Objective To explore the mechanism of Tongluo Baoshen Decoction in improving podocyte injury in rats with IgA nephropathy based on Gprc5b/NF-κB/NLRP3 pathway.Methods Totally 130 SPF-grade male SD rats were randomly divided into a normal group(n=20)and a modeling group(n=110).The IgA nephropathy model was established using a compound modeling method,and 100 modeling rats were randomly divided into model group,losartan potassium group(5 mg/kg),and Tongluo Baoshen Decoction low-,medium-,and high-dosage groups(5.3,10.6,21.2 g/kg),with 20 rats in each group.The administration group was given the corresponding dosage of medication by gavage,while the normal group and model group were given an equal amount of distilled water by gavage once a day.After 4 and 8 weeks of administration,urine samples were collected for 24 hours,and blood and kidney tissue specimens were collected.24-hour urinary protein quantification(24 h-UTP),urinary Nephrin,serum creatinine(SCr),blood urea nitrogen(BUN)and blood uric acid(BUA)contents were detected;RT-qPCR and Western blot were used to detect the expressions of G protein coupled receptor C-family 5b(Gprc5b),nuclear factor(NF)-κB p50,NOD like receptor protein 3(NLRP3),Caspase-1,interleukin(IL)-1β,Nephrin mRNA and protein in renal tissue,respectively;HE,PAS,PASM,Masson staining were used to observe the morphology of renal tissue,immunofluorescence was used to observe IgA deposition in the mesangial area of renal tissue,and transmission electron microscopy was used to observe the ultrastructure of podocytes.Results Compared with the normal group,the model group rats showed significantly increased contents of 24 h-UTP,urinary Nephrin and BUA(P<0.01),the mRNA and protein expressions of Gprc5b,NF-κB p50,NLRP3,Caspase-1 and IL-1β in renal tissue were significantly increased(P<0.01),while the mRNA and protein expressions of Nephrin were significantly decreased(P<0.01),with mild to moderate proliferation of mesangial cells in the glomerulus,increased mesangial matrix,and immunofluorescence showed clustered and linear deposition of IgA in the mesangial area,electron microscopy showed partial fusion of the foot processes.Compared with the model group,the 24 h-UTP and urinary Nephrin contents in different dosage groups of Tongluo Baoshen Decoction and the losartan potassium group after 4 and 8 weeks administration significantly decreased(P<0.01),with a decrease in BUA content in Tongluo Baoshen Decoction high-dosage group(P<0.05),the mRNA and protein expressions of Gprc5b,NF-κB p50,NLRP3,Caspase-1 and IL-1β in renal tissue of Tongluo Baoshen Decoction groups and losartan potassium group decreased(P<0.05,P<0.01),while the mRNA and protein expressions of Nephrin increased(P<0.05,P<0.01),with the proliferation of mesangial cells,the increase of mesangial matrix,the deposition of IgA in the mesangial area,and the fusion of foot processes in renal tissue were alleviated to varying degrees in different dosage groups of Tongluo Baoshen Decoction,with the most significant improvement observed in the high-dosage group.Compared with the 4-week administration,Tongluo Baoshen Decoction high-dose group showed further reductions in 24 h-UTP and urinary Nephrin contents after 8 weeks of administration(P<0.01),further decreases in the mRNA and protein expressions of Gprc5b,NF-κB p50,NLRP3,Caspase-1 and IL-1β in renal tissue(P<0.05,P<0.01),and further increases in the mRNA and protein expressions of Nephrin(P<0.01).Conclusion Tongluo Baoshen Decoction can reduce proteinuria,alleviate renal tissue lesions and improve podocyte injury in IgA nephropathy rats,and its mechanism may be related to the inhibition of Gprc5b/NF-κB/NLRP3 pathway in renal tissue.

Objective To explore the mechanism of Tongluo Baoshen Decoction in improving podocyte injury in rats with IgA nephropathy based on Gprc5b/NF-κB/NLRP3 pathway.Methods Totally 130 SPF-grade male SD rats were randomly divided into a normal group(n=20)and a modeling group(n=110).The IgA nephropathy model was established using a compound modeling method,and 100 modeling rats were randomly divided into model group,losartan potassium group(5 mg/kg),and Tongluo Baoshen Decoction low-,medium-,and high-dosage groups(5.3,10.6,21.2 g/kg),with 20 rats in each group.The administration group was given the corresponding dosage of medication by gavage,while the normal group and model group were given an equal amount of distilled water by gavage once a day.After 4 and 8 weeks of administration,urine samples were collected for 24 hours,and blood and kidney tissue specimens were collected.24-hour urinary protein quantification(24 h-UTP),urinary Nephrin,serum creatinine(SCr),blood urea nitrogen(BUN)and blood uric acid(BUA)contents were detected;RT-qPCR and Western blot were used to detect the expressions of G protein coupled receptor C-family 5b(Gprc5b),nuclear factor(NF)-κB p50,NOD like receptor protein 3(NLRP3),Caspase-1,interleukin(IL)-1β,Nephrin mRNA and protein in renal tissue,respectively;HE,PAS,PASM,Masson staining were used to observe the morphology of renal tissue,immunofluorescence was used to observe IgA deposition in the mesangial area of renal tissue,and transmission electron microscopy was used to observe the ultrastructure of podocytes.Results Compared with the normal group,the model group rats showed significantly increased contents of 24 h-UTP,urinary Nephrin and BUA(P<0.01),the mRNA and protein expressions of Gprc5b,NF-κB p50,NLRP3,Caspase-1 and IL-1β in renal tissue were significantly increased(P<0.01),while the mRNA and protein expressions of Nephrin were significantly decreased(P<0.01),with mild to moderate proliferation of mesangial cells in the glomerulus,increased mesangial matrix,and immunofluorescence showed clustered and linear deposition of IgA in the mesangial area,electron microscopy showed partial fusion of the foot processes.Compared with the model group,the 24 h-UTP and urinary Nephrin contents in different dosage groups of Tongluo Baoshen Decoction and the losartan potassium group after 4 and 8 weeks administration significantly decreased(P<0.01),with a decrease in BUA content in Tongluo Baoshen Decoction high-dosage group(P<0.05),the mRNA and protein expressions of Gprc5b,NF-κB p50,NLRP3,Caspase-1 and IL-1β in renal tissue of Tongluo Baoshen Decoction groups and losartan potassium group decreased(P<0.05,P<0.01),while the mRNA and protein expressions of Nephrin increased(P<0.05,P<0.01),with the proliferation of mesangial cells,the increase of mesangial matrix,the deposition of IgA in the mesangial area,and the fusion of foot processes in renal tissue were alleviated to varying degrees in different dosage groups of Tongluo Baoshen Decoction,with the most significant improvement observed in the high-dosage group.Compared with the 4-week administration,Tongluo Baoshen Decoction high-dose group showed further reductions in 24 h-UTP and urinary Nephrin contents after 8 weeks of administration(P<0.01),further decreases in the mRNA and protein expressions of Gprc5b,NF-κB p50,NLRP3,Caspase-1 and IL-1β in renal tissue(P<0.05,P<0.01),and further increases in the mRNA and protein expressions of Nephrin(P<0.01).Conclusion Tongluo Baoshen Decoction can reduce proteinuria,alleviate renal tissue lesions and improve podocyte injury in IgA nephropathy rats,and its mechanism may be related to the inhibition of Gprc5b/NF-κB/NLRP3 pathway in renal tissue.

IgA nephropathy is the most common primary glomerular disease in China. Its clinical manifestations are mainly proteinuria， hematuria， hypertension， edema， hyperuricemia， etc. Most patients have hidden onset. 30%-40% of patients develop into end stage renal disease 10-20 years after diagnosis and rely on dialysis or kidney transplantation to maintain their lives， which is extremely harmful. Proteinuria is a common clinical manifestation of this disease， and most patients have small-to-moderate amounts of proteinuria， while 10%-24% of patients have large amounts of proteinuria. Proteinuria is the main risk factor affecting the progression of renal function in IgA nephropathy. Podocytes are the terminal part of the glomerular filtration barrier， and various factors can affect the fusion and detachment of podocyte processes that occur after podocyte injury. They are common histological lesions in IgA nephropathy and are key factors leading to proteinuria and the continuous progression of the disease. At present， Western medicine lacks targeted treatment for podocyte injury， with limited intervention methods. Drugs such as glucocorticoids are often used for treatment， and there are many adverse reactions. Based on the physiological function of podocytes， pathological and physiological changes after injury， and histological morphology of this disease， it is believed that it is closely related to traditional Chinese medicine's "Xuanfu Theory" "Kidney Collateral Syndrome" "Collateral Disease Theory"， and "Dry Blood Theory". More and more studies have shown that traditional Chinese medicine， which has the characteristics of multiple links， pathways， and targets， has a significant therapeutic effect on podocyte injury in IgA nephropathy. It can protect podocytes and reduce proteinuria and has good application and research prospects. This article systematically summarizes the mechanism and morphological changes of podocyte injury in IgA nephropathy， the understanding of podocyte injury in traditional Chinese medicine theory， and the research progress in traditional Chinese medicine treatment of podocyte injury in IgA nephropathy， so as to provide a reference for further research and application of traditional Chinese medicine intervention in podocyte injury in IgA nephropathy.

AIM:To explore the role and molecular mechanism of Men1 gene in regulating mouse renal fibro-sis.METHODS:A unilateral ureteral obstruction(UUO)-induced renal fibrosis model was established using C57BL/6 mice,and the mice were randomly divided into 4 groups:sham,UUO-3 d,UUO-7 d,and UUO-14 d,with 15 mice in each group.The C57BL/6 mice with Men1 knockout were randomly divided into 4 groups:sham-Men1-WT,sham-Men1-CKO,UUO-Men1-WT,and UUO-Men1-CKO,with 8 mice in each group.HE staining,Masson staining,and Sirius red staining were used to detect UUO-induced renal injury and renal fibrosis.Human renal tubular epithelial HK-2 cells with MEN1 knockout were constructed.RT-qPCR,Western blot,immunohistochemistry and immunoflurorescnence were per-formed to detect the mRNA and protein expression of MEN1,fibrosis markers(α-smooth muscle actin,collagen type Ⅲ and fibronectin 1)and m6A-related proteins[methyltransferase-like 3(METTL3),METTL14,YTH domain family pro-tein 2(YTHDF2),AlkB homolog 5(ALKBH5),and fat mass and obesity-associated protein(FTO)]in UUO mouse kid-ney tissues and transforming growth factor-β(TGF-β;10 μg/L)-treated HK-2 cells.Dot blot analysis was conducted to measure m6A methylation levels in both mouse kidney tissuess and HK-2 cells.RESULTS:The expression of Men1 de-creased with the aggravation of renal fibrosis(P＜0.01).Men1 inhibited the expression of fibrosis markers in renal tis-sues,and MEN1 knockout increased the accumulation of collagen induced by UUO and TGF-β(P＜0.01).The expres-sion of FTO and ALKBH5 in mouse kidney tissues and HK-2 cells was down-regulated by MEN1 knockout(P＜0.01),and the methylation level of m6A was increased(P＜0.01).Overexpression of FTO significantly reduced the accumulation of m6A modifications and renal fibrosis caused by MEN1 loss,and the methylation level of m6A was increased(P＜0.01).CONCLUSION:Loss of Men1 gene promotes renal fibrosis in mice,and Men1 suppresses renal fibrosis in mice by pro-moting the expression of FTO/ALKBH5 to reduce m6A modifications.

AIM:To explore the role and molecular mechanism of Men1 gene in regulating mouse renal fibro-sis.METHODS:A unilateral ureteral obstruction(UUO)-induced renal fibrosis model was established using C57BL/6 mice,and the mice were randomly divided into 4 groups:sham,UUO-3 d,UUO-7 d,and UUO-14 d,with 15 mice in each group.The C57BL/6 mice with Men1 knockout were randomly divided into 4 groups:sham-Men1-WT,sham-Men1-CKO,UUO-Men1-WT,and UUO-Men1-CKO,with 8 mice in each group.HE staining,Masson staining,and Sirius red staining were used to detect UUO-induced renal injury and renal fibrosis.Human renal tubular epithelial HK-2 cells with MEN1 knockout were constructed.RT-qPCR,Western blot,immunohistochemistry and immunoflurorescnence were per-formed to detect the mRNA and protein expression of MEN1,fibrosis markers(α-smooth muscle actin,collagen type Ⅲ and fibronectin 1)and m6A-related proteins[methyltransferase-like 3(METTL3),METTL14,YTH domain family pro-tein 2(YTHDF2),AlkB homolog 5(ALKBH5),and fat mass and obesity-associated protein(FTO)]in UUO mouse kid-ney tissues and transforming growth factor-β(TGF-β;10 μg/L)-treated HK-2 cells.Dot blot analysis was conducted to measure m6A methylation levels in both mouse kidney tissuess and HK-2 cells.RESULTS:The expression of Men1 de-creased with the aggravation of renal fibrosis(P＜0.01).Men1 inhibited the expression of fibrosis markers in renal tis-sues,and MEN1 knockout increased the accumulation of collagen induced by UUO and TGF-β(P＜0.01).The expres-sion of FTO and ALKBH5 in mouse kidney tissues and HK-2 cells was down-regulated by MEN1 knockout(P＜0.01),and the methylation level of m6A was increased(P＜0.01).Overexpression of FTO significantly reduced the accumulation of m6A modifications and renal fibrosis caused by MEN1 loss,and the methylation level of m6A was increased(P＜0.01).CONCLUSION:Loss of Men1 gene promotes renal fibrosis in mice,and Men1 suppresses renal fibrosis in mice by pro-moting the expression of FTO/ALKBH5 to reduce m6A modifications.