717解毒合剂对蝮蛇毒所致局部组织损伤的保护作用与机制研究

Study of protective effect and mechanism of 717 Jiedu Decoction on local tissue damage caused by Agkistrodon halys venom bite

目的研究717解毒合剂对蝮蛇咬伤动物模型局部组织损伤的保护作用及其可能的机制.方法将59只健康SD大鼠按随机数字表法分为生理盐水(NS)对照组(n=12)、蝮蛇毒模型组(n=9)、抗蝮蛇毒血清组(n=11)和717解毒合剂25、60、150 mg/kg组(均n=9).采用皮下注射50 g/L蛇毒溶液建立蝮蛇毒模型,NS对照组予以等量NS.制模成功后2 h,抗蝮蛇毒血清组于尾静脉注射抗蝮蛇毒血清(每只0.6 mL),717解毒合剂组分别予以25、60、150 mg/kg 717解毒合剂生药灌胃(每日1次,连用6 d),NS对照组和蝮蛇毒模型组予以等量NS灌胃,6 d后取各组大鼠血清及大腿腓肠肌.另取15只健康KM小鼠随机分为NS对照组、蝮蛇毒模型组和717解毒合剂10 mg/kg组,每组5只,制模方法同SD大鼠,制模后717解毒合剂组以10 mg/kg 717解毒合剂生药灌胃(每日1次,连用3 d),3 d后处死.观察各组大鼠局部组织损伤情况以及小鼠腹部皮下出血情况,光镜下观察各组大鼠腓肠肌的病理学改变,采用放射免疫法(RIA)测定血清细胞外基质透明质酸(HA)、Ⅳ型胶原(ColⅣ)及层黏连蛋白(LN)水平,采用酶联免疫吸附试验(ELISA)检测血清白细胞介素-2(IL-2)、肿瘤坏死因子-α(TNF-α)、核转录因子-κB(NF-κB)和转化生长因子-β1(TGF-β1)水平,采用蛋白质免疫印迹试验(Western blotting)检测各组腓肠肌诱导型一氧化氮合酶(iNOS)和环氧合酶-2(COX-2)蛋白表达情况.结果蝮蛇毒制模后大鼠创面均出现典型中毒现象,且随时间延长逐渐加重,72 h后717解毒合剂60 mg/kg组大鼠的局部肿胀程度明显轻于同期蝮蛇毒模型组.制模4 h后KM小鼠腹部皮下可见弥漫性出血,为典型局部中毒,72 h后717解毒合剂10 mg/kg组小鼠的恢复情况明显优于同期蝮蛇毒模型组.蝮蛇毒模型组大鼠血清HA、ColⅣ和LN水平均明显高于NS对照组,而抗蝮蛇毒血清组及717解毒合剂60 mg/kg和150 mg/kg组上述指标水平则较蝮蛇毒模型组明显改善,其中HA和ColⅣ水平以717解毒合剂150 mg/kg组改善最为明显〔HA(μg/L):112.83±19.89比162.52±16.29,ColⅣ(μg/L):120.04±21.44比158.77±36.64,均P<0.01〕,LN水平以60 mg/kg组改善最为明显(μg/L:69.09±15.22比96.66±15.72,P<0.01).蝮蛇毒模型组大鼠血清IL-2、TNF-α、NF-κB和TGF-β1水平均明显高于NS对照组,而抗蝮蛇毒血清组及717解毒合剂60 mg/kg和150 mg/kg组上述指标水平则较蝮蛇毒模型组明显改善,其中NF-κB水平以717解毒合剂60 mg/kg组改善最为明显(ng/L:14.46±4.42比20.81±2.59,P<0.01).苏木素-伊红(HE)染色结果显示,NS对照组大鼠腓肠肌结构正常;蝮蛇毒模型组大鼠腓肠肌严重弥漫性肌纤维损伤与炎性浸润,可见大量细胞坏死;抗蝮蛇毒血清组大鼠腓肠肌损伤减轻,但炎性浸润显著;717解毒合剂组大鼠炎性细胞浸润减轻,正常细胞数增多.蝮蛇毒模型组大鼠腓肠肌iNOS和COX-2蛋白表达均较NS对照组明显增强,717解毒合剂60 mg/kg和150 mg/kg组的iNOS表达较蝮蛇毒模型组明显减弱,抗蝮蛇毒血清组及717解毒合剂各剂量组COX-2表达均较蝮蛇毒模型组明显减弱,其中以717解毒合剂150 mg/kg组变化最明显(iNOS/β-tubulin:0.71±0.13比0.95±0.14,COX-2/β-tubulin:0.33±0.10比0.67±0.11,均P<0.01).结论717解毒合剂能显著改善小鼠腹部皮下出血症状,促进新生肉芽组织生长,加快创面愈合,减轻腓肠肌病理损害,同时能明显改善大鼠局部组织结构,降低血清中细胞外基质成分含量与炎性因子水平,抑制腓肠肌iNOS和COX-2蛋白表达.

Objective To study the protective effect of 717 Jiedu Decoction on animal model with local tissue damage caused by the bite of Agkistrodon halys venom and its possible mechanism.Methods Fifty-nine healthy SD rats were randomly divided into 4 groups:normal saline(NS)control group(n=12),Agkistrodon halys bite model group(n=9),anti-venom serum group(n=11)and 717 Jiedu Decoction group consisting of 3 subgroups of 25,60 and 150 mg/kg(each n=9).Agkistrodon halys bite model was established by subcutaneous injection of 50 g/L snake venom solution,while the NS control group was given the same amount of NS at the same site.After successful establishment of the model for 2 hours,anti-venom serum(0.6 mL)was injected into the tail vein of each rat in the anti-venom group,in the 717 Jiedu Decoction group,25,60 and 150 mg/kg 717 Jiedu Decoction crude drug were given by gastric irrigation respectively in the corresponding subgroups(the drug was used once a day for 6 days),NS control group and Agkistrodon halys bite model group were given the same amount of NS by intragastric administration.After 6 days,the serum and thigh gastrocnemius muscle of each rat of all the groups were collected.Other 15 healthy KM mice were divided into NS control group,Agkistrodon halys bite model group and 717 Jiedu Decoction group,with 5 mice in each group.The model establishing method was the same as that of SD rats.After modeling,717 Jiedu Decoction 10 mg/kg group was given 10 mg/kg 717 Jiedu Decoction crude drug gavage(once a day for 3 days),and the mice were killed after 3 days.The rats'local tissue damage situations and the mice abdominal subcutaneous bleeding conditions were observed in each group.The pathological changes of rats'gastrocnemius muscle were investigated under light microscope in various groups.The levels of serum extracellular matrix hyaluronic acid(HA),type Ⅳ collagen(Col Ⅳ)and laminin(LN)were determined by radioimmunoassay(RIA).The levels of serum interleukin-2(IL-2),tumor necrosis factor-α(TNF-α),nuclear factor-κB(NF-κB)and transforming growth factor-β1(TGF-β1)were detected by enzyme-linked immunosorbent assay(ELISA).The protein expressions of inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)in gastrocnemius muscle were detected by Western blotting.Results After modeling,the typical poisoning phenomena appeared in all the wounds of rats in Agkistrodon halys bite model group,and became ingravescent with time.After 72 hours,the local swelling degree of rats in 717 Jiedu Decoction 60 mg/kg group was significantly milder than that in Agkistrodon halys bite model group at the same time.After modeling for 4 hours,there was subcutaneous diffuse bleeding occurred at the abdomen in KM mice,which was the typical local poisoning.After 72 hours,the recovery of rats in 717 Jiedu Decoction 10 mg/kg group was significantly better than that in Agkistrodon halys bite model group at the same time.The serum levels of HA,Col Ⅳ and LN in the Agkistrodon halys bite model group were significantly higher than those in the NS control group,while the levels of above indexes in the anti-venom serum group and the 717 Jiedu Decoction 60 mg/kg and 150 mg/kg groups were obviously improved compared with those in the Agkistrodon halys bite model group.Among them,the levels of HA and Col Ⅳ in the 717 Jiedu Decoction 150 mg/kg group were improved the most evidently[HA(μg/L):112.83±19.89vs.162.52±16.29,Col Ⅳ(μg/L):120.04±21.44vs.158.77±36.64,both P<0.01],andthe level of LN in the 60 mg/kg group was improved the most significantly(μg/L:69.09±15.22 vs.96.66±15.72,P<0.01).The serum levels of IL-2,TNF-α,NF-κB and TGF-β1 in the Agkistrodon halys bite model group were significantly higher than those in the NS control group,while those in the anti-venom serum group and the 717 Jiedu Decoction 60 mg/kg and 150 mg/kg groups were markedly improved compared with those in the Agkistrodon halys bite model group,and the NF-κB level in the 717 Jiedu Decoction 60 mg/kg group was the most significantly improved(ng/L:14.46±4.42 vs.20.81±2.59,P<0.01).Hematoxylin-eosin(HE)staining results showed that the structure of gastrocnemius muscle in NS control group was normal,in Agkistrodon halys bite model group,the gastrocnemius muscle was severely damaged showing diffuse muscular fiber injury and inflammatory infiltration with a large number of cell necrosis;the rats in the anti-venom serum group,the gastrocnemius muscle injuiy was milder,but the inflammatory infiltration was obvious;the inflammatory cell infiltration in rats in 717 Jiedu Decoction group was ameliorated and the normal cell number was increased.The protein expressions of iNOS and COX-2 in gastrocnemius muscle of rats in Agkistrodon halys bite model group were significantly strengthened compared with those in NS control group.The expressions of iNOS in 717 Jiedu Decoction 60 mg/kg and 150 mg/kg groups were obviously weakened compared with the expression in Agkistrodon halys bite model group.The expressions of COX-2 in serum group and 717 Jiedu Decoction various groups were markedly weakened compared with the expression in Agkistrodon halys bite model group,Among them,the most obvious change was seen in 717 Jiedu Decoction 150 mg/kg group(iNOS/β-tubulin:0.71±0.13 vs.0.95±0.14,COX-2/β-tubulin:0.33±0.10 vs.0.67±0.11,both P<0.01).Conclusion 717 Jiedu Decoction can significantly improve the abdomen subcutaneous bleeding situation of mice,and in experimental model rats,it can promote the growth of new granulation tissue,accelerate wound healing,reduce the pathological damage of gastrocnemius muscle,in the mean time it can obviously improve the local tissue structure,decrease the content of extracellular matrix and inflammatory factor level in serum and inhibit the protein expressions of iNOS and COX-2 in gastrocnemius muscle.