Yishen Huashi Granules Inhibit Diabetic Nephropathy Through Ameliorating Podocyte Injury

Objective:Diabetic nephropathy(DN),the most common and severe complication of diabetes,has been identified as the main pathogenic factor in the development of chronic kidney disease and terminal renal failure.About 30-40% of diabetic patients in the world gradually progress to end-stage renal disease(ESRD).From the perspective of pathophysiology,both macrophage-mediated inflammation and inflammation-induced podocyte apoptosis play a vital role in the evolution of DN.To observe the therapeutic effect of Yishen Huashi Granules(YSHS)in podocyte damage,diabetic nephropathy(DN)proteinuria and to explore the corresponding mechanism.More and more experimental data show that traditional Chinese medicine is beneficial to the treatment of diabetic nephropathy because of its various effects and overall regulatory effects.Studies have found that Yishen Huashi Granules can inhibit proteinuria,podocyte damage and inflammatory factor secretion in diabetic nephropathy,and delay the progression of diabetic kidneys.However,the corresponding mechanism of Yishen Huashi Granules has not been reported yet.Methods:(1)The db/db mice were used to establish the diabetic neuropathy model.Serum creatinine,urea nitrogen and 24 h urinary proteinuria were detected with specific kits.Glomerular structural lesions and glomerular cell apoptosis were detected through HE staining and TUNEL assay.(2)The medicated Serum of Yishen Huashi Granules(YSHS-Serum)or control serum was prepared from SD rats.(3)Macrophage-derived exosomes were extracted by exosome extraction kit.(4)Morphology and the protein concentration of exosomes were evaluated by transmission electron microscope and BCA kit.(5)The activity and apoptosis of podocyte MPC5 cells,the M1 macrophage polarization and the protein expression of exosome marker were detected by CCK-8experiment,flow cytometry and Western blot respectively.(6)The miR-21a-5p expression in podocytes and the exosomes from macropahge were measured by q RT-PCR.(7)The effect of YSHS on the infiltration of M1 macrophages in the kidney tissue in db/db mice were measured by immunofluorescence.Results:1.YSHS could improve renal function,reduce proteinuria and inhibit glomerular structural lesions and glomerular cell apoptosis in db/db mice.2.In addition,YSHS-Serum could inhibit high glucose induced M1 polarization of macrophages and M1 macrophage infiltration in renal tissue.3.M1 polarization of macrophages could change the content of miR-21a-5p in macrophage-derived exosomes.4.miR-21a-5p mimics could reduce podocyte activity and promote caspase 3 shearing.5.Macrophage-derived exosomes could inhibit the cell viability and increase apoptosis of podocytes,especially the exosomes from macrophages treated with high glucose and control serum.6.Compared with the exosomes secreted by macrophages after high glucose treatment,the exosome from macrophages treated with high glucose and YSHS-Serum showed lower inhibitory effects on podocyte activity,accompanied by the decreased up-regulating effects of macrophage-derived exosomes on the miR-21a-5p in podocytes.7.High glucose stimulation and YSHS-Serum treatment did not affect the protein concentration in macrophage-derived exosomes,But it can change the miRNA content in macrophage exosomes.Conclusions:1.This regulation might be related to the inhibition of M1 macrophage polarization by YSHS and the reduction of the miR-21a-5p content in macrophage-derived exosomes.2.This study suggests that YSHS could improve the podocyte injury induced by macrophage-derived exosomes and alleviate the progression of diabetic nephropathy.