Based On JNK Signaling Pathway, The Mechanism Of Danzhiyin On Pelvic Bloody Microenvironment In EMs Was Explored

Endometriosis(EMs)is a chronic gynecological disease characterized by sustained painful symptoms which are responsible for a decline in the quality of life of sufferers.Conventional treatment includes surgical and pharmacological therapy,but there is a high recurrence rate.Although the pathogenesis of EMs remains unclear,studies have shown that chronic pelvic pain(CPP)is closely related to "immune-inflammatory effects" in EMs,including immune cytokines,inflammatory channels,and chemotactic inflammatory factors.Professor Jin proposed a brand-new theory of "the blood stasis microenvironment of EMs pelvic" and considered it as a bridge linking "blood stasis" and "immune-inflammatory effect".Dan-Zhi-Yin(DZY)is a mixture based on an empirical traditional Chinese medicine formula which can promote blood circulation and remove blood stasis.Our preliminary studies had shown that DZY could decrease the level of VEGF expression in the cytoplasm of endometrial glandular epithelial cells,which demonstrated a therapeutic effect of DZY on the chronic pelvic pain of EMs.However,the precise mechanisms of DZY on treatment for CCP of EMs are not fully understood.Accordingly,to solve the above issues,our present study was designed in three aspects including clinical trial,in vivo and in vitro experiment.Part 1 Effect of Dan-Zhi-Yin on patients with the chronic pelvic pain of EMs——a randomized controlled clinical trial.Objective:To observe the effect of DZY on the visual analogue scale(VAS)of pelvic pain,traditional Chinese medicine(TCM)symptom score,and the endometriosis health profile-30(EHP-30).Method:Between May 2016 and October 2017,a randomized placebo-controlled study was undertaken.Patients meeting inclusion criteria(confirmed endometriosis,?6 months CPP,pain score on VAS>4cm,TCM syndrome of Qi stagnation and Blood stasis)were randomly assigned using the table of random number,sequence to receive either Dan-Zhi-Yin(2 times a day)or Dan'e Fukang(10-15g/times,2 times a day).Response to treatment was assessed using subjective data for scores on VAS at baseline and at 1,2,and 3 months of follow-up.Additionally,women completed TCM symptom score and the EHP-30 at 3 months.Result:31 patients were assigned to the DZY group,and 32 patients were assigned to the Dan'e Fukang group.In the DZY group,VAS scores were significantly lower at 1,2,3 months than at baseline(P<0.05 for all).Additionally,scores were significantly lower in the DZY group than in the Dan'e Fukang group(P<0.05).In the DZY group,the total TCM symptom scores were significantly lower at 1,2,3 months than at baseline(P<0.05 for all).However,there was no difference between DZY group and Dan'e Fukang group(P>0.05).In the TCM symptoms,non-menstrual pain,the obstruction of menstrual flow,and breast tenderness scores were significantly lower in the DZY group than in the Dan'e Fukang group(P<0.05).At 3 months,the women in the DZY group significantly improve the quality of life,compared with the baseline(P<0.05).What's more,pain and emotion control of EHP-30 were significantly lower in the DZY group than in the Dan'e Fukang group(P<0.05).Conclusion:Dan-Zhi-Yin could be used to manage endometriosis-associated CPP.Part 2 in vitro experiment of DZY affecting "immune-inflammatory effect" on rat Endometriosis model.Objective:To observe the effect of DZY on the expression levels of MCP-1?MIP-2?IL-17A?IL-17F and JNK phosphorylation.Method:A total of 30 female Sprague-Dawley(SD)rats were divided into the blank control group(n=6)and endometriosis group(EMs group,n=24),in which endometriosis was surgically induced in model rats by auto-transplantation of EMs tissues.After 4 weeks,the EMs model rats randomly divided into four subgroups(n=6),including the untreated model group,and three groups administered low,middle,high DZY decoction.Following 21 days of treatment,the associated proteins were analyzed using immunohistochemistry and enzyme linked immune-sorbent assay(ELISA)to investigate the underlying mechanisms.Result:Compared with the model group,the expression levels of immune cytokines(IL-17A and IL-17F)and chemotactic inflammatory factors(MCP-1 and MIP-2)were significantly decreased in DZY-treated groups(P<0.05).Furthermore,the level of JNK phosphorylation was significantly lower in the DZY group than in the model group(P<0.05).Conclusion:DZY may be partially attributed to the decrease of MCP-1 and MIP-2 expression levels via JNK pathway in rat Endometriosis model.Part 3 in vivo experiment of DZY affecting "immune-inflammatory effect" on NIH 3T3 cells.Objective:The aim of the present study was to observe the mechanism of DZY on IL-17A/F-induced chemokines in the mouse embryonic fibroblasts(NIH 3T3).Method:The viability of cells was determined by CCK8 assay.We evaluated the expression of MCP-1 and MIP-2 by ELISA and real-time RT-PCR.The phosphor JNK was assessed by Cytometric Bead Array.IL-17RA and IL-17RC were inhibited by small interfering RNA(siRNA).Result:Our study demonstrated that IL-17A/F were able to increase the gene levels and protein secretion of MCP-1 and MIP-2.However,the DZY could disrupt the expression of MCP-1 and MIP-2 stimulated by IL-17A/F.We observed that IL-17A/F could induce JNK activation and that pharmacological inhibitors of JNK(SP600125)blocked the IL-17A/F mediated MCP-1 and MIP-2 release.DZY could inhibited JNK expression at each time point,compared with Il-17A/F stimulated group alone.In addition,we also found that NIH 3T3 cells expressed IL-17 receptors.The expression of MCP-1 and MIP-2 stimulated by IL-17A/F could be reduced by inhibiting IL-17RA or IL-17RC expression via siRNA.Conclusion:IL-17A/F could induce the expression of MCP-1 and MIP-2 through JNK pathway in NIH 3T3 cells.However,DZY can inhibit IL-17A/F-induce MCP-1 and MIP-2 expression which might be regulated by JNK pathway.All these above indicate that DZY plays a role in anti-inflammatory process in NIH 3T3 cells.