Study on the effect mechanism of spleen-strengthening and purifying formula in improving focal segmental glomerulosclerosis
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 ex-perimental 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 cre-atinine,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 micros-copy(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 for-mula 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 cytome-try 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:Zmmu-noblotting was done to evaluate the effect of JAK2 agonist coumermycin A1(C-A1)on p-JAK2/JAK2,p-STAT3/STAT3 pro-tein expression.Results:1.In vivo experiments:compared with the control group,glomerulosclerosis,basement membrane thick-ening,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 ex-pression 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 re-duced,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 de-creased,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 relation-ship(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.
Focal segmental glomerulosclerosisPodocyte injurySpleen-enhancing and cleansing formulaJAK2/STAT3
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维普
