朱氏妇炎净方调控TNF/JNK/Bcl-2-Bax信号轴改善湿热瘀结型慢性盆腔痛大鼠的机制研究OA

Objective To investigate the mechanism of action of Zhu's Fuyanjing Formula(ZSFYJ)on chronic pelvic pain(CPP)with dampness-heat and blood stasis pattern in rats via the modulation of the TNF/JNK/Bcl-2-Bax signaling axis.Methods Six of twenty-six rats were randomly selected as the normal control(NC)group,while the remaining twenty rats were used to establish rat model of CPP with dampness-heat and blood stasis pattern by chemical burn method(intraperitoneal injection of 25%phenol paste into the right uterine horn of rats).After successful modeling,eighteen rats were randomly divided into the model(M)group,the ZSFYJ group,and the Fuke Qianjin Capsule(FKQJ)group,with six rats in each group.The ZSFYJ group received intragastric administration of ZSFYJ at a dose of 11.7 g/kg per day,while the FKQJ group were given Fuke Qianjin Capsules at 0.22 g/kg per day.The NC and M groups were administered an equal volume of distilled water via gavage.All treatments were continued for 21 consecutive days.Pain behavior was assessed using the hot plate test.Uterine histopathology was observed via HE staining.Serum levels of the inflammatory cytokines IL-10,IL-1β,and TNF-α,as well as the pain-associated chemokines CCL2 and CCL5,were measured using ELISA.The mRNA expression levels of JNK,Bcl-2,and Bax,as well as the protein expression levels of TNF-α,p-JNK,JNK,p-c-Jun,c-Jun,Bcl-2,Cleaved Caspase-3,Caspase-3,and Bax in uterine tissues were detected using qPCR and Western blotting,respectively.Apoptosis in uterine tissue was evaluated by TUNEL staining.Results Compared with the NC group,following changes were observed in the M group:the thermal pain threshold was significantly decreased(P<0.01).The rat uterus exhibited evident inflammatory lesions,characterized by severe congestion and edema.The endometrial structure was severely damaged,showing glandular atrophy and disarray,diffuse inflammation,and prominent edema with hyperemia.Serum levels of the upstream pro-inflammatory cytokines IL-1β,and TNF-α were significantly upregulated(P<0.01),while the chemokines CCL2 and CCL5 were simultaneously highly expressed(P<0.01).At the mRNA level,JNK expression was significantly upregulated(P<0.01),alongside marked increases in the pro-apoptotic gene Bax(P<0.01)and a significant decrease in the anti-apoptotic gene Bcl-2(P<0.01).Regarding protein expression,the upstream pro-inflammatory cytokine TNF-α was significantly elevated(P<0.01),accompanied by notable increases in the ratios of p-JNK/JNK and p-c-Jun/c-Jun(P<0.01).The pro-apoptotic protein Bax was aberrantly highly expressed(P<0.01),whereas the anti-apoptotic protein Bcl-2 was significantly reduced(P<0.01),leading to a marked elevation in the Cleaved Caspase-3/Caspase-3 ratio(P<0.01).Consequently,the percentage of TUNEL-positive cells in uterine tissues was significantly increased(P<0.01).Compared with the M group,the ZSFYJ and FKQJ groups exhibited significantly prolonged thermal pain thresholds(P<0.01,P<0.05),alongside alleviated inflammatory infiltration and markedly improved pathological damage in uterine tissues.Serum release of the pro-inflammatory cytokines IL-1β and TNF-α was significantly inhibited(P<0.01,P<0.05),while expression of the chemokines CCL2 and CCL5 was notably reduced(P<0.001),accompanied by a significant rebound in the anti-inflammatory cytokine IL-10(P<0.01).Both treatments significantly downregulated JNK mRNA transcription(P<0.001),suppressed the aberrant overexpression of Bax mRNA,and upregulated Bcl-2 mRNA expression(P<0.001).Furthermore,they inhibited the abnormal release of TNF-α protein(P<0.05),blocked the phosphorylation and activation of JNK and c-Jun(P<0.05),significantly reduced Bax protein levels,increased Bcl-2 protein levels(P<0.05,P<0.01),and diminished Caspase-3 cleavage(P<0.05).The percentage of TUNEL-positive cells was significantly reduced in both treatment groups(P<0.001).Conclusion ZSFYJ can effectively alleviate pain behavior,reduce uterine inflammation,and mitigate tissue damage in rats model of CPP with dampness-heat and blood stasis pattern.Its mechanism may be associated with modulating the TNF/JNK/Bcl-2-Bax signaling axis,thereby suppressing excessive inflammatory response and inhibiting abnormal tissue apoptosis.