机构地区:[1]西南医科大学附属中医医院中西医结合研究中心,四川泸州646000 [2]北京中医医院顺义医院肾病科,北京101320 [3]西南医科大学附属中医医院泌尿外科,四川泸州646000 [4]北京中日友好医院临床医学研究所免疫介导炎症疾病北京重点实验室,北京100029
出 处:《中药新药与临床药理》2023年第12期1658-1667,共10页Traditional Chinese Drug Research and Clinical Pharmacology
基 金:国家自然科学基金项目(82274489,81904174);四川省自然科学基金项目(2022YFS0621);西南医科大学附属中医医院学科创新团队项目(2022-CXTD-03)。
摘 要:目的研究柴黄益肾颗粒对小鼠慢性肾病模型肾脏纤维化的影响及潜在作用机制。方法将36只C57BL/6雄性小鼠随机均分为假手术组、模型组、柴黄益肾颗粒低剂量(3.835 g·kg^(-1))组、柴黄益肾颗粒高剂量(7.67 g·kg^(-1))组、阳性对照厄贝沙坦组和柴黄益肾颗粒高剂量+Mincle激动剂海藻糖-6,6-二苯甲酸酯(trehalose-6,6-dibehenate,TDB)组(术后1 h,一次性腹腔注射TDB 10 mg·kg^(-1))。采用单侧输尿管结扎法(UUO)建立小鼠肾脏纤维化模型。术后1 h,各药物组给小鼠灌胃相应药物,假手术组和模型组灌胃生理盐水,每日1次,连续7 d。通过苏木精-伊红染色和天狼星红染色观察各组小鼠肾脏病理变化和纤维化改变,采用Real-time PCR和ELISA法检测各组肾脏中炎症因子白细胞介素(IL)-1β、IL-6和肿瘤坏死因子(TNF)-α的表达和分泌,免疫组化和Western Blot法检测纤维化指标α-SMA和Fn的蛋白水平,免疫荧光和Western Blot法检测Mincle及其下游信号Syk/NF-κB的蛋白水平,流式细胞术分析Mincle阳性的巨噬细胞比例。结果与模型组比,柴黄益肾颗粒低、高剂量组均能有效改善UUO诱导的肾脏损伤和纤维化,抑制肾脏α-SMA和Fn的蛋白水平(P<0.01)。同时,柴黄益肾颗粒高剂量组可有效降低UUO模型肾脏炎症因子IL-1β、IL-6和TNF-α的表达和分泌(P<0.01),抑制NF-κB的磷酸化(P<0.01)。机制研究表明,柴黄益肾颗粒高剂量组可明显下调UUO小鼠肾脏中巨噬细胞Mincle的蛋白水平以及下游信号Syk的活性(P<0.01)。采用Mincle激动剂TDB激活Mincle后,柴黄益肾颗粒高剂量组改善的肾脏损伤、炎症因子分泌和纤维化效果明显减弱(P<0.05,P<0.01)。结论柴黄益肾颗粒可改善UUO诱导的小鼠肾损伤、炎症和纤维化,其作用机制可能与下调巨噬细胞Mincle/Syk/NF-κB信号有关。Objective To study the effect and potential mechanism of Chaihuang Yishen Granules on renal fibrosis in mice with chronic kidney disease.Methods Thirty-six C57BL/6 male mice were randomly divided into sham group,model group,low-,and high-dose Chaihuang Yishen Granules groups(3.835 g·kg^(-1)and 7.67 g·kg^(-1)),positive control group and Mincle ligand trehalose-6,6-dibehenate(TDB 10 mg·kg^(-1))group.Unilateral ureteral obstruction(UUO)was used to establish the model of renal fibrosis in mice.One hour after operation,mice in each drugtreated group were given corresponding drugs by gavage,and sham operation group and model group were given saline by gavage,once a day for 7 consecutive days.The pathological changes and fibrosis in the kidneys of mice in all groups were observed by hematoxylin eosin staining and Sirius red staining.The expressions and secretions of inflammatory factor IL-1β,IL-6 and TNF-α in kidneys of all groups were detected by Real-time PCR and ELISA.Immunohistochemistry and Western Blot were used to detect fibrosis indicators,includingα-SMA and Fn protein levels.Immunofluorescence and Western Blot were further used to determine protein levels of Mincle and its downstream signal Syk/NF-κB.The proportion of Mincle positive macrophages was analyzed by flow cytometry.Results Compared with the model group,low-and high-dose Chaihuang Yishen Granules can effectively improve UUO-induced renal injury and fibrosis,and inhibitα-SMA and Fn protein levels in the kidney(P<0.01).Moreover,high-dose Chaihuang Yishen Granules can effectively reduce the expressions and secretions of inflammatory factor IL-1β,IL-6 and TNF-α of kidney in UUO model(P<0.01),as well as inhibit phosphorylation of NF-κB(P<0.01).The mechanism study showed that Chaihuang Yishen Granules could significantly reduce the protein level of Mincle and the activity of its downstream signal Syk in the kidney of UUO mice(P<0.01).After TDB was used to activate Mincle,the effect of Chaihuang Yishen Granules on improving renal injury,inflammato
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