nright sciatic nerve. After the surgical procedure, \n6.0-grade silk thread was used to suture the in -\ncision, and to prevent the infection gentamicin  \n(50 mg/ml, 0.2 ml, Thermo Fisher Scientific, \nWaltham, MA) was injected into the stomach. In \ngroup 3, the dose of LY294002 was 15 mg/ml, \nwhich was dissolved in DMSO. A dose of 30 mg/kg  \nwas administered to the animals via a microsy -\nringe into the i.t. tubing. In group 4, 100 mg/kg \nliriodendrin was pretreated about 3 days before \nthe surgery. The animal experiment procedures \nwere approved by the institutional ethical com -\nmittee. \nBehavioral testing \nTo assess the behavior of the rats, the von Frey \nfilament test was used. Briefly, the von Frey filament \ntest was performed following the standard proto-\ncol, which involves applying mechanical stimuli to \nmeasure the hypersensitivity to mechanical stress. \nThe key factor to measure was the paw withdraw-\nal threshold. A  series of ten von Frey filaments of \nequal logarithmic bending force was used in a se -\nquence. To perform the test, the rats were allowed \nto habituate on the wire gauge surface for 15 min. \nTo analyze the thermal hyperalgesia, a heat source \nwas applied to the hind paw of the rats. The heat \nsource was made up of a lamp fitted with a high-in-\ntensity bulb of power 50 W, and a voltage of 8 V . The \nexposure limit was decided to be 20 s so that the \nrats did not get skin burns. Following these behavior \ntests, the effects of LY294002 following the sciatic \nendometriosis procedure were evaluated.\nProcedure for abdominal puncture \nThe medicine was administered orally to rats \nfor 21 days and after that 10 ml of PBS buffer was \nfilled into the stomach cavity of rats under anes -\nthesia with the help of a  syringe. The rats were \nshaken gently for 10 min and a  puncture was \nmade into the abdominal wall of the rats using \na needle. The peritoneal fluid was extracted with \nthe help of a needle. Later, the rats were eutha -\nnized, and the samples were collected. The upper \nclear layer of the peritoneal fluid and blood sam -\nples were obtained and refrigerated at −80°C.\nEnzyme-linked immunosorbent assay \nThe peritoneal fluid samples and blood sam -\nples were extracted and stored at 2–8°C for \na maximum of 5 days. Then, substance P , IL-1, IL-2, \nIL-6, TNF-α, PGE2, and CGRP levels were analyzed \nusing the protocol provided with the kit in accor -\ndance with published methods [23]. The antibody \nwas incubated and procedures for washing were \nperformed based on the published methods. The \nabsorbance of the ELISA experiments was record-\ned using the absorbance microplate reader (Mo -\nlecular Devices, San Jose, CA).\nCell culture \nIn this study, we established our cell models \nwith 12Z cells (Applied Biological Materials (abm) \nInc., Canada), which were an immortalized human \nendometriotic cell line. The cells were cultured in \n37°C and 5% CO 2 in Roswell Park Memorial In -\nstitute 1640 medium containing 10 mM HEPES \nand other antibiotics according to previously pub-\nlished methods [24]. The cells were transferred \ninto 6-well plates and cultured until they were \nconfluent. To these cells, the drugs at the de -\nsired concentration were added to the wells. The \ncells were divided into four groups: 1) negative \ncontrol group, 2) LPS group; the cells were stimu -\nlated with 100 ng/ml of lipopolysaccharide (LPS), \n3) LPS + LY294002 group; the cells were treated \ninitially with 10 μM LY294002 and 6 h later with  \n100 ng/ml of LPS, and 4) LPS + liriodendrin group; \nthe cells were pretreated with 50 μM liriodendrin \nand 6 h later treated with 100 ng/ml of LPS. All \nexperimental procedures were done four times \nusing the above-mentioned protocol.\nRNA isolation and real-time PCR\nThe authors performed a polymerase chain re -\naction test using the RNA extraction kit obtained \nfrom Thermo Fisher Scientific. The genetic mate -\nrial was removed from the samples using the kit. \nThe reverse transcription of the RNA material in \nthe samples into the cDNA was carried out using \n\nJianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing\n4 Arch Med Sci\nthe iScript cDNA Synthesis Kit (Bio-Rad, Hercules, \nCA). Later, the quantitative RT-PCR was carried \nout using the iTaq Universal SYBR Green Super -\nmix (Bio-Rad, Hercules, CA). The polymerase re -\naction was performed with Applied Biosystems \nReal-Time PCR Instruments (Thermo Fisher Sci -\nentific, Waltham, MA). The relative expression of \nsubstance P , IL-6, TNF-α, IL-1 β, NF- κB and CGRP \nwas calculated relative to the reference standard \ngenes by the 2 ∆∆Ct method following published \nprotocols [25].\nWestern blot analysis\nThe Western blot analysis was performed ac -\ncording to published protocols [26]. The cultured \ncells and tissue samples obtained from the decap-\nitated animals were first washed with PBS buffer. \nRadioimmunoprecipitation buffer was used as \na lysed buffer to treat the cells and tissues. This \nprocedure was used to extract the enclosed pro -\nteins from the cells and tissue samples. The mix -\nture of proteins obtained from the samples was \nseparated into bands using 10% SDS-polyacryl -\namide gel. Then, the total protein content isolat -\ned from the cells was resolved on a 10% sodium \ndodecyl sulfate-polyacrylamide gel using an elec -\ntrophoresis system (VWR, Radnor, PA). Later, the \nseparated bands were expanded on a PVDF mem-\nbrane and the reaction was blocked by skim milk. \nSubsequently, antibodies for CGRP (Cat#ab47207, \nAbcam, Cambridge, UK), PI3K (Cat#ab32089, Ab -\ncam, Cambridge, UK), AKT (Cat#ab8805, Abcam, \nCambridge, UK), mTOR (Cat#ab134903, Abcam, \nCambridge, UK), p-PI3K (Cat#ab278545, Abcam, \nCambridge, UK), p-AKT (Cat#ab38449, Abcam, \nCambridge, UK), p-mTOR (Cat#ab109268, Abcam, \nCambridge, UK), substance P (Cat#ab14184, Ab -\ncam, Cambridge, UK), TNF- α (Cat#ab183218, Ab-\ncam, Cambridge, UK) and IL-1 β (Cat#ab254360, \nAbcam, Cambridge, UK) were applied to the pro -\ntein bands followed by the secondary antibodies.\nStatistical analysis \nStatistical analysis was carried out by making use \nof SPSS 19.0 software (IBM, Chicago, IL). One-way \nANOVA and Student’s t-tests were used to com -\npare intergroup differences. Tukey’s test was used \nas the post hoc test following one-way ANOVA.  \nAll statistical tests were two-sided, while the p-val-\nues of < 0.05 indicated statistical significance.\nResults\nLY294002 or liriodendrin administration \nattenuated the increased pain  \nin endometriosis rat models \nThe paw withdrawal threshold was significant-\nly lower in ENDO rats when compared with the \ncontrol. LY294002 and liriodendrin administra -\ntion remarkably maintained the paw withdrawal \nthreshold in ENDO treated rats, indicating the ef -\nficiency of acute drug administration (Figure 1 A). \nMoreover, daily drug administration efficiency was \nevaluated every three days using the von Frey fila-\nment test. LY294002 and liriodendrin administra-\ntion showed considerable efficiency in restoring \nthe reduction in the paw withdrawal threshold in \nthe ENDO group (Figure 1 B). Additionally, a ther -\nmal hyperalgesia test was used to examine the ef-\nficiency of acute and chronic drug administration. \nLY294002 and liriodendrin administration showed \nconsiderable efficiency in restoring the paw with-\ndrawal latency loss in rats from the ENDO group \nin both acute drug administration (Figure 2 A) and \ndaily drug administration (Figure 2 B) efficiency \nevaluation.\nFigure 1. Chemical structure of liriodendrin and the efficiency of acute/daily drug administration evaluated by von \nFrey filament test (*p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – Acute LY294002 and liriodendrin ad -\nministration remarkably maintained the paw withdrawal threshold in ENDO-treated rats. B – Daily LY294002 and \nliriodendrin administration remarkably maintained the paw withdrawal threshold in ENDO-treated rats\n25\n20\n15\n10\n5\n0\n25\n20\n15\n10\n5\n0\nA B\nPaw withdrawal threshold [g]\nPaw withdrawal threshold [g]\n BL 0 1 2 3 4 5 6 7 8\nTime [h]\n SHAM        \n  ENDO\n ENDO + LY294002        \n  ENDO + liriodendrin\n 3 6 9 12 15 18 21\nDays after surgery\n SHAM        \n  ENDO\n ENDO + LY294002        \n  ENDO + liriodendrin\n\nLiriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response  \nand regulating the signaling pathway of Pl3K/Akt/mToR\nArch Med Sci 5\nFigure 2. Acute/daily drug administration efficiency evaluated using thermal hyperalgesia test (*p < 0.05 vs. SHAM \ngroup; #p < 0.05 vs. ENDO group). A – Acute LY294002 and liriodendrin administration remarkably maintained \nthe paw withdrawal latency in ENDO-treated rats. B – Daily LY294002 and liriodendrin administration remarkably \nmaintained the paw withdrawal latency in ENDO-treated rats \n20\n15\n10\n5\n0\n20\n15\n10\n5\n0\nA B\nPaw withdrawal latency [s]\nPaw withdrawal latency [s]\n BL 0 1 2 3 4 5 6 7 8\nTime [h]\n SHAM        \n  ENDO\n ENDO + LY294002        \n  ENDO + liriodendrin\n 3 6 9 12 15 18 21\nDays after surgery\n SHAM        \n  ENDO\n ENDO + LY294002        \n  ENDO + liriodendrin\nLY294002 and liriodendrin administration \nrestored the up-regulation of p-PI3K/ \np-AKT/p-mTOR in the endometrium of rats \nwith endometriosis\nThe expression of PI3K/AKT/mTOR and p-PI3K/\np-AKT/p-mTOR in the endometrium of rat groups \nwas investigated (Figure 3 A). No obvious differ -\nence was observed for the expression of PI3K (Fig-\nure 3 B), AKT (Figure 3 C) and mTOR (Figure 3 D) \nin the endometrium of rats under distinct treat -\nments. However, the expression levels of p-PI3K \n(Figure 3 E), p-AKT (Figure 3 F) and p-mTOR (Fig-\nure 3 G) were remarkably higher in the endometri-\num of ENDO rats when compared with the control. \nLY294002 and liriodendrin administration notably \ndecreased the up-regulation of p-PI3K (Figure 3 E), \np-AKT (Figure 3 F) and p-mTOR (Figure 3 G) in the \nendometrium of rats with endometriosis.\nLiriodendrin administration restored  \nthe elevation of IL-1, IL-2, IL-6, TNF-α  \nand PGE2 in rats with endometriosis\nThe IL-1, IL-2, IL-6, TNF- α and PGE2 levels in \nthe peritoneal fluid of rats with endometriosis \nwere significantly higher when compared with the \ncontrol. LY294002 and liriodendrin administra -\ntion notably decreased the elevation of IL-1, IL-2,  \nIL-6, TNF-α and PGE2 in the peritoneal fluid of rats \nwith endometriosis. Moreover, the therapeutic ef-\nficiency of liriodendrin administration at the dose \ninvestigated was stronger than LY294002 admin -\nistration at the dose investigated in restoring the \nlevels of IL-1 (Figura 4 A), IL-2 (Figure 4 B), IL-6 \n(Figure 4 C), TNF- α (Figure 4 D) and PGE2 (Fig-  \nure 4 E) in the peritoneal fluid of rats. Further -\nmore, the PGE2 levels in the blood of rats were \nexamined under distinct conditions. The serum \nlevel of PGE2 in ENDO rats was significantly high-\ner when compared with the control. Liriodendrin \nadministration notably decreased the elevation of \nPGE2 in the serum of the ENDO group, but no ther-\napeutic efficiency was observed for LY294002 on \nrestoring the PGE2 levels in the blood of rats from \nthe ENDO group (Figure 4 F).\nLiriodendrin administration restored  \nthe elevation of CGRP and substance P  \nin the ventral horn of the spinal cord  \nof rats with endometriosis\nLY294002 and liriodendrin administration nota-\nbly decreased the up-regulated substance P (Fig -\nure 5 A) and CGRP levels (Figure 5 B) in the ENDO \ngroup. Moreover, LY294002 and liriodendrin admin-\nistration notably decreased the up-regulated sub-\nstance P (Figures 5 C, F , G) and CGRP (Figures 5 D, \nF , H) mRNA and protein in the ventral horn of the \nspinal cord in the ENDO rats. \nIt is worth noting that the efficiency of lirioden-\ndrin administration at the dose investigated was \nmore apparent than LY294002 administration at \nthe dose investigated for maintaining the expres-\nsion of CGRP and substance P in the ventral horn of \nthe spinal cord. Also NF-κB mRNA level (Figure 5 E) \nshowed the same tendency as the gene expression \nlevel of CGRP and substance P. \nLY294002 and liriodendrin administration \nrestored the LPS-induced up-regulation  \nof p-PI3K/p-AKT/p-mTOR in 12Z cells\nThe expression of PI3K/AKT/mTOR and p-PI3K/ \np-AKT/p-mTOR in 12Z cells treated with LPS followed \nby LY294002 and liriodendrin administration was \ninvestigated (Figure 6 A). No obvious difference was \nobserved for the expression of PI3K (Figure 6 B),  \n\nJianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing\n6 Arch Med Sci\nFigure 3. LY294002 and liriodendrin administration restored \nthe up-regulated p-PI3K/p-AKT/p-mTOR level in the endo -\nmetrium of endometriosis rat models (* p < 0.05 vs. SHAM \ngroup; #p < 0.05 vs. ENDO group). A – Western blot analysis \nof PI3K/AKT/mTOR and p-PI3K/p-AKT/p-mTOR in the endo-\nmetrium of rats under distinct conditions. B – Quantitative \nanalysis indicated that no obvious difference was observed \nfor the expression of PI3K in the endometrium of rats un -\nder distinct treatments. C – Quantitative analysis indicated \nthat no obvious difference was observed for the expression \nof AKT in the endometrium of rats under distinct treatments.  \nD – Quantitative analysis indicated that no obvious differ -\nence was observed for the expression of mTOR in the endo-\nmetrium of rats under distinct treatments. E – Quantitative \nanalysis indicated that LY294002 and liriodendrin adminis-\ntration restored the up-regulated p-PI3K level in the ENDO \ngroup. F – Quantitative analysis indicated that LY294002 \nand liriodendrin administration restored the up-regulated \np-AKT level in the ENDO group. G – Quantitative analysis \nindicated that LY294002 and liriodendrin administration \nrestored the up-regulated p-mTOR level in the ENDO group\n6\n4\n2\n0\n4\n3\n2\n1\n0\n8\n6\n4\n2\n0\nE\nG\nF\nRelative density of p-PI3KRelative density of p-mTOR\nRelative density of p-AKT\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n2.0\n1.5\n1.0\n0.5\n0\n1.5\n1.0\n0.5\n0\n1.5\n1.0\n0.5\n0\nA B\nC D Relative density of PI3K\nRelative density of AKT\nRelative density of mTOR\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\nPI3K\np-PI3K\nAKT\np-AKT\nmTOR\np-mTOR\nβ-actin\nSHAM ENDOENDO + LY294002ENDO + \nliriodendrin\n\nLiriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response  \nand regulating the signaling pathway of Pl3K/Akt/mToR\nArch Med Sci 7\nAKT (Figure 6 C) and mTOR (Figure 6 D) in 12Z cells \nunder differential conditions. However, the expres-\nsion levels of p-PI3K (Figure 6 E), p-AKT (Figure 6 F) \nand p-mTOR (Figure 6 G) were remarkably higher \nin LPS-treated 12Z cells when compared with the \ncontrol. LY294002 and liriodendrin administration \nnotably decreased the LPS-induced up-regula -\ntion of p-PI3K (Figure 6 E), p-AKT (Figure 6 F) and \np-mTOR (Figure 6 G) in LPS-treated 12Z cells.\nLiriodendrin administration restored  \nthe LPS-induced up-regulation of IL-6,  \nTNF-α, and IL-1β mRNA and protein \nexpression in 12Z cells\nLiriodendrin administration at the dose inves -\ntigated showed greater efficiency than LY294002  \nadministration at the dose investigated in restoring \nthe LPS-induced up-regulation of IL-6 (Figure 7 A),  \nTNF-α (Figure 7 B), and IL-1 β (Figure 7 C) mRNA \n300\n200\n100\n0\n250\n200\n150\n100\n50\n0\n800\n600\n400\n200\n0\n3000\n2000\n1000\n0\n600\n400\n200\n0\n600\n400\n200\n0\nA\nC\nE\nB\nD\nF\nLevel of IL-1 [ng/l]Level of IL-6 [ng/l]Peritoneal fluid level of PGE2 [ng/l]\nLevel of IL-2 [ng/l]Level of TNF-α [ng/l]Serum level of PGE2 [ng/l]\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\nFigure 4. Liriodendrin administration restored the elevation of IL-1, IL-2, IL-6, TNF-α and PGE2 in endometriosis rat \nmodels (*p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – Liriodendrin administration restored the elevated \nperitoneal fluid IL-1 level in the ENDO group. B – Liriodendrin administration restored the elevated peritoneal fluid \nIL-2 level in the ENDO group. C – Liriodendrin administration restored the elevated peritoneal fluid IL-6 level in the \nENDO group. D – Liriodendrin administration restored the elevated peritoneal fluid TNF-α level in the ENDO group. \nE – Liriodendrin administration restored the elevated peritoneal fluid PGE2 level in the ENDO group. F – Lirioden-\ndrin administration restored the elevated serum PGE2 level in the ENDO group\n\n8 Arch Med Sci\n400\n300\n200\n100\n0\n600\n400\n200\n0\nA B\nLevel of substance P [pg/l]\nLevel of CGRP [pg/l]\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n6\n4\n2\n0\n4\n3\n2\n1\n0\nC D\nExpression level of substance P mRNA\nExpression level of CGRP mRNA\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n6\n4\n2\n0\n5\n4\n3\n2\n1\n0\nG H\nRelative density of substance P\nRelative density of CGRP\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n6\n4\n2\n0\nE F\nExpression level of NF-κB mRNA\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\n SHAM ENDO ENDO +  ENDO +\n   LY294002  liriodendrin\nSubstance P\nCGRP\nβ-actin\nFigure 5. Liriodendrin administration restored the increased CGRP and substance P in the ventral horn of spinal \ncord samples collected from rats with endometriosis (* p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – \nELISA analysis indicated that liriodendrin administration restored the elevated substance P in the ENDO rats.  \nB – ELISA analysis indicated that liriodendrin administration restored the elevated CGRP in the ENDO rats. C – \nQPCR analysis indicated that liriodendrin administration restored the elevated substance P in the ENDO rats.  \nD – QPCR analysis indicated that liriodendrin administration restored the elevated CGRP in the ENDO rats. E – \nQPCR analysis indicated that liriodendrin administration restored the elevated NF- κB in the ENDO rats. F – West-\nern blot analysis of CGRP and substance P expression in the ventral horn of the spinal cord of rats under distinct \nconditions. G – ELISA analysis indicated that liriodendrin administration restored the elevated substance P in the \nENDO rats. H – ELISA analysis indicated that liriodendrin administration restored the elevated CGRP in the ENDO rats\n\nLiriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response  \nand regulating the signaling pathway of Pl3K/Akt/mToR\nArch Med Sci 9\nFigure 6. LY294002 and liriodendrin administration re -\nstored the LPS-induced up-regulation of p-PI3K/p-AKT/  \np-mTOR in 12Z cells (* p < 0.05 vs. NC group; #p < 0.05 vs. \nLPS group). A  – Western blot analysis of PI3K/AKT/mTOR \nand p-PI3K/p-AKT/p-mTOR in 12Z cells under distinct con -\nditions. B – Quantitative analysis indicated that no obvious \ndifference was observed for the expression of PI3K in 12Z \ncells under distinct treatments. C – Quantitative analysis \nindicated that no obvious difference was observed for the \nexpression of AKT in 12Z cells under distinct treatments.  \nD – Quantitative analysis indicated that no obvious differ -\nence was observed for the expression of mTOR in 12Z cells \nunder distinct treatments. E – Quantitative analysis indicat-\ned that LY294002 and liriodendrin administration restored \nthe LPS-induced up-regulation of p-PI3K in 12Z cells. F – \nQuantitative analysis indicated that LY294002 and lirioden-\ndrin administration restored the LPS-induced up-regulation \nof p-AKT in 12Z cells. G – Quantitative analysis indicated \nthat LY294002 and liriodendrin administration restored the \nLPS-induced up-regulation of p-mTOR in 12Z cells\n5\n4\n3\n2\n1\n0\n5\n4\n3\n2\n1\n0\n6\n4\n2\n0\nE\nG\nF\nRelative density of p-PI3KRelative density of p-mTOR\nRelative density of p-AKT\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n1.5\n1.0\n0.5\n0\n1.5\n1.0\n0.5\n0\n1.5\n1.0\n0.5\n0\nA B\nC D Relative density of PI3K\nRelative density of AKT\nRelative density of mTOR\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\nPI3K\np-PI3K\nAKT\np-AKT\nmTOR\np-mTOR\nβ-actin\nSHAM ENDOENDO + LY294002ENDO + \nliriodendrin\n\nJianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing\n10 Arch Med Sci\nFigure 7. Liriodendrin administration restored the LPS-in -\nduced up-regulation of IL-6, TNF- α, and IL-1 β mRNA and \nprotein expression in 12Z cells (* p < 0.05 vs. NC group;  \n#p < 0.05 vs. LPS group). A – Liriodendrin administration re -\nstored the LPS-induced up-regulation of IL-6 mRNA expres-\nsion in 12Z cells. B – Liriodendrin administration restored \nthe LPS-induced up-regulation of TNF- α mRNA expression \nin 12Z cells. C – Liriodendrin administration restored the \nLPS-induced up-regulation of IL-1β mRNA expression in 12Z \ncells. D – Liriodendrin administration restored the LPS-in -\nduced up-regulation of NF-κB mRNA expression in 12Z cells. \nE – ELISA analysis indicated that liriodendrin administration \nrestored the LPS-induced up-regulation of IL-6 protein ex -\npression in 12Z cells. F – Western blot analysis indicated \nthat liriodendrin administration restored the LPS-induced \nup-regulation of TNF- α protein expression in 12Z cells.  \nG – Western blot analysis indicated that liriodendrin ad -\nministration restored the LPS-induced up-regulation of  \nIL-1β protein expression in 12Z cells\n4\n3\n2\n1\n0\n8\n6\n4\n2\n0\n4000\n3000\n2000\n1000\n0\n1500\n1000\n500\n0\n4\n3\n2\n1\n0\n4\n3\n2\n1\n0\n6000\n4000\n2000\n0\nA\nC\nE\nG\nB\nD\nF\nExpression level of IL-6 mRNAExpression level of IL-1β mRNALevel of IL-6 [pg/ml]Level of IL-1β [pg/ml]\nExpression level of TNF-α mRNAExpression level of NF-κB mRNALevel of TNF-α [pg/ml]\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n NC  LPS LPS +  LPS + \n   LY294002  liriodendrin\n\nLiriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response  \nand regulating the signaling pathway of Pl3K/Akt/mToR\nArch Med Sci 11\nin 12Z cells. Liriodendrin administration at the \ndose investigated showed more apparent effi -\nciency than LY294002 administration at the dose \ninvestigated in restoring the LPS-induced up-reg -\nulation of IL-6 (Figure  7 E), TNF- α protein (Fig-  \nure 7 F), and IL-1β protein (Figure 7 G) in 12Z cells. \nAlso the NF- κB mRNA level (Figure 7 D) showed \nthe same tendency as the gene expression level of \nCGRP and substance P.\nDiscussion\nA  disaccharide lignan named liriodendrin, \n(+)-syringaresinol di-O-b-D-glucopyranoside, is the  \nbioactive component of Sargentodoxa cuneata  \n(Oliv.) Rehd. Et Wils. A  plethora of biological ef -\nfects against inflammation, pain, and arrythmia \nhave been observed for the medicine [14, 15, 27].  \nIn this study, we evaluated the efficiency of acute \ndrug administration and daily drug administra -\ntion of liriodendrin in attenuating pain in endo -\nmetriosis rats. Liriodendrin has been reported to \nsuppress inflammatory damage in the treatment \nof ulcerative colitis and sepsis-associated tissue \ninjuries [28, 29]. However, its effect in the treat -\nment of endometriosis-associated pain was not \ninvestigated. Therefore, we aimed to explore and \ncompare its effect in the treatment of endometrio-\nsis-associated pain with LY294002 in our study. As \na result, we found that, although both LY294002 \nand liriodendrin functioned as PI3K inhibitors, \nonly liriodendrin alleviated inflammation, thus \nmaking liriodendrin a better therapeutic method \ncompared with LY294002. In addition, the authors \ncompared the distinct expression of neuropep -\ntides mRNA and protein in the ventral horn of the \nspinal cord of ENDO rats. Liriodendrin administra-\ntion restored the elevation of neuropeptides in the \nventral horn of the spinal cord of ENDO rats. Upon \noral administration, liriodendrin is transformed \nto syringaresinol, which is attributed to the anti-  \ninflammatory effect [15]. In various hormone-  \ndependent conditions, the phytoestrogen syringa-\nresinol binds selectively to estrogen receptors [21].\nPrevious studies have shed light on the function \nof the mTOR/AKT pathway in endometriosis. One \nof the studies revealed that the P3KCA mutation, \nwhich is important for Akt activation, is frequently \nobserved in patients with ovarian clear cell carci -\nnoma [30, 31]. Higher AKT activity has been ob -\nserved in ovarian endometriosis compared to the \nnormal endometrium, which could be possibly due \nto the estrogen in endometriotic cells [32]. The \nauthors found that DIE patients showed higher \nactivity of AKT in endometriotic lesions similar to \novarian endometriosis. A PI3K inhibitor, LY294002, \nwas found to inhibit the PI3K/Akt/mTOR signaling \npathway in animals. The aim of the study was to \ninvestigate the acute and chronic effects of intra -\nthecal injection of LY294002 on various signaling \npathways in sciatic nerve endometriosis models \nin rats including PI3K/Akt/mTOR. The effects of \nLY294002 in the animal models began at 2-3 h. \nThe peak effect was seen at four to five hours and \nwas four hours long. Daily injections of LY294002 \nfor 3 weeks greatly reduced mechanical and ther-\nmal hyperalgesia caused by sciatic nerve endome-\ntriosis (Figures 1 and 2). The therapeutic effects of \nLY294002 in sciatic nerve endometriosis models \nmight be related to reduced expression levels of \nPI3K/Akt/mTOR signaling pathway-related fac -\ntors (Figure 3). The authors performed western \nblot analysis to determine the expression of the \nsignaling proteins and their phosphorylated coun-\nterparts in ENDO-treated rats and LPS-treated 12Z \ncells. Treatment with the drugs LY294002 and lir -\niodendrin reestablished the lower levels of phos -\nphorylated signaling proteins in the animal and \ncellular models. In addition, an ELISA assay was \nperformed to measure the IL-1, IL-2, IL-6, TNF- α \nand PGE2 levels in stomach fluid of ENDO-treated \nrats. Previously, it was reported that injection of \nLY294002 significantly reduced lung inflammation \nand airway hyperresponsiveness in an asthma an-\nimal model [33, 34]. \nEven though the mechanism is still unclear, \nrecent studies have shown that swelling and \nmodified nerves in the endometriotic lesions are \nconnected to the pathology of endometriosis. \nThe aberrant stimulation causes modification in \nneurotransmitter levels and also abnormal secre -\ntion of inflammatory factors. The PI3K inhibitors \nreduced the levels of IL-6, MCP-1 (also known as \nCCL2), TNF-α and nitric oxide, which are the key \nfactors regulating the inflammatory process. The \nPI3K/Akt signaling pathway has been shown to \nregulate NF- κB activation and phosphorylation \nof p65 units of NF- κB [35]. In this study, we an -\nalyzed the differential expression of IL-6, TNF- α, \nand IL-1β in LPS-treated 12Z cells. Liriodendrin ad-\nministration restored the LPS-induced elevation of \nIL-6, TNF-α, and IL-1β in LPS-treated 12Z cells. The \n(+)-syringaresinol, produced in situ from lirioden -\ndrin by bacteria in intestines, enhanced FOXO3 \nand SIRT1 activity, which further modulated the \nactivity of HIF-1 [36–38]. Hence, the authors hy -\npothesized that liriodendrin degrades HIF-1 α by \nFOXO3 and SIRT1 activation, in turn reducing \nVEGF expression. \nIn conclusion, we found that compared with \nLY294200, the administration liriodendrin exerted \na more evident therapeutic effect in the treatment \nof endometriosis-associated pain by suppressing \nthe secretion of pro-inflammatory cytokines.\nConflict of interest\nThe authors declare no conflict of interest.\n\nJianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing\n12 Arch Med Sci\nReferences\n1. Sang Q, Sun D, Chen Z, Zhao W. NGF and PI3K/Akt sig -\nnaling participate in the ventral motor neuronal pro -\ntection of curcumin in sciatic nerve injury rat models. \nBiomed Pharmacother 2018; 103: 1146-53.\n2. Fraser IS. Recognising, understanding and managing \nendometriosis. J Hum Reprod Sci 2008; 1: 56-64.\n3. Alvarez P , Giudice LC, Levine JD. 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