清热除湿凝胶对HPV 16阳性的宫颈癌荷瘤小鼠线粒体凋亡蛋白的影响

Effect of Qingre Dehumidification Gel on mitochondria apoptosis protein of HPV 16 positive cervical cancer tumor-bearing mice

目的研究外用中药清热除湿凝胶对人乳头瘤病毒(HPV)16阳性的宫颈癌荷瘤小鼠线粒体凋亡蛋白的影响。方法培养HPV16阳性人宫颈癌SiHa细胞株,制成浓度为2×107/mL的细胞悬液。选取5周龄雌性裸小鼠,用1 mL注射器抽取充分混匀的细胞悬液,注入裸小鼠右前腿外上方皮下,造成HPV16阳性移植瘤模型。造模成功后采用随机数字表法将荷瘤小鼠分为5组,每组10只。即模型组、卡波姆940组、中药组、顺铂组及联合用药组。中药组于成瘤部位给予清热除湿凝胶外涂(每天每50 mm3瘤体体积外涂凝胶0.3 mL);顺铂组:将10 mg顺铂溶于66.7 mL生理盐水中制成顺铂注射液腹腔注射(每10 g体重注射0.2 mL,每3天注射1次);卡波姆940组(凝胶中辅料):卡波姆940外涂(每天每50 mm3体积外涂0.15 mL);联合用药组:同时给予中药外涂和顺铂腹腔注射,各组共给药28 d。而后麻醉剥取小鼠右前腿肿瘤组织,免疫荧光法进行检测细胞凋亡,同时Western blot法检测组织中凋亡相关蛋白Bax、Bcl-2、Caspase-9、Caspase-3。结果与模型组、卡波姆940组比较,中药组、顺铂组、联合用药组细胞凋亡率均有升高,以联合用药凋亡率最高,差异有统计学意义(P<0.05);而卡波姆940组与模型组细胞凋亡率比较,差异无统计学意义(P>0.05)。与模型组、卡波姆940组比较,中药组、顺铂组、联合用药组Bax、Caspase-3表达均上调,Bcl-2表达均下降,且联合用药组表达最明显,差异有统计学意义(P<0.05)。与模型组、卡波姆940组比较,顺铂组、联合用药组Caspase-9表达上调,差异有统计学意义(P<0.05)。结论清热除湿凝胶能改变HPV16阳性的宫颈癌荷瘤小鼠的细胞凋亡,该外用药发挥抗毒抑瘤作用的机制可能与上调肿瘤组织线粒体凋亡相关蛋白Bax、Caspase-9、Caspase-3,下调Bcl-2有关。

Objective To study the effect of topical Chinese medicine Qingre Dehumidification Gel on the mitochondrial apoptosis protein of human papillomavirus(HPV)16-positive cervical cancer tumor-bearing mice.Methods The HPV 16 positive human cervical cancer SiHa cell line to prepare a cell suspension with a concentration of 2×107/mL was cultured.Five-week-old female nude mice were selected,1 mL syringe to draw a well-mixed cell suspension were used and injected under the skin of the upper right front leg of the nude mice,resulted in an HPV 16-positive xenograft model.After successful modeling,the tumor-bearing mice were divided into five groups with ten mice in each group by random number method.Namely model group,carbomer 940 group,traditional Chinese medicine group,Cisplatin group and combination medication group.The traditional Chinese medicine group was given a Qingre Dehunidification Gel external coating(0.3 mL of gel per 50 mm3 tumor volume per day)at the tumor formation site;in the Cisplatin group:10 mg of cisplatin was dissolved in 66.7 mL of normal saline to make Cisplatin injection liquid intraperitoneal injection(0.2 mL per 10 g body weight,once every three days);carbomer 940 group(excipients in the gel):carbomer 940 externally applied(0.15 mL per 50 mm3 volume per day);combination medication group:both external application of Chinese medicine and intraperitoneal injection of Cisplatin were given for 28 days in each group.Then the tumor tissue of the right front leg of the mouse was anesthetized and removed,and the cell apoptosis was detected by immunofluorescence method.At the same time,the apoptosis-related proteins Bax,Bcl-2,Caspase-9 and Caspase-3 in the tissue were detected by Western blot method.Results Compared with the model group and the carbomer 940 group,the apoptosis rate of the Chinese medicine group,the Cisplatin group,and the combination medication group were all increased,while the combination medication had the highest apoptosis rate,and the differences were statistically significant(P<0.05);there was no statistically significant difference in the apoptosis rate between the carbomer 940 group and the model group(P>0.05).Compared with the model group and the carbomer 940 group,the expressions of Bax and Caspase-3 in the traditional Chinese medicine group,the Cisplatin group,and the combination group were all up-regulated,while the expression of Bcl-2 was decreased,while the expression in the combination group was the most obvious,and the differences were statistically significant(P<0.05).Compared with the model group and the carbomer 940 group,the expression of Caspase-9 in the Cisplatin group and the combination group was up-regulated,and the difference was statistically significant(P<0.05).Conclusion Qingre Dehumidification Gel can change the apoptosis of HPV 16-positive cervical cancer tumor-bearing mice.The mechanism of the anti-toxin and anti-tumor effect of the topical drug may be related to the up-regulation of mitochondrial apoptosis-related proteins Bax,Caspase-9,and Caspase-3 in tumor tissues,and the down-regulation of Bcl-2.