龙胆苦苷腹腔注射对单侧输尿管梗阻小鼠肾间质纤维化的改善作用及其机制

Effect and mechanism of intraperitoneal injection of gentiopicroside on renal interstitial fibrosis in UUO mice

目的 探讨龙胆苦苷(GPS)对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的改善作用及其机制。方法将25只C57BL/6雄性小鼠随机分为假手术组、模型对照组、GPS低剂量组、GPS高剂量组、厄贝沙坦组,每组各5只。假手术组正常饲养,其余四组采用结扎单侧输尿管的方式建立UUO模型。造模成功后,GPS低、高剂量组分别给予0.1、0.2 g/kg GPS腹腔注射,厄贝沙坦组给予0.02 g/kg厄贝沙坦腹腔注射,模型组和假手术组给予等体积生理盐水腹腔注射,连续7 d。处死小鼠,取肾脏组织,采用HE染色法观察肾组织形态学变化,Masson染色法观察肾组织纤维化情况,采用Western blotting法、RT-qPCR法检测肾组织纤连蛋白(Fn)、α平滑肌肌动蛋白(α-SMA)蛋白及mRNA表达。另取6只小鼠,随机分为模型组和干预组各3只,建立UUO模型后,干预组给予0.2 g/kg GPS腹腔注射,模型组给予等体积生理盐水腹腔注射,连续7 d。处死小鼠,取肾脏组织进行转录组测序,分析两组差异表达基因,并进行GO和KEGG通路富集,最后采用RT-qPCR法进行验证。结果 与假手术组比较,模型对照组肾小管明显扩张,肾组织大量炎性细胞浸润,肾间质大量胶原纤维沉积;与模型对照组比较,GPS低、高剂量组肾脏组织肾小管扩张、炎性细胞浸润有一定程度减轻,肾间质纤维化改善,其中GPS高剂量组改善效果更明显。与假手术组比较,模型对照组肾脏组织Fn、α-SMA蛋白及mRNA表达水平均升高;与模型对照组比较,GPS低、高剂量组肾脏组织Fn、α-SMA蛋白及mRNA表达水平均降低,且GPS高剂量组低于低剂量组(P均<0.05)。测序分析结果显示,模型组与干预组存在710个差异表达基因。GO富集分析显示,差异表达基因主要集中在细胞进程、生物调节、生物进程调控等方面;KEGG通路富集分析发现,差异表达基因涉及Ras、Rap1、PI3K/AKT、MAPK信号通路等。筛选出2个有效基因HGF、CCDC3进行验证,干预组肾组织HGF、CCDC3表达水平较模型组升高(P均<0.05),与测序结果一致。结论 GPS能够有效改善UUO小鼠肾脏纤维化,且高剂量GPS改善作用更明显;其作用机制可能与上调HGF及CCDC3表达作用于Rap1、Ras、PI3K/AKT、MAPK信号通路等生物学途径有关。

Objective To investigate the effect and mechanism of gentiopicroside(GPS)on renal interstitial fibrosis in unilateral ureteral obstruction(UUO)mice.Methods Twenty-five C57BL/6 male mice were randomly divided into the sham operation group,model control group,low-dose GPS group,high-dose GPS group,and irbesartan group,with 5 mice in each group.Mice in the sham operation group were fed normally,while in the other four groups,we established the UUO models by ligating one side of the ureter in mouse.After successful modeling,mice in the low-dose and high-dose GPS groups were given 0.1 and 0.2 g/kg GPS through intraperitoneal injection,mice in the irbesartan group were given 0.02 g/kg irbesartan intraperitoneally,and mice in the model group and sham operation group were given equal volume of normal saline through intraperitoneal injection for 7 consecutive days.The mice were executed to take the kidney tissues to observe the morphological changes of kidney tissues using HE staining,and to observe the fibrosis of kidney tissues using Masson staining.Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of fibronectin(Fn)andα-smooth muscle actin(α-SMA)in the renal tissues.Another six mice were randomly divided into the model group and intervention group,with 3 mice in each.After the UUO models were established,the intervention group was giv-en 0.2 g/kg GPS through intraperitoneal injection,while the model group was given equal volume of normal saline intraper-itoneally for 7 consecutive days.Mice were euthanized,the kidney tissues were taken for transcriptome sequencing,two sets of differentially expressed genes were analyzed,and GO and KEGG pathways enrichment were conducted.Finally,RT-qPCR was used for validation.Results Compared with the sham operation group,the model control group showed significant dilation of renal tubules,a large number of inflammatory cells in the renal tissues,and deposition of a large amount of collagen fibers in the renal interstitium.Compared with the model control group,the low-dose and high-dose GPS groups showed a certain degree of reduction in the renal tubular dilation and inflammatory cell infiltration in the kid-ney tissues,and improved renal interstitial fibrosis,with more significant effect in the high-dose GPS group.Compared with the sham operation group,the Fn,α-SMA protein and mRNA expression levels increased in the renal tissues of the model control group.Compared with the model control group,the Fn,α-SMA protein and mRNA expression levels de-creased in the renal tissues of the low-dose and high-dose GPS groups(all P<0.05),and those in the high-dose group were lower than those of the low-dose group.The sequencing analysis results showed that there were 710 differentially expressed genes between the model group and the intervention group.GO enrichment analysis showed that differentially expressed genes were mainly concentrated in cell processes,biological regulation,and biological process regulation;KEGG pathway enrichment analysis revealed that differentially expressed genes involved Ras,Rap1,PI3K/AKT,MAPK signaling path-ways,etc.Two effective genes HGF and CCDC3 were screened for validation.Compared with the model group,the expres-sion of HGF and CCDC3 in the intervention group increased(both P<0.05),which was consistent with the sequencing re-sults.Conclusion GPS can effectively alleviate renal fibrosis in UUO mice,and its mechanism of action may be related to up-regulating the expression of HGF and CCDC3,Rap1,Ras,PI3K/AKT,MAPK signaling pathways,and other bio-logical pathways.