兔豆状囊尾蚴Ⅰ型胱抑素对脂多糖诱导小鼠脓毒症防治机制的研究

Studies on the effect of Cysticercus pisiformis type Ⅰ cystatin on lipopolysaccharide-induced sepsis in mice

为深入探究兔豆状囊尾蚴半胱氨酸蛋白酶抑制剂(CPI)超家族成员I型胱抑素(Stefin)对脂多糖(LPS)诱导的小鼠脓毒症的治疗效果,本研究将重组菌pGEX-4T-CpStefin/BL21经IPTG诱导表达重组Stefin蛋白(r-Stefin),采用GST试剂盒纯化上述蛋白,并去除内毒素后,采用SDS-PAGE检测r-Stefin的纯化效果。结果显示,r-Stefin在大肠杆菌中获得了表达且纯化效果较好。将雄性BALB/c小鼠随机分为6组,每组16只,分别为A组(生理盐水100μL/只,阴性对照组)、B组:GST蛋白对照组(25μg/只)、C组:r-Stefin组(25μg/只)、D组:脓毒症组(0.2 mg/100μL LPS/只)、E组:GST蛋白干预组(0.2 mg/100μL/只LPS+GST蛋白25μg/只)、F组:r-Stefin干预组(0.2 mg/100μL LPS/只+r-Stefin 25μg/只)。经腹腔注射后观察各组小鼠的临床症状、绘制各组小鼠在LPS诱导后不同时间的生存率曲线。在LPS诱导后12 h和24 h对各组小鼠采血,采用ELISA检测各组小鼠血清中各炎性细胞因子的含量,通过全自动生化仪测定各组小鼠血清中肝、肾功能指标。结果显示,D、E组小鼠经LPS诱导后很快出现被毛发凌乱、活动量减少、精神沉郁等症状。F组小鼠上述症状在一定程度上得到改善。A、B、C组小鼠均健活。生存率曲线显示,D组小鼠在36 h内全部死亡,E组小鼠在48 h~60 h时的生存率由75%快速降至72 h时的37.5%,F组小鼠的生存率在72 h时达75%。A~C组小鼠生存率均达100%。ELISA结果显示,LPS诱导12 h时,D组、E组和F组小鼠血清中促炎细胞因子(IL-6、TNF-α和TNF-β)及抑炎细胞因子(IL-10)的含量与A组相比均极显著升高;D组小鼠血清中IL-10含量显著低于F组。TNF-α及TNF-β含量分别极显著和显著高于F组。LPS诱导24 h时,D组小鼠血清中IL-6、TNF-α和TNF-β含量极显著高于F组,IL-10含量极显著低于F组。经LPS诱导后12 h,D组、E组、F组小鼠血清中肝脏指标ALB的含量与A组相比均极显著下降(P<0.001),ALT含量均极显著升高。D组小鼠血清中ALB和ALT含量显著或极显著高于F组;LPS诱导24 h时,D组小鼠血清中ALB的含量极显著低于F组,ALT的含量极显著高于F组。LPS诱导12 h时,D组、E组、F组小鼠血清中肾脏指标BUN和CREA的含量与A组相比均极显著升高,D组小鼠血清中BUN和CREA的含量均显著高于F组。LPS诱导24 h时,D组小鼠血清中BUN和CREA的含量均极显著高于F组。病变观察结果显示,LPS诱导后12 h,各组小鼠各脏器组织病变均无差别;LPS诱导后24 h,D组和E组小鼠肺、肝、肾组织均出现较严重的病理改变,而F组小鼠上述各组织病变均得到了改善。A、B、C组小鼠各组织均无明显病变。上述结果表明,r-Stefin干预可减轻脓毒症小鼠的炎症反应,使其肝肾功能明显好转,各组织病变明显减轻。综上所述,本研究首次证实Stefin蛋白对LPS诱导的小鼠脓毒症有明显的治疗作用,具有成为治疗脓毒症候选药物的潜力,为深入探究脓毒症的发病机制及开发新的治疗药物提供实验依据。

In order to deeply investigate the therapeutic effect of Cysticercus pisiformis type I cystatin(Stefin),a member of the cysteine protease inhibitor(CPI)superfamily,on lipopolysaccharide(LPS)-induced sepsis in mice.In this study,recombinant bacterium pGEX-4T-CpStefin/BL21 was induced by IPTG to express recombinant Stefin protein(r-Stefin),and the protein were purified by using GST kit.The expression and purification of r-Stefin was detected by SDS-PAGE after removalendotoxin.The results showed that r-Stefin was expressed in E.coli.There was one band at 36ku(r-Stefin),indicating that r-Stefin had a good purification effect.Male BALB/c mice were randomly divided into 6 groups with 16 mice in each group,namely,Group A(100μL of saline injection per mouse,negative control group),Group B:GST protein control group(25μg of GST protein per mouse),Group C:r-Stefin group(25μg of r-Stefin protein per mouse),Group D:sepsis group[100μL of LPS(containing 0.2mg LPS,hereinafter)],Group E:GST protein intervention group(100μL LPS and 25μg of GST protein per mouse),and Group F:r-Stefin intervention group(100μL LPS and 25μg r-Stefin per mouse).All groups were administered by intraperitoneal injection.The clinical symptoms of the mice in each group were observed during the 72 hours observation period,and the survival rate curves of mice in each group were plotted at different time after LPS induction.At 12 hours and 24 hours after LPS induction,blood samples were collected from each group of mice and the contents of inflammatory cytokine in serum of mice in each group were detected by ELISA,and the liver and kidney function indexes in the serum of mice in each group were measured by fully automatic biochemistry instrument.The results showed that mice in group D and E showed dishevelled coat,reduced activity and depression after LPS induction,and mice in group F showed some improvement of the above symptoms,while mice in groups A,B and C were all healthy.The survival rate curve showed that all mice in group D died within 36 hours,the survival rate of mice in group E decreased rapidly from 75%at 48 hours to 60 hours to 37.5%at 72 hours,and the survival rate of mice in group F increased significantly,reaching 75%at 72 hours.The survival rate of mice in group A to C was 100%.ELISA results showed that the serum levels of pro-inflammatory cytokines(IL-6,TNF-αand TNF-β)and anti-inflammatory cytokines(IL-10)in mice of groups D,E and F were significantly increased compared with those in group A after LPS induction 12 hours.The contens of IL-10 in mice serum of group D were significantly lower than that in group F.The contents of TNF-αand TNF-βwere highly significantly higher than those in group F,respectively.At 24 hours after LPS induction,the serum levels of IL-6,TNF-α,and TNF-βin mice in group D were highly significant higher than those in group F,while the levels of IL-10 were highly significant lower than those in group F.The serum levels of IL-10 in mice serum in group D were significantly lower than those in group A.At 12 hours after LPS induction,the liver indicators in serum of ALB in mice in groups D,E,and F were all significantly lower and ALT was significantly higher compared with those in group A.The ALB and ALT in serum of mice in group D were significantly higher than those in group F.After 24 hours of LPS induction,the serum levels of ALB in mice of group D were significantly lower than those in group F,while the levels of ALT were significantly higher than those in group F.At 12 hours after LPS induction,the kidney indicators in serum of BUN and CREA in mice in groups D,E,and F were all significantly higher than those in group A,and the BUN and CREA in serum of mice in group D were all significantly higher than those in group F.At 24 hours after LPS induction,the kidney indicators in serum of BUN and CREA in group D were all significantly higher than those in group F.The lesion observation results showed that at 12 hours after LPS induction,there was no difference in tissue lesions of all organs in all groups of mice;at 24 hours after LPS induction,the lung,liver and kidney tissues of the mice in groups D and E showed more serious pathological changes,while the lesions of the above mentioned tissues of the mice in group F were improved,and the tissue lesions of the mice in groups A,B,and C had no obvious lesions.The above results indicate that the LPS-induced mouse sepsis model was established in the present study,and the inflammatory reaction of septic mice was reduced,the liver and kidney functions were significantly improved,and the lesions of lungs,livers,and kidneys were also significantly reduced and improved after r-Stefin intervention.In conclusion,the present study demonstrated for the first time that Stefin protein,as an effective immunomodulator in vivo,has a clear therapeutic effect on LPS-induced sepsis in mice,and has the potential to be a drug of choice for the treatment of sepsis symptoms,providing experimental basis for further exploration of the pathogenesis of sepsis and the development of new therapeutic drugs.