基于Bax、Bcl-2、与Caspase-3探讨凉血固元汤对大鼠急性胃辐射伤的作用机制

Mechanism of Liangxue Guyuan decoction on acute gastric radiation injury in rats based on Bax, Bcl-2 and Caspase-3

目的通过动物实验,探讨中药复方凉血固元汤对急性辐射伤大鼠胃黏膜细胞凋亡的作用及机制。方法选用雌性SD大鼠30只,取5只作为空白组,其余大鼠用60 Co-γ射线照射制造大鼠急性胃辐射伤模型,取造模成功20只,随机分为4组:单纯照射组、地塞米松组及中药低、高剂量组,每日观察大鼠生存状况,在照射后第3天麻醉、处死大鼠,行胃组织HE染色并评分,采用TUNEL荧光染色法检测细胞凋亡,Western Blot法检测B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)和半胱氨酸天冬氨酸蛋白酶-3(cysteinyl aspartate specific proteinase 3,Caspase-3)的蛋白表达,PCR法检测Bax、Bcl-2与Caspase-3 mRNA表达量。结果照射后各组大鼠均出现不同程度的稀便,以单纯照射组明显,中药高剂量组呈不成型的软便。病理观察见单纯照射组细胞间隙扩大,腺体排列不整齐,黏膜充血、水肿明显,各治疗组损伤程度有所减轻,损伤评分均高于空白组(P<0.01),中药高剂量组胃黏膜轻度充血、水肿,损伤评分低于单纯照射组(P<0.05)。TUNEL表达结果显示,中药高剂量组TUNEL阳性细胞表达数低于单纯照射组(P<0.05)。在蛋白表达方面,单纯照射组Bax表达量明显高于空白组(P<0.01),中药高剂量组较单纯照射组降低(P<0.05);单纯照射组、地塞米松组及中药低剂量组Caspase-3及Bax/Bcl-2较空白组均升高(P<0.05或P<0.01),中药高剂量组低于单纯照射组(P<0.05或P<0.01)。各组mRNA表达比较,单纯照射组、地塞米松组和中药低剂量组中Bax mRNA相对表达量及单纯照射组和地塞米松组Caspase-3 mRNA相对表达量较空白组均升高(P<0.05或P<0.01),而中药低、高剂量组能降低Bax mRNA和Caspase-3 mRNA表达,以中药高剂量组最为显著(P<0.01)。结论凉血固元汤能减轻由60 Co-γ射线引发的大鼠急性胃辐射伤,其作用机制可能与其抑制Bax与Caspase-3表达,降低Bax/Bcl-2,从而抑制细胞凋亡有关。

Objective To explore the effect and mechanism of Liangxue Guyuan decoction on apoptosis of gastric mucosa cells in rats with acute radiation injury through animal experiments.Methods 5 of 30 female SD rats were selected as the blank group.The other rats were irradiated with 60 Co-gamma rays to establish acute gastric radiation injury model.A total of 20 rats were successfully modeled and randomly divided into 4 groups:irradiation group,dexamethasone group,low and high dose of Liangxue Guyuan decoction(LGD)group.The survival situation of rats were observed daily.On the 3rd day after irradiation,rats were anesthetized and sacrificed.The gastric tissues were taken for HE staining and scoring.The apoptotic cells was detected by TUNEL fluorescence staining.The protein expressions of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)and Cysteinyl aspartate specific proteinase 3(Caspase-3)were detected by Western Blot.The mRNA expressions of Bax,Bcl-2 and Caspase-3 were detected by PCR.Results After irradiation,the rats in each group showed different degrees of loose stool,which was obvious in the irradiation group,while the rats in the high dose Chinese medicine group were unformed soft stool.Pathological observation showed that the cell gap was enlarged,glands were irregularly arranged,mucosal congestion and edema were obvious in the simple irradiation group.The injury degree of each treatment group was alleviated,and the injury score was higher than that of the blank group(P<0.01),while the gastric mucosa of the high dose Chinese medicine group was slightly congested and edema,and the injury score was lower than that of the simple irradiation group(P<0.05).The expression of TUNEL showed that positive cells in high dose Chinese medicine group was lower than that in simple irradiation group(P<0.05).In terms of the Bax protein expression level,the simple irradiation group was significantly higher than that of the blank group(P<0.01),and the Chinese medicine high dose group was lower than that of the simple irradiation group(P<0.05).In terms of the protein expression levels of Caspase-3 and Bax/Bcl-2,the simple irradiation group,Dexamethasone group and low dose Chinese medicine group were higher than the blank control group(P<0.05 or P<0.01);and the high dose of Chinese medicine group was lower than that of the simple irradiation group(P<0.05 or P<0.01).Compared with the blank group,the relative expression of mRNA Bax in the simple irradiation group,Dexamethasone group and the Chinese medicine low dose group increased(P<0.05 or P<0.01);the relative expression of mRNA Caspase-3 in the simple irradiation group and Dexamethasone group incresed(P<0.05 or P<0.01).The low and high dose Chinese medicine group could decrease the mRNA expression of Bax and Caspase-3,which was the most significant in the high dose Chinese medicine group(P<0.01).Conclusion Liangxue Guyuan decoction can alleviate acute gastric radiation injury induced by 60 Co-gamma rays in rats.Its mechanism may be related to inhibiting the expression of Bax and Caspase-3,decreasing Bax/Bcl-2,and inhibiting apoptosis.