组织蛋白酶B抑制剂CA-074Me对多发性肌炎豚鼠模型肌纤维保护机制的研究

Protective mechanisms of a cathepsin B inhibitor, CA-074 Me, on muscle fibers in coxsackievirus B1-induced polymyositis in guinea pigs： an experimental study

目的探讨组织蛋白酶B抑制剂CA-074Me对组织蛋白酶B的影响，以阐明其对肌纤维的保护机制。方法40只健康雌性短毛英国种豚鼠，随机分为5组，每组8只，分别为：阳性对照叫干扰素组、PM模型组、健康组、假干预组（即生理氯化钠溶液对照组）和阴性对照CA-074Me组。相应时间点处死动物，留取血液检测肌酸激酶（CK）及其同工酶（CK-MM）、门冬氨酸氨基转移酶（AST）、乳酸脱氢酶（LDH）变化；取肌肉组织进行组织病理学观察（HE染色）；用免疫组化法（Envision二步法）分别检测CD8＋细胞、组织蛋白酶B的表达水平；原位末端标记法（TUNEL）检测各组豚鼠骨骼肌细胞的凋亡变化。肌酶、肌肉炎症程度评分、肌细胞凋亡的比较均采用单因素方差分析（ANOVA）；CD8＋T细胞、组织蛋白酶B表达的结果采用Pearsonr检验。结果PM模型组、假干预组、γ干扰素组、CA-074Me组肌酶谱（CK、CK-MM、AST和LDH）均异常升高，以CK升高最为明显，4个组的CK分别为（3537．3±2141．6）U／L、（2222．0±226．9）U／L、（973．8±423．2）U／L、（814．0±268．4）U／L，均高于健康组（410．7±167．9）U／L（P〈0．05）；1干扰素组、CA-074Me组的肌酶谱呈低水平升高，与假干预组比较，差异均有统计学意义（P〈0．05）。PM模型组、假干预组、y干扰素组、CA-074Me组的骨骼肌炎症程度评分分别为1．75±0．50、1．40±0．55、2．38±0．74和1．20±0．45，均较健康组明显增加，尤其以1干扰素组炎症细胞浸润明显（P〈O．05）。与健康组相比，PM模型组、假干预组、1干扰素组、CA一074Me组的CD8’细胞、组织蛋白酶B表达增加，凋亡指数升高，差异均有统计学意义（P〈0．05）：CA-074Me组的CD8＋细胞（42．3±27．4）个、组织蛋白酶B表达（31．3±6．7）个均低于假干预组[分别为（68．0±13．2）个和（37．5±9．2）个]，P值均〈0．05；干扰素组组织蛋白酶B表达（49．3±17．0）个、凋亡指数（40．1±6．7）个均较假干预组[分别为（37．5±9．2）和（25．4±5．0）个]升高（P〈0．05）。结论组织蛋白酶B在PM豚鼠模型中存在着高表达，CA-074Me通过抑制组织蛋白酶B表达减少其介导的炎症和细胞凋亡，起到对PM模型豚鼠肌组织的保护作用。

Objective To evaluate the effect of a specific cathepsin B inhibitor, CA-074 Me, on the expression of cathepsin B in coxsackievirus Bl-induced polymyositis in guinea pigs, and to elucidate the protective mechanisms of CA-074 Me on muscle fibers. Methods Polymyositis model was established in 32 guinea pigs by infection with coxsackievirus B1, which were then equally divided into 4 groups： y-interferon group treated with intraperitoneal y-interferon （150 000 IU per kilogram per day） from week 5 to week 8, polymyositis model group receiving no treatment, pseudo-intervention group treated with intraperitoneal sodium chloride physiological solution, CA-074 Me group treated with intraperitoneal CA-074 Me （4 mg per kilogram per day） for 7 days, after the infection with coxsackievirus B1. Eight guinea pigs receiving no infection or treatment served as the healthy control group. Blood samples and muscle tissue samples were obtained from the guinea pigs in the y- interferon group on week 8 and in the other 4 groups on week 5. The serum level of muscle enzymes including creatine kinase （CK）, CK-MM, aspartic transaminase （AST） and l-lactate dehydrogenase （LDH） were determined. Muscle tissue samples were studied by hematoxylin and eosin （HE） staining, and Envision two-step method was used to quantify the expression of cathepsin B and to numerate CD8＋ T cells. The apoptosis in muscle cells was detected by terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labeling （TUNEL）.One-way analysis of variance （ANOVA） was conducted to compare the serum level of muscle enzymes, inflammation score of muscle and apoptosis index of muscular cells, and Pearson ehi-square test to compare the count of CD8＋ T cells and cathepsin B expression, among these groups. Results Polymyositis model was successfully established by infection with coxsaekievirus B1 in the 32 guinea pigs with a marked increase in the serum level of the tested enzymes, especially in that of CK. In detail, the serum level of CK was （410.7 ± 167.9） U/L in the healthy control group, significantly lower than that in the polymyositis model group （（3537.3 ± 2141.6） U/L, P 〈 0.05）, pseudo-intervention group （（2222.0 ± 226.9） U/L, P 〈 0.05）, y-interferon group （（973.8 ±423.2） U/L, P〈 0.05） and CA-074 Me group （（814.0 ± 268.4） U/L, P〈 0.05）. Compared with the pseudo-intervention group, the 5＇-interferon group and CA-074 Me group showed a slight increase in the serum level of all the four enzymes （all P 〈 0.05）. There was a significant elevation in the inflammation score of skeletal muscles in the polymyositis model group, pseudo-intervention group, y-interferon group and CA-074 Me group compared with the healthy control group （1.75 ± 0.50, 1.40 ± 0.55, 2.38 ± 0.74 and 1.20± 0.45 vs. 0.00 ± 0.00, all P 〈 0.05）, with the most intense infiltration of inflammatory cells observed in the y-interferon group. Moreover, the number of CD8 ＋ T cells, cathepsin B expression and muscular cell apoptosis index were all significantly higher in the polymyositis model group, pseudo-intervention group, 5,-interferon group and CA-074 Me group than in the healthy control group （all P 〈 0.05）. Compared with the pseudo-intervention group, the CA-074 Me group showed less CD8＋ T cells （42.3± 27.4 vs. 68.0 ± 13.2, P 〈 0.05） and lower expression of cathepsin B （31.3 ±6.7 vs. 37.5 ± 9.2, P 〈 0.05）, whereas the 5,-interferon group exhibited elevated cathepsin B expression （49.3 ± 17.0 vs. 37.5 ± 9.2, P〈 0.05） and apoptosis index （40.1 ±6.7 vs. 25.4 ± 5.0, P〈 0.05）. Conclusions Cathepsin B is highly expressed in the guinea pig model of polymyositis, while CA-074 Me may protect muscle tissue in this model by downregulating the expression of cathepsin B and attenuating the inflammation and apoptosis induced by cathepsin B.