Abstract:
Objective:To observe the effects of strengthening spleen and cleansing formula on adriamycin‑induced balb/c mice and mouse renal podocytes (MPC‑5) to explore the potential effect mechanism of its anti‑oxidative stress against focal segmental glomerulosclerosis (FSGS) to ameliorate podocyte injury. Methods:(1) In vivo experiments: balb/c mice were randomly divided into five groups:the blank group (CON),the model group (MOD),the low‑dose group (L: 1.13 g/kg),the medium‑dose group (Z: 2.26 g/kg) and the high‑dose group (H: 4.52 g/kg) of the strengthening spleen and clearing harmonizing formula. In the experimental group of balb/c mice, FSGS animal model was established by single tail vein injection of adriamycin (10.5 mg/kg), and the drug was administered by continuous gavage for 6 weeks after modeling. The 24‑h urine protein (quantitative), blood creatinine, and the levels of superoxide dismutase (SOD), malonaldehyde (MDA), catalase (CAT) in renal tissues were measured. H&E, Masson and PAS staining were used to observe the degree of renal histopathological damage. Transmission electron microscopy (TEM) was used to observe the morphological changes of podocytes. Immunofluorescence nephrine/p‑JAK2 double staining was used to observe the expression of p‑JAK2 on podocytes. The protein expression levels of p‑JAK2/JAK2, p‑STAT3/STAT3, and TGF‑β1 in renal tissues were detected by immunoblotting. (2) In vitro experiments: Experiment 1: The podocytes were divided into 5 groups:the control group (CON),the model group (MOD),the low‑dose group (L),the medium‑dose group (M), and the high‑dose group (H) of JianSheng QingHua Fang. Experiment 2: Foot cells were divided into the CON group (CON),the model group (ADR),the high dose group of strengthening the spleen and cleansing and harmonizing formula (ADR+H), and the C‑A1 group and the ADR+H+C‑A1 group. Experiment 1: The effects of adriamycin and spleen‑health clearing formula on cell viability were detected by CCK‑8 assay, and the intervention concentrations of modeling and spleen‑health clearing formula were screened. DCFH‑DA fluorescent probe was used to detect the content of cellular ROS in each group. Flow cytometry was used to determine the apoptosis of podocytes.Zmmunoblotting detected the protein expression levels of p‑JAK2/JAK2, p‑STAT3/STAT3, and TGF‑β1 as well as RT‑qPCR detected the downstream of STAT3 for the apoptotic factors BAX, BAD, Caspase3, p21, Blc‑2, and the inflammatory factors IL‑6, IL‑1β, and TNF‑α mRNA expression, Experiment 2: Zmmunoblotting was done to evaluate the effect of JAK2 agonist coumermycin A1 (C‑A1) on p‑JAK2/JAK2,p‑STAT3/STAT3 protein expression. Results:1. In vivo experiments: compared with the control group, glomerulosclerosis, basement membrane thickening, extensive fusion of podocytes, and p‑JAK2 fluorescence expression in renal tissues were significantly increased in the model group. 24 h urinary protein, blood creatinine, and MDA were elevated, and SOD, and CAT were decreased (P<0.05). The expression of p‑JAK2/JAK2, p‑STAT3/STAT3 and TGF‑β1 were elevated. Compared with the model group, foot cell injury was alleviated after the intervention of spleen‑enhancing and purifying formula, renal tissue p‑JAK2 fluorescence expression was reduced, 24 h urine protein, blood creatinine,and MDA were reduced as well as, SOD,and CAT were elevated. p‑JAK2/JAK2, p‑STAT3/STAT3,and TGF‑β1 expressions were reduced (P<0.05). 2. In vitro experiments: Experiment (1): CCK‑8 analysis showed that the modeling concentration of adriamycin was 0.4 μg/mL, and the intervening low, medium and high concentrations of the strengthening spleen and clearing harmonization formula were 1 mg/mL, 2 mg/mL and 4 mg/mL, respectively. compared with the control group, the apoptotic cell number and ROS content increased in the model group, and the expressions of p‑JAK2/JAK2, p‑STAT3/STAT3 and TGF‑β1 were elevated (P<0.05),mRNA expressions of BAX, BAD, Caspase3, p21, IL‑6, TNF‑α,and IL‑1β were decreased, and Blc‑2 mRNA expression was elevated (P<0.05); compared with the model group, after the intervention of the spleen‑strengthening and cleansing formula, the number of apoptotic cells,and the ROS content were decreased, JAK2/STAT3, TGF‑ β1 protein expressions were decreased,as well as mRNA expressions of BAX, BAD, Caspase3, p21, IL‑6, TNF‑α,and IL‑1β were elevated, while mRNA expression of Blc‑2 was decreased with a quantitative effect relationship (P<0.05). Experiment (2): Compared with the control group, p‑JAK2/JAK2 protein expression was elevated in the C‑A1 group (P<0.05); compared with the ADR+H group, p‑STAT3/STAT3 protein expression was elevated in the ADR+H+C‑A1 group (P<0.05). Conclusion:Strengthening spleen and clearing formula may improve podocyte injury in mice with focal segmental glomerulosclerosis by reducing oxidative stress through inhibiting the activation of TGF‑β1/JAK2/STAT3 signaling pathway.