Liriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing inflammatory response and regulating the signaling pathway of Pl3K/Akt/mToR

Introduction: LY294002 has been validated as a PI3K pan-inhibitor in the pathogenesis of airway inflammation, which attenuated IL-25-induced asth - ma-like AHR. Liriodendrin was proved to play essential roles in attenuating endometriosis-associated pain. This work aimed to gain a  mechanical in - sight into the therapeutic efficiency of liriodendrin administration in atten - uating endometriosis-associated pain.

The von Frey filament test and thermal hyperalgesia test were carried out to evaluate the acute and daily efficiency of drug ad - ministration. Western blot was used to analyze the expression of substance P , PI3K/AKT/mTOR, p-PI3K/p-AKT/p-mTOR, and CGRP. ELISA was performed to examine interleukin (IL)-1, IL-2, IL-6, TNF- α and PGE2 levels.

As the results showed, liriodendrin significantly attenuated pain in endometriosis rats and restored the up-regulation of p-PI3K/p-AKT/ p-mTOR in the endometriosis rats. The increased IL-1, IL-2, IL-6, TNF- α, and PGE2 levels were remarkably restored by liriodendrin in endometriosis rats. Moreover, the activated expression of substance P in the ventral horn of the spinal cord of endometriosis rats was notably restored by liriodendrin in addition to CGRP. Furthermore, liriodendrin effectively restored the lipopoly - saccharide (LPS)-induced up-regulation of p-PI3K/p-AKT/p-mTOR protein as well as the LPS activated IL-6, TNF- α, and IL-1 β mRNA and protein expres - sion in 12Z cells.

We found that compared with LY294200, liriodendrin exerted a more evident therapeutic effect in the treatment of endometriosis-associ - ated pain by suppressing the secretion of pro-inflammatory cytokines. Key words: liriodendrin, endometriosis, pain, inflammation, Pl3K, Akt, mToR. Jianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing 2 Arch Med Sci

The presence of stromal or glandular cells on the edge of the uterine cavity is a defining char - acteristic of endometriosis [1]. Chronic pain such as radicular pain, back pain, dyspareunia, and dys- menorrhea affects about 5–10% of young women [2, 3]. Endometriosis affecting the sciatic nerve is a very rare condition [4]. The current treatment op- tions include extracting the ectopic lesions and ad- ministration of hormones and non-steroidal anti- inflammatory drugs (NSAIDs) [5]. Nonetheless, the current treatment options have limited efficacy and unwanted side effects [6]. Normal cellular functions such as metabolism, survival and growth are regulated by two signal - ing pathways: the phosphatidylinositol-3 kinases (PI3Ks) and the mammalian target of rapamycin (mTOR) [7, 8]. Both the pathways are intercon - nected by a  type of serine/threonine kinase-like AKT [9]. Protein kinase B, also known as AKT, plays a  role in several cellular functions via PI3K and mTOR [10]. Stimuli from different types of recep - tors, such as antigen receptors, cytokine receptors, insulin receptors, or the insulin-like growth factor I receptor, activate the PI3K and mTOR pathways. Upon activation, PI3K causes phosphorylation of the phosphorylate phosphatidylinositol 4,5-bis - phosphate (PIP2), which produces a second mes - senger, phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 further stimulates downstream path - ways such as AKT. Elevated levels of the PI3K/Akt/ mTOR pathway components were observed in the eutopic endometrium of women with endometri - osis [11]. The multilevel signaling pathway involv- ing PI3K, Akt and mTOR correlated well with pain and endometriosis [12]. Deying Dai, an expert in Chinese medicine, invented the prescription formula of Caulis Sar - gentodoxae prescription. The formula was found to be effective against EMs (90%), menstrual fe - ver (90.63%) and dysmenorrhea (96.25%) [13]. Extensive research has been performed to assess its effects in ulcerative colitis in humans. Sargent- odoxa cuneata is very effective for treating several inflammation conditions affecting the appendix, stomach and rheumatic arthritis. However, the mechanism of action of the medicine remains elusive. Another example of traditional medi - cine called liriodendrin, a type of lignin, has been studied for its therapeutic effects. This disaccha - ride lignan named liriodendrin, (+)-syringaresinol di-O-b-D-glucopyranoside, is the bioactive com - ponent of Sargentodoxa cuneata (Oliv.) Rehd. Et Wils, and a plethora of biological effects against inflammation, pain, and arrhythmia have been observed for the medicine [14–16]. This medicine has shown efficacy against disorders such as ar - rhythmia, myocardial ischemia, inflammation and oxidative stress [14, 17]. Moreover, according to previous findings, liriodendrin administration re - stored the elevation of neuropeptides in the ven - tral horn of the spinal cord of ENDO rat models. Upon oral administration liriodendrin is trans - formed to syringaresinol, which is attributable to the anti-inflammatory effect [17]. In various hor - mone-dependent conditions, the phytoestrogen syringaresinol binds selectively to the estrogen receptors [18]. The proposed mechanism of action of lirio - dendrin involved reduced secretion of inflamma - tion-inducing cytokines such as interleukin (IL)-6, tumor necrosis factor α (TNF-α) and IL-1 β in the colon tissues. A similar decrease in proinflamma - tory cytokines was observed in DSS-induced co - lon damage models. In the intestine, a  link was observed between the nuclear factor κB (NF- κB) cascade and the AKT signaling at transcription and translation levels [19, 20]. Liriodendrin blocks protein-1 and/or NF-κB in SW982 human synovial sarcoma cells [21]. Based on the above-mentioned evidence, LY294002 may alleviate endometriosis-associated pain via regulating PI3K signaling, and liriodendrin as an extract of Sargentodoxa cuneata may also inhibit PI3K signaling, and in addition, liriodendrin may also suppress the inflammatory response which is also involved in endometriosis-associat - ed pain. We aimed to validate our hypothesis that the administration of liriodendrin could exhibit a therapeutic effect in the treatment of endome - triosis-associated pain, the efficacy of which is supposed to be better than LY294002 in endome- triosis. And the novelty of our study was to com - pare the efficacy of liriodendrin and LY294002 in the treatment of endometriosis.

Animal and treatment Female SD rats with ages ranging between fourteen and eighteen weeks and weights in the range of 180–220 g were purchased from Charles River Laboratories Portage, MI. The rats were housed under alternating cycles of dark (12 h) and light (12 h) at room temperature and humidi- ty near 70%. The rats were allowed access to food and water. Animals were divided into four groups with 8 rats in each group, i.e.: 1) SHAM group (group of animals received the sham operation), 2) ENDO group (group of animals were subject - ed to the surgical procedure and established as endometriosis rat model), 3) ENDO + LY294002 group (endometriosis rat models subjected to the surgical procedure to create a rat model of endo - metriosis and received i.p. injection of LY294002) and 4) ENDO + liriodendrin group (endometriosis Liriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response and regulating the signaling pathway of Pl3K/Akt/mToR Arch Med Sci 3 rat models subjected to the surgical procedure to create a rat model of endometriosis and received oral administration of liriodendrin). Models of sci- atic nerve endometriosis were created using pre - viously reported procedures [22]. Irrespective of the estrous cycle of the female rats, the autologous uterine tissue was grafted near the site of the sciatic nerve. Initially, the rats were put under anesthesia using isoflurane then a 2 cm long incision was created near the lower abdomen which exposed the right uterine horn. Later, a 5 mm length of uterus tissue was dissected and extracted from the horn region of the uterus. It was kept in a Petri dish which was preloaded with a phosphate buffer solution containing 1% penicillin and streptomycin. Sub - sequently, the right sciatic nerve was revealed on the rats positioned on the left side and an inci - sion of 1 cm long near the thigh was made. The connective tissues near the desired area were cautiously separated. The longitudinal section of the uterus was opened and tied around the nerve with uterus endometrial tissue next to the nerve. In the case of rats that received the Sham opera - tions, no implant was installed near the exposed right sciatic nerve. After the surgical procedure, 6.0-grade silk thread was used to suture the in - cision, and to prevent the infection gentamicin (50 mg/ml, 0.2 ml, Thermo Fisher Scientific, Waltham, MA) was injected into the stomach. In group 3, the dose of LY294002 was 15 mg/ml, which was dissolved in DMSO. A dose of 30 mg/kg was administered to the animals via a microsy - ringe into the i.t. tubing. In group 4, 100 mg/kg liriodendrin was pretreated about 3 days before the surgery. The animal experiment procedures were approved by the institutional ethical com - mittee. Behavioral testing To assess the behavior of the rats, the von Frey filament test was used. Briefly, the von Frey filament test was performed following the standard proto- col, which involves applying mechanical stimuli to measure the hypersensitivity to mechanical stress. The key factor to measure was the paw withdraw- al threshold. A  series of ten von Frey filaments of equal logarithmic bending force was used in a se - quence. To perform the test, the rats were allowed to habituate on the wire gauge surface for 15 min. To analyze the thermal hyperalgesia, a heat source was applied to the hind paw of the rats. The heat source was made up of a lamp fitted with a high-in- tensity bulb of power 50 W, and a voltage of 8 V . The exposure limit was decided to be 20 s so that the rats did not get skin burns. Following these behavior tests, the effects of LY294002 following the sciatic endometriosis procedure were evaluated. Procedure for abdominal puncture The medicine was administered orally to rats for 21 days and after that 10 ml of PBS buffer was filled into the stomach cavity of rats under anes - thesia with the help of a  syringe. The rats were shaken gently for 10 min and a  puncture was made into the abdominal wall of the rats using a needle. The peritoneal fluid was extracted with the help of a needle. Later, the rats were eutha - nized, and the samples were collected. The upper clear layer of the peritoneal fluid and blood sam - ples were obtained and refrigerated at −80°C. Enzyme-linked immunosorbent assay The peritoneal fluid samples and blood sam - ples were extracted and stored at 2–8°C for a maximum of 5 days. Then, substance P , IL-1, IL-2, IL-6, TNF-α, PGE2, and CGRP levels were analyzed using the protocol provided with the kit in accor - dance with published methods [23]. The antibody was incubated and procedures for washing were performed based on the published methods. The absorbance of the ELISA experiments was record- ed using the absorbance microplate reader (Mo - lecular Devices, San Jose, CA). Cell culture In this study, we established our cell models with 12Z cells (Applied Biological Materials (abm) Inc., Canada), which were an immortalized human endometriotic cell line. The cells were cultured in 37°C and 5% CO 2 in Roswell Park Memorial In - stitute 1640 medium containing 10 mM HEPES and other antibiotics according to previously pub- lished methods [24]. The cells were transferred into 6-well plates and cultured until they were confluent. To these cells, the drugs at the de - sired concentration were added to the wells. The cells were divided into four groups: 1) negative control group, 2) LPS group; the cells were stimu - lated with 100 ng/ml of lipopolysaccharide (LPS), 3) LPS + LY294002 group; the cells were treated initially with 10 μM LY294002 and 6 h later with 100 ng/ml of LPS, and 4) LPS + liriodendrin group; the cells were pretreated with 50 μM liriodendrin and 6 h later treated with 100 ng/ml of LPS. All experimental procedures were done four times using the above-mentioned protocol. RNA isolation and real-time PCR The authors performed a polymerase chain re - action test using the RNA extraction kit obtained from Thermo Fisher Scientific. The genetic mate - rial was removed from the samples using the kit. The reverse transcription of the RNA material in the samples into the cDNA was carried out using Jianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing 4 Arch Med Sci the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Later, the quantitative RT-PCR was carried out using the iTaq Universal SYBR Green Super - mix (Bio-Rad, Hercules, CA). The polymerase re - action was performed with Applied Biosystems Real-Time PCR Instruments (Thermo Fisher Sci - entific, Waltham, MA). The relative expression of substance P , IL-6, TNF-α, IL-1 β, NF- κB and CGRP was calculated relative to the reference standard genes by the 2 ∆∆Ct method following published protocols [25]. Western blot analysis The Western blot analysis was performed ac - cording to published protocols [26]. The cultured cells and tissue samples obtained from the decap- itated animals were first washed with PBS buffer. Radioimmunoprecipitation buffer was used as a lysed buffer to treat the cells and tissues. This procedure was used to extract the enclosed pro - teins from the cells and tissue samples. The mix - ture of proteins obtained from the samples was separated into bands using 10% SDS-polyacryl - amide gel. Then, the total protein content isolat - ed from the cells was resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel using an elec - trophoresis system (VWR, Radnor, PA). Later, the separated bands were expanded on a PVDF mem- brane and the reaction was blocked by skim milk. Subsequently, antibodies for CGRP (Cat#ab47207, Abcam, Cambridge, UK), PI3K (Cat#ab32089, Ab - cam, Cambridge, UK), AKT (Cat#ab8805, Abcam, Cambridge, UK), mTOR (Cat#ab134903, Abcam, Cambridge, UK), p-PI3K (Cat#ab278545, Abcam, Cambridge, UK), p-AKT (Cat#ab38449, Abcam, Cambridge, UK), p-mTOR (Cat#ab109268, Abcam, Cambridge, UK), substance P (Cat#ab14184, Ab - cam, Cambridge, UK), TNF- α (Cat#ab183218, Ab- cam, Cambridge, UK) and IL-1 β (Cat#ab254360, Abcam, Cambridge, UK) were applied to the pro - tein bands followed by the secondary antibodies. Statistical analysis Statistical analysis was carried out by making use of SPSS 19.0 software (IBM, Chicago, IL). One-way ANOVA and Student’s t-tests were used to com - pare intergroup differences. Tukey’s test was used as the post hoc test following one-way ANOVA. All statistical tests were two-sided, while the p-val- ues of < 0.05 indicated statistical significance.

LY294002 or liriodendrin administration attenuated the increased pain in endometriosis rat models The paw withdrawal threshold was significant- ly lower in ENDO rats when compared with the control. LY294002 and liriodendrin administra - tion remarkably maintained the paw withdrawal threshold in ENDO treated rats, indicating the ef - ficiency of acute drug administration (Figure 1 A). Moreover, daily drug administration efficiency was evaluated every three days using the von Frey fila- ment test. LY294002 and liriodendrin administra- tion showed considerable efficiency in restoring the reduction in the paw withdrawal threshold in the ENDO group (Figure 1 B). Additionally, a ther - mal hyperalgesia test was used to examine the ef- ficiency of acute and chronic drug administration. LY294002 and liriodendrin administration showed considerable efficiency in restoring the paw with- drawal latency loss in rats from the ENDO group in both acute drug administration (Figure 2 A) and daily drug administration (Figure 2 B) efficiency evaluation. Figure 1. Chemical structure of liriodendrin and the efficiency of acute/daily drug administration evaluated by von Frey filament test (*p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – Acute LY294002 and liriodendrin ad - ministration remarkably maintained the paw withdrawal threshold in ENDO-treated rats. B – Daily LY294002 and liriodendrin administration remarkably maintained the paw withdrawal threshold in ENDO-treated rats 25 20 15 10 5 0 25 20 15 10 5 0 A B Paw withdrawal threshold [g] Paw withdrawal threshold [g] BL 0 1 2 3 4 5 6 7 8 Time [h] SHAM ENDO ENDO + LY294002 ENDO + liriodendrin 3 6 9 12 15 18 21 Days after surgery SHAM ENDO ENDO + LY294002 ENDO + liriodendrin Liriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response and regulating the signaling pathway of Pl3K/Akt/mToR Arch Med Sci 5 Figure 2. Acute/daily drug administration efficiency evaluated using thermal hyperalgesia test (*p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – Acute LY294002 and liriodendrin administration remarkably maintained the paw withdrawal latency in ENDO-treated rats. B – Daily LY294002 and liriodendrin administration remarkably maintained the paw withdrawal latency in ENDO-treated rats 20 15 10 5 0 20 15 10 5 0 A B Paw withdrawal latency [s] Paw withdrawal latency [s] BL 0 1 2 3 4 5 6 7 8 Time [h] SHAM ENDO ENDO + LY294002 ENDO + liriodendrin 3 6 9 12 15 18 21 Days after surgery SHAM ENDO ENDO + LY294002 ENDO + liriodendrin LY294002 and liriodendrin administration restored the up-regulation of p-PI3K/ p-AKT/p-mTOR in the endometrium of rats with endometriosis The expression of PI3K/AKT/mTOR and p-PI3K/ p-AKT/p-mTOR in the endometrium of rat groups was investigated (Figure 3 A). No obvious differ - ence was observed for the expression of PI3K (Fig- ure 3 B), AKT (Figure 3 C) and mTOR (Figure 3 D) in the endometrium of rats under distinct treat - ments. However, the expression levels of p-PI3K (Figure 3 E), p-AKT (Figure 3 F) and p-mTOR (Fig- ure 3 G) were remarkably higher in the endometri- um of ENDO rats when compared with the control. LY294002 and liriodendrin administration notably decreased the up-regulation of p-PI3K (Figure 3 E), p-AKT (Figure 3 F) and p-mTOR (Figure 3 G) in the endometrium of rats with endometriosis. Liriodendrin administration restored the elevation of IL-1, IL-2, IL-6, TNF-α and PGE2 in rats with endometriosis The IL-1, IL-2, IL-6, TNF- α and PGE2 levels in the peritoneal fluid of rats with endometriosis were significantly higher when compared with the control. LY294002 and liriodendrin administra - tion notably decreased the elevation of IL-1, IL-2, IL-6, TNF-α and PGE2 in the peritoneal fluid of rats with endometriosis. Moreover, the therapeutic ef- ficiency of liriodendrin administration at the dose investigated was stronger than LY294002 admin - istration at the dose investigated in restoring the levels of IL-1 (Figura 4 A), IL-2 (Figure 4 B), IL-6 (Figure 4 C), TNF- α (Figure 4 D) and PGE2 (Fig- ure 4 E) in the peritoneal fluid of rats. Further - more, the PGE2 levels in the blood of rats were examined under distinct conditions. The serum level of PGE2 in ENDO rats was significantly high- er when compared with the control. Liriodendrin administration notably decreased the elevation of PGE2 in the serum of the ENDO group, but no ther- apeutic efficiency was observed for LY294002 on restoring the PGE2 levels in the blood of rats from the ENDO group (Figure 4 F). Liriodendrin administration restored the elevation of CGRP and substance P in the ventral horn of the spinal cord of rats with endometriosis LY294002 and liriodendrin administration nota- bly decreased the up-regulated substance P (Fig - ure 5 A) and CGRP levels (Figure 5 B) in the ENDO group. Moreover, LY294002 and liriodendrin admin- istration notably decreased the up-regulated sub- stance P (Figures 5 C, F , G) and CGRP (Figures 5 D, F , H) mRNA and protein in the ventral horn of the spinal cord in the ENDO rats. It is worth noting that the efficiency of lirioden- drin administration at the dose investigated was more apparent than LY294002 administration at the dose investigated for maintaining the expres- sion of CGRP and substance P in the ventral horn of the spinal cord. Also NF-κB mRNA level (Figure 5 E) showed the same tendency as the gene expression level of CGRP and substance P. LY294002 and liriodendrin administration restored the LPS-induced up-regulation of p-PI3K/p-AKT/p-mTOR in 12Z cells The expression of PI3K/AKT/mTOR and p-PI3K/ p-AKT/p-mTOR in 12Z cells treated with LPS followed by LY294002 and liriodendrin administration was investigated (Figure 6 A). No obvious difference was observed for the expression of PI3K (Figure 6 B), Jianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing 6 Arch Med Sci Figure 3. LY294002 and liriodendrin administration restored the up-regulated p-PI3K/p-AKT/p-mTOR level in the endo - metrium of endometriosis rat models (* p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – Western blot analysis of PI3K/AKT/mTOR and p-PI3K/p-AKT/p-mTOR in the endo- metrium of rats under distinct conditions. B – Quantitative analysis indicated that no obvious difference was observed for the expression of PI3K in the endometrium of rats un - der distinct treatments. C – Quantitative analysis indicated that no obvious difference was observed for the expression of AKT in the endometrium of rats under distinct treatments. D – Quantitative analysis indicated that no obvious differ - ence was observed for the expression of mTOR in the endo- metrium of rats under distinct treatments. E – Quantitative analysis indicated that LY294002 and liriodendrin adminis- tration restored the up-regulated p-PI3K level in the ENDO group. F – Quantitative analysis indicated that LY294002 and liriodendrin administration restored the up-regulated p-AKT level in the ENDO group. G – Quantitative analysis indicated that LY294002 and liriodendrin administration restored the up-regulated p-mTOR level in the ENDO group 6 4 2 0 4 3 2 1 0 8 6 4 2 0 E G F Relative density of p-PI3KRelative density of p-mTOR Relative density of p-AKT SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin 2.0 1.5 1.0 0.5 0 1.5 1.0 0.5 0 1.5 1.0 0.5 0 A B C D Relative density of PI3K Relative density of AKT Relative density of mTOR SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin PI3K p-PI3K AKT p-AKT mTOR p-mTOR β-actin SHAM ENDOENDO + LY294002ENDO + liriodendrin Liriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response and regulating the signaling pathway of Pl3K/Akt/mToR Arch Med Sci 7 AKT (Figure 6 C) and mTOR (Figure 6 D) in 12Z cells under differential conditions. However, the expres- sion levels of p-PI3K (Figure 6 E), p-AKT (Figure 6 F) and p-mTOR (Figure 6 G) were remarkably higher in LPS-treated 12Z cells when compared with the control. LY294002 and liriodendrin administration notably decreased the LPS-induced up-regula - tion of p-PI3K (Figure 6 E), p-AKT (Figure 6 F) and p-mTOR (Figure 6 G) in LPS-treated 12Z cells. Liriodendrin administration restored the LPS-induced up-regulation of IL-6, TNF-α, and IL-1β mRNA and protein expression in 12Z cells Liriodendrin administration at the dose inves - tigated showed greater efficiency than LY294002 administration at the dose investigated in restoring the LPS-induced up-regulation of IL-6 (Figure 7 A), TNF-α (Figure 7 B), and IL-1 β (Figure 7 C) mRNA 300 200 100 0 250 200 150 100 50 0 800 600 400 200 0 3000 2000 1000 0 600 400 200 0 600 400 200 0 A C E B D F Level of IL-1 [ng/l]Level of IL-6 [ng/l]Peritoneal fluid level of PGE2 [ng/l] Level of IL-2 [ng/l]Level of TNF-α [ng/l]Serum level of PGE2 [ng/l] SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin Figure 4. Liriodendrin administration restored the elevation of IL-1, IL-2, IL-6, TNF-α and PGE2 in endometriosis rat models (*p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – Liriodendrin administration restored the elevated peritoneal fluid IL-1 level in the ENDO group. B – Liriodendrin administration restored the elevated peritoneal fluid IL-2 level in the ENDO group. C – Liriodendrin administration restored the elevated peritoneal fluid IL-6 level in the ENDO group. D – Liriodendrin administration restored the elevated peritoneal fluid TNF-α level in the ENDO group. E – Liriodendrin administration restored the elevated peritoneal fluid PGE2 level in the ENDO group. F – Lirioden- drin administration restored the elevated serum PGE2 level in the ENDO group 8 Arch Med Sci 400 300 200 100 0 600 400 200 0 A B Level of substance P [pg/l] Level of CGRP [pg/l] SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin 6 4 2 0 4 3 2 1 0 C D Expression level of substance P mRNA Expression level of CGRP mRNA SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin 6 4 2 0 5 4 3 2 1 0 G H Relative density of substance P Relative density of CGRP SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin 6 4 2 0 E F Expression level of NF-κB mRNA SHAM ENDO ENDO + ENDO + LY294002 liriodendrin SHAM ENDO ENDO + ENDO + LY294002 liriodendrin Substance P CGRP β-actin Figure 5. Liriodendrin administration restored the increased CGRP and substance P in the ventral horn of spinal cord samples collected from rats with endometriosis (* p < 0.05 vs. SHAM group; #p < 0.05 vs. ENDO group). A – ELISA analysis indicated that liriodendrin administration restored the elevated substance P in the ENDO rats. B – ELISA analysis indicated that liriodendrin administration restored the elevated CGRP in the ENDO rats. C – QPCR analysis indicated that liriodendrin administration restored the elevated substance P in the ENDO rats. D – QPCR analysis indicated that liriodendrin administration restored the elevated CGRP in the ENDO rats. E – QPCR analysis indicated that liriodendrin administration restored the elevated NF- κB in the ENDO rats. F – West- ern blot analysis of CGRP and substance P expression in the ventral horn of the spinal cord of rats under distinct conditions. G – ELISA analysis indicated that liriodendrin administration restored the elevated substance P in the ENDO rats. H – ELISA analysis indicated that liriodendrin administration restored the elevated CGRP in the ENDO rats Liriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response and regulating the signaling pathway of Pl3K/Akt/mToR Arch Med Sci 9 Figure 6. LY294002 and liriodendrin administration re - stored the LPS-induced up-regulation of p-PI3K/p-AKT/ p-mTOR in 12Z cells (* p < 0.05 vs. NC group; #p < 0.05 vs. LPS group). A  – Western blot analysis of PI3K/AKT/mTOR and p-PI3K/p-AKT/p-mTOR in 12Z cells under distinct con - ditions. B – Quantitative analysis indicated that no obvious difference was observed for the expression of PI3K in 12Z cells under distinct treatments. C – Quantitative analysis indicated that no obvious difference was observed for the expression of AKT in 12Z cells under distinct treatments. D – Quantitative analysis indicated that no obvious differ - ence was observed for the expression of mTOR in 12Z cells under distinct treatments. E – Quantitative analysis indicat- ed that LY294002 and liriodendrin administration restored the LPS-induced up-regulation of p-PI3K in 12Z cells. F – Quantitative analysis indicated that LY294002 and lirioden- drin administration restored the LPS-induced up-regulation of p-AKT in 12Z cells. G – Quantitative analysis indicated that LY294002 and liriodendrin administration restored the LPS-induced up-regulation of p-mTOR in 12Z cells 5 4 3 2 1 0 5 4 3 2 1 0 6 4 2 0 E G F Relative density of p-PI3KRelative density of p-mTOR Relative density of p-AKT NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin 1.5 1.0 0.5 0 1.5 1.0 0.5 0 1.5 1.0 0.5 0 A B C D Relative density of PI3K Relative density of AKT Relative density of mTOR NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin PI3K p-PI3K AKT p-AKT mTOR p-mTOR β-actin SHAM ENDOENDO + LY294002ENDO + liriodendrin Jianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing 10 Arch Med Sci Figure 7. Liriodendrin administration restored the LPS-in - duced up-regulation of IL-6, TNF- α, and IL-1 β mRNA and protein expression in 12Z cells (* p < 0.05 vs. NC group; #p < 0.05 vs. LPS group). A – Liriodendrin administration re - stored the LPS-induced up-regulation of IL-6 mRNA expres- sion in 12Z cells. B – Liriodendrin administration restored the LPS-induced up-regulation of TNF- α mRNA expression in 12Z cells. C – Liriodendrin administration restored the LPS-induced up-regulation of IL-1β mRNA expression in 12Z cells. D – Liriodendrin administration restored the LPS-in - duced up-regulation of NF-κB mRNA expression in 12Z cells. E – ELISA analysis indicated that liriodendrin administration restored the LPS-induced up-regulation of IL-6 protein ex - pression in 12Z cells. F – Western blot analysis indicated that liriodendrin administration restored the LPS-induced up-regulation of TNF- α protein expression in 12Z cells. G – Western blot analysis indicated that liriodendrin ad - ministration restored the LPS-induced up-regulation of IL-1β protein expression in 12Z cells 4 3 2 1 0 8 6 4 2 0 4000 3000 2000 1000 0 1500 1000 500 0 4 3 2 1 0 4 3 2 1 0 6000 4000 2000 0 A C E G B D F Expression level of IL-6 mRNAExpression level of IL-1β mRNALevel of IL-6 [pg/ml]Level of IL-1β [pg/ml] Expression level of TNF-α mRNAExpression level of NF-κB mRNALevel of TNF-α [pg/ml] NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin NC LPS LPS + LPS + LY294002 liriodendrin Liriodendrin alleviates sciatic endometriosis-associated pain in rats via suppressing the inflammatory response and regulating the signaling pathway of Pl3K/Akt/mToR Arch Med Sci 11 in 12Z cells. Liriodendrin administration at the dose investigated showed more apparent effi - ciency than LY294002 administration at the dose investigated in restoring the LPS-induced up-reg - ulation of IL-6 (Figure  7 E), TNF- α protein (Fig- ure 7 F), and IL-1β protein (Figure 7 G) in 12Z cells. Also the NF- κB mRNA level (Figure 7 D) showed the same tendency as the gene expression level of CGRP and substance P.

A  disaccharide lignan named liriodendrin, (+)-syringaresinol di-O-b-D-glucopyranoside, is the bioactive component of Sargentodoxa cuneata (Oliv.) Rehd. Et Wils. A  plethora of biological ef - fects against inflammation, pain, and arrythmia have been observed for the medicine [14, 15, 27]. In this study, we evaluated the efficiency of acute drug administration and daily drug administra - tion of liriodendrin in attenuating pain in endo - metriosis rats. Liriodendrin has been reported to suppress inflammatory damage in the treatment of ulcerative colitis and sepsis-associated tissue injuries [28, 29]. However, its effect in the treat - ment of endometriosis-associated pain was not investigated. Therefore, we aimed to explore and compare its effect in the treatment of endometrio- sis-associated pain with LY294002 in our study. As a result, we found that, although both LY294002 and liriodendrin functioned as PI3K inhibitors, only liriodendrin alleviated inflammation, thus making liriodendrin a better therapeutic method compared with LY294002. In addition, the authors compared the distinct expression of neuropep - tides mRNA and protein in the ventral horn of the spinal cord of ENDO rats. Liriodendrin administra- tion restored the elevation of neuropeptides in the ventral horn of the spinal cord of ENDO rats. Upon oral administration, liriodendrin is transformed to syringaresinol, which is attributed to the anti- inflammatory effect [15]. In various hormone- dependent conditions, the phytoestrogen syringa- resinol binds selectively to estrogen receptors [21]. Previous studies have shed light on the function of the mTOR/AKT pathway in endometriosis. One of the studies revealed that the P3KCA mutation, which is important for Akt activation, is frequently observed in patients with ovarian clear cell carci - noma [30, 31]. Higher AKT activity has been ob - served in ovarian endometriosis compared to the normal endometrium, which could be possibly due to the estrogen in endometriotic cells [32]. The authors found that DIE patients showed higher activity of AKT in endometriotic lesions similar to ovarian endometriosis. A PI3K inhibitor, LY294002, was found to inhibit the PI3K/Akt/mTOR signaling pathway in animals. The aim of the study was to investigate the acute and chronic effects of intra - thecal injection of LY294002 on various signaling pathways in sciatic nerve endometriosis models in rats including PI3K/Akt/mTOR. The effects of LY294002 in the animal models began at 2-3 h. The peak effect was seen at four to five hours and was four hours long. Daily injections of LY294002 for 3 weeks greatly reduced mechanical and ther- mal hyperalgesia caused by sciatic nerve endome- triosis (Figures 1 and 2). The therapeutic effects of LY294002 in sciatic nerve endometriosis models might be related to reduced expression levels of PI3K/Akt/mTOR signaling pathway-related fac - tors (Figure 3). The authors performed western blot analysis to determine the expression of the signaling proteins and their phosphorylated coun- terparts in ENDO-treated rats and LPS-treated 12Z cells. Treatment with the drugs LY294002 and lir - iodendrin reestablished the lower levels of phos - phorylated signaling proteins in the animal and cellular models. In addition, an ELISA assay was performed to measure the IL-1, IL-2, IL-6, TNF- α and PGE2 levels in stomach fluid of ENDO-treated rats. Previously, it was reported that injection of LY294002 significantly reduced lung inflammation and airway hyperresponsiveness in an asthma an- imal model [33, 34]. Even though the mechanism is still unclear, recent studies have shown that swelling and modified nerves in the endometriotic lesions are connected to the pathology of endometriosis. The aberrant stimulation causes modification in neurotransmitter levels and also abnormal secre - tion of inflammatory factors. The PI3K inhibitors reduced the levels of IL-6, MCP-1 (also known as CCL2), TNF-α and nitric oxide, which are the key factors regulating the inflammatory process. The PI3K/Akt signaling pathway has been shown to regulate NF- κB activation and phosphorylation of p65 units of NF- κB [35]. In this study, we an - alyzed the differential expression of IL-6, TNF- α, and IL-1β in LPS-treated 12Z cells. Liriodendrin ad- ministration restored the LPS-induced elevation of IL-6, TNF-α, and IL-1β in LPS-treated 12Z cells. The (+)-syringaresinol, produced in situ from lirioden - drin by bacteria in intestines, enhanced FOXO3 and SIRT1 activity, which further modulated the activity of HIF-1 [36–38]. Hence, the authors hy - pothesized that liriodendrin degrades HIF-1 α by FOXO3 and SIRT1 activation, in turn reducing VEGF expression. In conclusion, we found that compared with LY294200, the administration liriodendrin exerted a more evident therapeutic effect in the treatment of endometriosis-associated pain by suppressing the secretion of pro-inflammatory cytokines. Conflict of interest The authors declare no conflict of interest. Jianfang Gong, Liangliang Xue, Mengling Wei, Wenli Han, Shen Jing 12 Arch Med Sci

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