717解毒合剂通过NETs-CYR61/CCN1信号通路对蝮蛇咬伤大鼠局部组织损伤修复作用

Effect of 717 Jiedu Decoction on Local Tissue Damage Repair of Rats After Agkistrodon halys Bite Through NETs-CYR61/CCN1 Signaling Pathway

目的探究717解毒合剂对蝮蛇咬伤大鼠局部组织中性粒细胞胞外陷阱(neutrophil extracellular traps,NETs)水平,以及对富含半胱氨酸蛋白61(CYR61/CCN1)表达的影响。方法将SD大鼠随机分为正常对照组、蝮蛇咬伤组及抗蝮蛇毒血清组及717解毒合剂高、低剂量组。收集血清、大腿腓肠肌,采用苏木素-伊红(Hematoxylin-Eosin staining,HE)和马松(Masson)染色观察各组腓肠肌病理学变化,透射电镜观察腓肠肌超微结构,原位末端凋亡(TUNEL)法检测各组大鼠腓肠肌细胞凋亡情况,PicoGreen荧光检测各组血清游离DNA(cf-DNA)水平,酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)法检测各组血清与腓肠肌中髓过氧化物酶(myeloperoxidase,MPO)的表达水平,免疫组化检测腓肠肌中性粒细胞弹性蛋白酶(neutrophil elastase,NE)的改变,免疫荧光法检测各组大鼠腓肠肌瓜氨酸化组蛋白H3(CitH3)水平,Western blot与聚合酶链式反应(polymerase chain reaction,PCR)技术检测腓肠肌组织CYR61/CCN1蛋白及其mRNA表达情况。结果与正常组比较,模型组出现显著的炎性浸润与出血反应,且胶原纤维化程度均升高;超微结构显示模型组大鼠腓肠肌细胞结构损伤严重,肌节呈非对称性分布,肌原纤维丝断裂;并分布有大量凋亡细胞,血清cf-DNA、MPO含量显著上升(P<0.01),腓肠肌MPO及CitH3蛋白表达均有升高(P<0.05或P<0.01),腓肠肌CYR61/CCN1蛋白及其mRNA表达均有上调;717解毒合剂各给药组均能显著改善大鼠局部炎性反应及出血症状,降低胶原纤维化程度,有效抑制蝮蛇咬伤大鼠腓肠肌细胞凋亡率,减少血清cf-DNA、MPO含量(P<0.05或P<0.01),717解毒合剂高剂量组抑制了腓肠肌NE、MPO的表达,717解毒合剂各给药组均能降低腓肠肌CitH3的表达(P<0.05或P<0.01),抑制了CYR61/CCN1蛋白及其mRNA高表达。结论蝮蛇毒诱导局部组织中性粒细胞NETs形成,CYR61/CCN1地过表达,717解毒合剂改善了大鼠腓肠肌超微结构,抑制了细胞凋亡,并可能通过调控NETs-CYR61/CCN1信号通路改善蛇伤症状,NETs信号分子及CYR61/CCN1有望成为蝮蛇毒致局部组织损伤防治的新靶点。

OBJECTIVE To investigate the effect of 717 Jiedu decoction on neutrophil extracellular traps(NETs)and CYR61/CCN1 expression in local tissue of rats after Agkistrodon halys bite.METHODS SD rats were randomly divided into normal control group,Agkistrodon halys bite group and high-dose and low-dose 717 Jiedu decoction groups.The serum and thigh gastrocnemius were collected,and the pathological changes of gastrocnemius in each group were observed by Hematoxylin-Eosin staining(HE)and Masson staining,the ultrastructure of gastrocnemius in each group was observed by transmission electron microscopy,the apoptosis of gastrocnemius cells in each group was detected by TUNEL method,and the serum circulating-free cell(cf-DNA)level was detected by PicoGreen fluorescence.The expression levels of myeloperoxidase(MPO)in serum and gastrocnemius were detected by enzyme-linked immunosorbent assay(ELISA),the change of neutrophil elastase(NE)in gastrocnemius was detected by immunohistochemistry,and the level of citcitinic histone H3(CitH3)in gastrocnemius was detected by immunofluorescence.The expressions of CYR61/CCN1 protein and its mRNA in gastrocnemius were detected by Western blot and PCR.RESULTS Compared with the normal group,the model group showed significant inflammatory infiltration and bleeding reaction,and the degree of collagen fibrosis increased.Ultrastructure showed that the structure of gastrocnemius cells in the model group was seriously damaged,and the distribution of muscle segments was asymmetrical.The contents of cf-DNA and MPO in serum were significantly increased(P<0.01),the protein expressions of MPO and CitH3 in gastrocnemius were increased(P<0.05 or P<0.01),and the protein and mRNA expressions of CYR61/CCN1 in gastrocnemius were up-regulated.All the administration groups of 717 Jiedu decoction significantly improved the local inflammatory response and bleeding symptoms of rats,reduced the degree of collagen fibrosis,effectively inhibited the apoptosis rate of gastrocnemius cells of pit viper bitten rats,and reduced the contents of cf-DNA and MPO in serum(P<0.05 or P<0.01).The expressions of NE and MPO in gastrocnemius were inhibited in the high-dose 717 Jiedu decoction group,and the expression of CitH3 in gastrocnemius muscle was decreased in all administration groups of 717 Jiedu decoction(P<0.05 or P<0.01),and the high expression of CYR61/CCN1 protein and its mRNA was inhibited.CONCLUSION The venom of Agkistrodon halys can induce the formation of neutrophil NETs in local tissue and the overexpression of CYR61/CCN1.The 717 Jiedu decoction could improve the ultrastructure of gastrocnemius muscle and inhibit cell apoptosis,which may also improve snitch symptoms by regulating the NETs-Cyr61/CCN1 pathway.The signaling molecules of NETs and CYR61/CCN1 are expected to be new targets for the prevention and treatment of local tissue damage caused by Agkistrodon halys venom.