Research On The Effect Of 717 Jiedu Decoction Regulated Local Inflammatory Microenvironment Homeostasis To Induce Tissue Regeneration In Agkistrodon Halys Bites

Objective:To explore the expression changes of 717 Jiedu decoction on local tissue inflammatory microenvironment of rats bitten by Agkistrodon halys bites,including neutrophil exagglutination(NETs)and CYR61/CCN1 expression in local tissue.Revealing the dynamic changes of local inflammatory microenvironment in the process of 717 Jiedu Mixture intervening local tissue injury from histomorphological,cellular and molecular levels,in order to find new targets for the treatment of venomous snakebite.Methods:SD rats were randomly divided into normal control group,Agkistrodon halys halys snake bite group,Agkistrodon halys venom serum group,717 Jiading mixture high and low dose groups(150,60 mg crude drug /kg).The serum and thigh gastrocnemius muscle were collected.The ultrastructural changes of gastrocnemius muscle were observed under transmission electron microscope.The pathological changes of gastrocnemius muscle were observed by HE and Masson staining.Pico Green fluorescence was used to detect the level of Circulating Free Cell DNA(cf-DNA)in the serum of each group.ELISA was used to detect the level of Neutrophil Elastase(NE)and Myeloperoxidase(MPO)expression levels,the changes of MPO in gastrocnemius muscle.The changes of Neutrophil Elastase(NE)in gastrocnemius muscle were detected by immunohistochemistry.The expression of Citrullinated Histone H3(Cit H3)in the gastrocnemius was detected by immunofluorescence.Western Blotting and PCR were used to detect the expression of CYR61/CCN1 protein and mRNA in the gastrocnemius.TUNEL method was used to detect the apoptosis of gastrocnemius muscle cells.Results:1.In terms of histomorphology:The anti-agkistrodon halys venom serum group showed slight swelling of local wounds and significant inflammatory reaction.In the 717 Jiedu mixture group,the inflammatory reaction of local wound was lighter,the granulation grew well,and the skin around the wound was repaired faster.The anti-inflammatory and repair effects of 717 Jiedu mixture in the high dose group were significantly higher than those in the low dose group.Under transmission electron microscope,the cell membrane of gastrocnemius muscle in the model group was disintegrated,a large area of muscle fiber was dissolved,and the number of organelles was significantly reduced.In the low-dose 717 Jiedu mixture group,the gastrocnemius muscle structure was slightly damaged,with hypertrophic changes,mild edema of cells,and neatly arranged muscle fibers.The gastrocnemius muscle structure in the high-dose 717 Jiading mixture group was slightly damaged,the cytoplasm was uniform,the number of organelles was normal,and the muscle fibers were arranged neatly and uniform in thickness.TUNEL assay showed that there were a large number of apoptotic cells in the model group,and the number was significantly higher than that in the normal control group.A small number of apoptotic cells were observed in all treatment groups,but the number of apoptotic cells in the 717 Jiedu mixture high dose group was less than that in the agkippers serum group and 717 Jiedu mixture low dose group.2.NETs markers: Compared with the normal group,there were significant inflammatory infiltration and bleeding reaction,and the degree of collagen fibrosis was increased in each snake injury group.The levels of serum cf-DNA,NE and MPO were significantly increased(P<0.05 or P < 0.01),the expressions of NE,MPO and Cit H3 proteins in the gastrocnemius were increased(P<0.05 or P<0.01),and the expressions of CYR61/CCN1 protein and mRNA in the gastrocnemius were up-regulated.717 Jiedu mixture significantly improved the local inflammatory response and bleeding symptoms of rats,reduced the degree of collagen fibrosis,reduced serum cf-DNA,MPO and NE contents(P<0.05 or P<0.01),and reduced the expression of NE,MPO and Cit H3 in gastrocnemium muscle(P<0.05 or P<0.01).The expression of CYR61/CCN1 protein and mRNA was inhibited,and the effect of high-dose group was significantly better than that of low-dose group in a dose-dependent manner.Conclusion:1.Viper bites in animal models can cause significant inflammatory infiltration and hemorrhagic reactions,and aggravate the degree of collagen fibrosis;it triggers the high expression of a variety of NETs key markers in the microenvironment,which is characterized by a significant increase in the content of cf-DNA,NE and MPO in rat serum and NE,MPO in Cit H3 content increased.2.717 Jiedu decoction can reduce the degree of collagen fibrosis of gastrocnemius muscle and the level of cf-DNA in rat blood.The levels of NE and MPO in serum can be significantly reduced.At the same time,the expression level of MPO,NE and Cit H3 protein in the gastrocnemius muscle can be significantly reduced,717 Jiedu decoction granules can significantly inhibit the high expression of viper bite rat serum and gastrocnemius muscle NETs markers.The regulation of NETs expression level in the inflammatory microenvironment is an important mechanism of717 antidote mixture in the treatment of local tissue damage of viper bites.