摘要
背景:尽管抗蛇毒血清为蛇伤治疗带来了一线希望,但这种药物对由毒蛇引起的局部组织损伤并不总是有效的。此外,抗蛇毒血清还存在超敏反应、“血清病”等免疫反应、部分地区难以获得等诸多局限性。因此,探寻蛇伤补充或替代疗法及治疗靶点已成为当前临床急需解决的问题。目的:观察717解毒合剂对蝮蛇伤大鼠局部组织细胞外基质蛋白及基质金属蛋白酶/组织金属蛋白酶抑制因子表达的影响。方法:将SD大鼠随机分为对照组、模型组、抗蝮蛇毒血清组及717解毒合剂低、中、高剂量组。除对照组外,其余各组于左后肢腓肠肌注射蛇毒1.25 mL/kg进行造模,717解毒合剂低、中、高剂量组在注射蛇毒前预防性灌胃1.98,3.96,7.92 g/kg 717解毒合剂,连续给药7 d,抗蛇毒血清组于注射蛇毒2 h后尾静脉注射0.6 mL抗蛇毒血清。注射蛇毒后24 h,苏木精-伊红染色观察腓肠肌病理学变化,ELISA及qPCR检测血清及腓肠肌中基质金属蛋白酶2、基质金属蛋白酶9、组织金属蛋白酶抑制因子1、组织金属蛋白酶抑制因子2蛋白及mRNA表达水平,免疫组化检测腓肠肌中Ⅰ型胶原、Ⅳ型胶原、纤连蛋白、层粘连蛋白、α-平滑肌肌动蛋白、转化生长因子β1、CD31及血管内皮生长因子的表达。结果与结论:①与对照组相比,模型组局部出现明显的炎细胞浸润及出血,肌肉结构紊乱,肌细胞变性坏死;血清及腓肠肌中基质金属蛋白酶2、基质金属蛋白酶9、组织金属蛋白酶抑制因子1蛋白及mRNA表达均升高(P<0.05或P<0.01),组织金属蛋白酶抑制因子2 mRNA表达降低(P<0.01);腓肠肌中Ⅰ型胶原、Ⅳ型胶原蛋白表达显著降低(P<0.01),层粘连蛋白、血管内皮生长因子及CD31蛋白表达升高(P<0.05或P<0.01),而纤连蛋白、α-平滑肌肌动蛋白、转化生长因子β1蛋白表达无统计学差异(P>0.05)。②与模型组相比,717解毒合剂各剂量组及抗蝮蛇毒血清组肌细胞损伤减轻,炎细胞浸润及出血现象改善;血清及腓肠肌中基质金属蛋白酶2、基质金属蛋白酶9、组织金属蛋白酶抑制因子1蛋白及mRNA表达明显降低(P<0.05或P<0.01);717解毒合剂高剂量组组织金属蛋白酶抑制因子2蛋白表达升高(P<0.05);Ⅰ型胶原、Ⅳ型胶原、层粘连蛋白、α-平滑肌肌动蛋白、转化生长因子β1表达升高(P<0.05或P<0.01),且存在剂量效应关系;血管内皮生长因子蛋白表达降低(P<0.05或P<0.01),纤连蛋白表达无显著差异(P>0.05);717解毒合剂低剂量组CD31蛋白表达升高(P<0.01)。结果表明,717解毒合剂可通过调控基质金属蛋白酶2、基质金属蛋白酶9与组织金属蛋白酶抑制因子1、组织金属蛋白酶抑制因子2的表达,促进α-平滑肌肌动蛋白、转化生长因子β1的产生,使细胞外基质的合成大于降解,维持细胞外基质稳态,进而促进蝮蛇咬伤局部组织损伤修复。
BACKGROUND:While antivenom has provided hope for treating snakebites,it is not always effective for local tissue damage caused by venomous snakes.Additionally,antivenom has several limitations,including the risk of hypersensitivity,immune reactions like“serum sickness,”and accessibility issues in some regions.Consequently,there is an urgent need to explore complementary or alternative therapies and therapeutic targets for snakebites in clinical settings.OBJECTIVE:To observe the effect of the 717 Jiedu Decoction on the expression of extracellular matrix proteins and matrix metalloproteinases/tissue inhibitors of metalloproteinases in the local tissues of rats bitten by Agkistrodon halys.METHODS:SD rats were randomly divided into a control group,a model group,an anti-viper venom serum group,and 717 Jiedu Decoction low-,medium-and high-dose groups.Except for the control group,all other groups received an injection of 1.25 mL/kg of viper venom into the gastrocnemius muscle of the left hind limb to create a model.The 717 Jiedu Decoction low-,medium-and high-dose groups were administered a preventive oral gavage of 1.98,3.96,and 7.92 g/kg daily for seven consecutive days prior to the viper venom injection.The anti-viper venom serum group received a tail vein injection of 0.6 mL of antivenom serum two hours after the venom injection.Twenty-four hours post-injection,the pathological changes in the gastrocnemius muscle were observed using hematoxylin-eosin staining.The expression levels of matrix metalloproteinase-2,matrix metalloproteinase-9,tissue inhibitor of metalloproteinase-1,and tissue inhibitor of metalloproteinase-2 proteins,as well as their mRNA in serum and gastrocnemius muscle,were evaluated using ELISA and qPCR.The expression levels of type I collagen,type IV collagen,fibronectin,laminin,α-smooth muscle actin,transforming growth factor-β1,CD31,and vascular endothelial growth factor in the gastrocnemius muscle were analyzed by immunohistochemistry.RESULTS AND CONCLUSION:(1)Compared to the control group,the model group displayed significant local inflammatory cell infiltration,hemorrhage,muscle structure disruption,and myocyte degeneration and necrosis.The protein and mRNA levels of matrix metalloproteinase-2,matrix metalloproteinase-9,and tissue inhibitor of metalloproteinase-1 in serum and gastrocnemius muscle were elevated(P<0.05 or P<0.01),while the mRNA expression of tissue inhibitor of metalloproteinase-2 was reduced(P<0.01).The protein levels of type I collagen and type IV collagen in the gastrocnemius muscle were significantly decreased(P<0.01),whereas the protein levels of laminin,vascular endothelial growth factor,and CD31 were increased(P<0.05 or P<0.01);however,there were no significant changes in the protein levels of fibronectin,α-smooth muscle actin,and transforming growth factor-β1(P>0.05).(2)In contrast to the model group,the 717 Jiedu Decoction low-,medium-and high-dose groups and the anti-viper venom serum group demonstrated alleviated myocyte damage,with improved reduction in inflammatory cell infiltration and hemorrhage.The levels of matrix metalloproteinase-2,matrix metalloproteinase-9,and tissue inhibitor of metalloproteinase-1 in serum and gastrocnemius muscle were significantly reduced(P<0.05 or P<0.01).In the high-dose 717 Jiedu Decoction group,the protein level of tissue inhibitor of metalloproteinase-2 was upregulated(P<0.05).Type I collagen,type IV collagen,laminin,α-smooth muscle actin,and transforming growth factor B1 protein expression were elevated(P<0.05 or P<0.01),and there was a dose-effect relationship;vascular endothelial growth factor expression was decreased(P<0.05 or P<0.01),there was no significant difference in the expression of fibronectin(P>0.05);the expression of CD31 protein was elevated in the 717 Jiedu Decoction low-dose group(P<0.01).The results showed that 717 Jiedu Decoction could promote the production ofα-smooth muscle actin and transforming growth factorβ1 by regulating the expression of matrix metalloproteinase 2,matrix metalloproteinase 9 with tissue metalloproteinase inhibitory factor 1,and tissue metalloproteinase inhibitory factor 2,so as to make the synthesis of extracellular matrix greater than the degradation,maintain the extracellular matrix homeostasis,and then promote the repair of local tissue damage in pit viper bites.
作者
王万春
易军
严张仁
杨悦
董德刚
李玉梅
Wang Wanchun;Yi Jun;Yan Zhangren;Yang Yue;Dong Degang;Li Yumei(Jiangxi University of Chinese Medicine,Nanchang 330004,Jiangxi Province,China;Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,Jiangxi Province,China)
出处
《中国组织工程研究》
北大核心
2025年第30期6457-6465,共9页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金地区项目(81360288,81960874),项目负责人:王万春
国家自然科学基金地区项目(82260937),项目负责人:董德刚
江西省自然科学基金重点项目(20202ACB206010),项目负责人:王万春
江西中医药大学首批科技创新团队资助项目(CXTD22009),项目负责人:王万春
江西省2023年度研究生创新专项资金项目(YC2023-B230),项目负责人:李玉梅。
关键词
蝮蛇咬伤
717解毒合剂
细胞外基质
腓肠肌
基质金属蛋白酶
Α-平滑肌肌动蛋白
转化生长因子β1
工程化组织构建
Agkistrodon halys bite
717 Jiedu Decoction
extracellular matrix
gastrocnemius muscle
matrix metalloproteinase
α-smooth muscle actin
transforming growth factorβ1
engineered tissue construction
