Abstract：Objective: To investigate the effects of Xinjiashenyuan formula on glomerular podocyte injury and RIPK1/RIPK3/MLKL pathway in diabetic nephropathy rats induced by high glucose, and to analyze its possible mechanism. Methods: A total of 70 SD rats were selected, and 12 were randomly selected as normal control group, and the remaining 58 rats were fed with high-sugar and high-fat diet for 8 weeks. Then, the DN rat model was established by intraderitoneal injection of 35 mg/kg streptozotocin, and 48 rats were successfully established. They were randomly divided into model group, positive control group, low-dose and high-dose Xinjiashenyuan Formula groups, with 12 rats in each group. Irbesartan solution was 0. 017g/kg in positive control group, 0. 72g/kg and 1. 44g/kg in Xinjiashenyuan formula group, and an equal volume of normal saline was the gavage for normal control group and model group. Renal function: urea nitrogen, serum creatinine, proteinuria; HE staining was used to observe the pathological morphology of renal tissue. Ultrastructural changes of glomerular podocyte were observed by transmission electron microscopy. TUNEL staining was used to detect the apoptosis of renal cells. The levels of TNF-α and IL-6 in renal tissue were detected by ELISA. The expression of RIPK1/RIPK3/MLKL pathway related proteins in renal tissues was detected by WB. The active ingredients contained in Xinjiashenyuan formula were collected and screened, and molecular docking was performed on the active ingredients with high degree values obtained from topological analysis of "active ingredient target". Results: Compared with normal control group, proteinuria, serum creatinine, urea nitrogen levels, apoptosis index, TNF-α, IL-6 levels, RIPK1, RIPK3 and MLKL protein expressions in model group were increased, while WT-1 protein expression was decreased (P<0. 05). Proteinuria, serum creatinine, urea nitrogen levels, apoptosis index, TNF-α, IL-6 levels, RIPK1, RIPK3 and MLKL protein expressions in positive control group, low and high dose groups were decreased compared with normal control group, while WT-1 protein expression was increased compared with normal control group (P<0. 05). In model group, the glomerular basement membrane was thickened, mesangial cells were hyperplasia, renal tubule turbidness and renal interstitial fibrosis were observed. The pathological morphological changes of renal tissues in positive control group, low dose group and high dose group were reduced in different degrees compared with model group. In model group, podocyte processes fused or disappeared and basement membrane was thickened. The pathological changes of renal ultrastructure in positive control group, low dose group and high dose group were improved in different degrees compared with model group. There are 22 active components of Panax ginseng in Xinjiashenyuanfang and 20 active components of zhihuangqi. Molecular docking results showed that Celabenzine in Ginseng and Mairin in prepared astragalus had better docking with key targets. The binding energies of Celabenzine in Ginseng and Mairin in prepared astragalus were -5. 3, -6. 42, -5. 48 and -5. 39, -7. 71, -5. 41 with RIPK1, RIPK3 and MLKL respectively. Conclusion: Xinjiashenyuan formula regulates RIPK1/RIPK3/MLKL signaling pathway through multiple components and multiple targets, reduces the levels of inflammatory factors TNF-α and IL-6, alleviates inflammatory response, and improves glomerular podocytic injury in diabetic nephropathy rats.
[1] Tang S C W,Yiu W H.Innate immunity in diabetic kidney disease[J].Nat Rev Nephrol,2020,16(4):206-222. [2] Bertheloot D,Latz E,Franklin B S.Necroptosis,pyroptosis and apoptosis:an intricate game of cell death[J].Cell Mol Immunol,2021,18(5):1106-1121. [3] 汤利华,方超,王浩然.山奈酚对高糖诱导的糖尿病肾病大鼠肾功能和组织病理损伤的保护作用[J].免疫学杂志,2018,34(12):1041-1046. [4] 牛晓静,张利芳,赵静,等.糖肾煎对糖尿病肾病大鼠足细胞自噬及mTOR/P70S6K通路的影响研究[J].广西医科大学学报,2021,38(11):2064-2070. [5] 靳贺超,于文涛,刘晓,等.补肾活血方对血管性痴呆大鼠脑海马细胞凋亡及ERK2,CREB表达的影响[J].中国实验方剂学杂志,2018,24(12):129-135. [6] Yuyin Xu,Hongyang Gao,Yuan Hu,et al.High glucose-induced apoptosis and necroptosis in podocytes is regulated by UCHL1 via RIPK1/RIPK3 pathway[J].Experimental Cell Research,2019,382(2):111463. [7] Newton K,Dugger D L,Maltzman A,et al.RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury[J].Cell Death Differ,2016,23(9):1565-1576. [8] Murphy J M,Czabotar P E,Hildebrand J M,et al.The pseudokinase MLKL mediates necroptosis via a molecular switch mechanism[J].Immunity,2013,39(3):443-453.
2022, Vol. 28 
冀公网安备13080202000677号