化瘀方对脓毒症大鼠脾脏损伤的保护作用机制研究

Study on the mechanism of protective effect of Huayu Decoction on spleen injury in rats with sepsis

目的观察化瘀方对脓毒症大鼠脾脏损伤的保护作用并探讨其机制。方法将50只健康SPF级Wistar雄性大鼠随机分为空白组、模型组、化瘀方低剂量组、化瘀方中剂量组、化瘀方高剂量组,每组10只。空白组、模型组给予生理盐水灌胃,化瘀方低、中、高剂量组分别给予0.25 g/mL、0.5 g/mL、1 g/mL化瘀方灌胃,连续7 d。除空白组外,其余组于实验第8天采用脂多糖尾静脉注射制备脓毒症模型。造模6 h后取血,ELISA法检测各组大鼠血清肿瘤坏死因子-α(TNF-α)水平;取脾脏标本,HE染色观察脾脏病理学变化,流式细胞仪检测脾脏组织中CD4^(+)T、CD8^(+)T细胞比例并计算CD4^(+)T/CD8^(+)T比值,RT-PCR法检测脾脏组织中TLR4 mRNA表达量。结果模型组大鼠脾窦扩张充血,内皮细胞水肿,巨噬细胞增多,淋巴小结结构破坏,脾细胞固缩凋亡。化瘀方各组大鼠脾脏损伤情况相比模型组大鼠轻,其中高剂量组减轻尤为明显。模型组大鼠血清TNF-α水平明显高于空白组(P<0.05);化瘀方各组大鼠血清TNF-α水平均明显低于模型组,且中、高剂量组明显低于低剂量组,高剂量组明显低于中剂量组,差异均有统计学意义(P均<0.05)。与空白组比较,模型组大鼠脾脏组织中CD4^(+)T和CD4^(+)T/CD8^(+)T比值明显降低(P均<0.05),CD8^(+)T和TLR4 mRNA表达量明显增高(P均<0.05)。化瘀方各组大鼠脾脏组织中CD4^(+)T和CD4^(+)T/CD8^(+)T比值均明显高于模型组(P均<0.05),且高剂量组明显高于其他组(P均<0.05);CD8^(+)T和TLR4 mRNA表达量均明显低于模型组(P均<0.05),且高剂量组明显低于其他组(P均<0.05)。结论化瘀方可以减轻脓毒症大鼠脾脏损伤,其机制可能与提高机体免疫力,调控TLR4信号通路,抑制炎症因子的产生有关。

Objective It is to observe the protective effect of Huayu Decoction on spleen injury in rats with sepsis and explore its mechanism.Methods Fifty healthy SPF male Wistar rats were randomly divided into a blank group,a model group,a low-dose Huayu Decoction group,a medium-dose Huayu Decoction group,and a high-dose Huayu Decoction group,with 10 rats in each group.The blank group and the model group were given normal saline by gavage,and the low,medium,and high-dose Huayu Decoction groups were respectively given 0.25 g/mL,0.5 g/mL,1 g/mL Huayu Decoction,all treated for 7 days.Except for the blank group,the other groups were given lipopolysaccharide by tail vein injection to prepare sepsis models on the 8th day of the experiment.The blood was collected in 6 hours after modeling,and the levels of serum tumor necrosis factor-α(TNF-α)were detected by ELISA method;the spleen samples were taken to observe the pathological changes of spleen by HE staining,the proportion of CD4^(+)T and CD8^(+)T cells in spleen tissue was detected by flow cytometry and the ratio of CD4^(+)T/CD8^(+)T was calculated,the expression of TLR4 mRNA in spleen tissue was detected by RT-PCR method.Results In the model group,the splenic sinus of the rats was dilated and congested,endothelial cells were edema,macrophages increased,the structure of lymph nodes was destroyed,and the spleen cells were pyknotic and apoptotic.The damage of spleen of rats in each group of Huayu Decoction was less than that of model group,and the relief was more obvious in the high-dose group.The serum level of TNF-αin the rats of the model group was significantly higher than that of the blank group(P<0.05);the serum levels of TNF-αin the rats of the Huayu Decoction groups were significantly lower than that in the model group,and the medium and high-dose groups were significantly lower than the low-dose group,the high-dose group was significantly lower than the medium-dose group,and the differences were statistically significant(all P<0.05).Compared with the blank group,the proportion of CD4^(+)T and CD4^(+)T/CD8^(+)T in the spleen tissue of the model group was significantly reduced(all P<0.05),and the expression of CD8^(+)T and TLR4 mRNA was significantly increased(all P<0.05).The levels of CD4^(+)T and CD4^(+)T/CD8^(+)T in the spleen tissue of rats in each group of Huayu Decoction were significantly higher than those in the model group(all P<0.05),and the high-dose group was significantly higher than the other groups(all P<0.05);the levels of CD8^(+)T and TLR4 mRNA expression in the spleen tissue of rats in each group of Huayu Decoction were significantly lower than the model group(all P<0.05),and the high-dose group was significantly lower than the other groups(all P<0.05).Conclusion Huayu Decoction can relieve the spleen injury of sepsis rats.The mechanism may be related to improving the body’s immunity,regulating the TLR4 signaling pathway,and inhibiting the production of inflammatory factors.