| 1 Objective:Research data from the November 2022 Epidemiological Survey shows that the number of people with diabetes in China exceeds 140 million,of which 95% are type 2diabetes mellitus(T2DM)patients,ranking first in the world.Diabetic nephropathy(DN)is one of the most serious complications of T2 DM and is a major cause of the development of chronic kidney disease.The pathological changes of glomeruli in the early stage of DN are dominated by glomerular basement membrane thickening,compensatory glomerular hypertrophy,and expansion of the thylakoid matrix.As the disease progresses,pathological changes such as segmental glomerulosclerosis and decreased glomerular filtration rate appear,and proteinuria can be detected clinically at this time.Podocytes ensure a stable filtration function,and damage to the podocyte is directly related to the occurrence of proteinuria.Inonotus obliquus(chaga)is a medicinal fungus parasitic on birch trees,with various pharmacological effects such as anti-inflammatory,antioxidant,anti-tumor,hypoglycemic and immune-increasing effects.Our preliminary work showed that chaga had pharmacological effects on reducing blood glucose and lipids,and reducing proteinuria in T2 DM rats.We speculate that the improvement of the above indices,especially proteinuria,by chaga is related to the protection of podocyte injury caused by T2 DM.Cyclic guanosine monophosphate(c GMP)is an important second messenger in renal tissue and is involved in regulating various physiological processes in the kidney.Studies have shown that renal injury is directly related to reduced c GMP levels and that increasing c GMP levels can effectively alleviate renal injury.c GMP levels are closely related to nitric oxide synthase(NOS)activity and phosphodiesterase(PDE)activity,with PDE5 inhibitors such as pentoxifylline,which have been used to treat DN patients with reliable clinical efficacy.Based on the previous work that chaga can reduce blood glucose,lipids and proteinuria in T2 DM rats,this study will further investigate the mechanism of action of chaga via NOS-c GMP-PDE5 signaling pathway to improve podocyte injury and reduce proteinuria in T2 DM rats.Among them,the study of three subtypes of NOS,namely endothelial(e NOS),inducible(i NOS),and neural(n NOS),will further our understanding of the characteristics of different subtypes that may exert different effects on c GMP levels.2 Methods:2.1 Establishment of T2 DM model rats and experimental grouping Five-week-old SD male rats were used as experimental subjects.Rats fed normal food were the normal control(NOR)group;the model group was fed a high-fat diet(HFD)for 8weeks followed by a single intraperitoneal injection of streptozotocin 35 mg/kg.Within one week of STZ injection,three consecutive measurements of random blood glucose(RBG)with levels greater than 16.7 mmol/L were determined as successful T2 DM modeling,and the model group continued to feed HFD.The pharmacological intervention group was treated with 50 and 100 mg/kg(DN+Ⅰ and DN+Ⅱ)of chaga on top of HFD for 8weeks,for a total of 17 weeks of the experiment.2.2 Effect of chaga on disorders of Glucolipid metabolism in T2 DM rats At the end of animal feeding,biochemical parameters were measured in all rats,including body weight(BW),kidneys weight(KW),random blood glucose(RBG),total cholesterol(TC),triglyceride(TG),fasting insulin(FINS),and fasting blood glucose(FBG).2.3 Improvement of renal function in T2 DM rats by chaga(1)Blood creatinine(SCr),urine creatinine(UCr),24 h urinary protein(24h-UP),and urea nitrogen(BUN)were measured by biochemical analyzer and kits.(2)The kidney tissues were sectioned and stained with Periodic Acid-Schiff stain(PAS),and the morphological changes of glomeruli and thylakoids in the kidney tissues of each group of rats were observed under light microscope,and the glomerular diameter was measured using K-viewer digital slice reading software,and the area of glomerular thylakoid area and capillary clusters were measured by Image-Pro plus image analysis software,and the relative area of thylakoid matrix was calculated according to the formula.2.4 Amelioration of podocyte injury in T2 DM rats by chaga(1)Electron microscopic sections were produced by ultrathin sectioning machine,and the morphology of podocytes was further observed by electron microscopy.The average thickness of glomerular basement membrane(GBM)and the length of GBM(μm)and the number of fissure pores on the basement membrane were measured in each group of rats by using RADIUS image analysis software,according to the formula The foot process width(FPW)was calculated according to the formula.(2)The m RNA and protein expression of the podocyte markers Nephrin and WT-1 were observed by Reverse Transcription-Polymerase Chain Reaction(RT-PCR)and Western blot(WB),respectively,to determine the damage of podocytes.2.5 Study on the mechanism of chaga to regulate the disorder of glucolipid metabolism and renal podocyte injury in T2 DM rats(1)The levels of serum c GMP were measured by Elisa kits at weeks 8,13 and 17 in each group of rats.(2)The levels of c GMP in the kidney of each group of rats at week 17 were measured by Elisa kit.(3)Three isoforms of nitric oxide synthase: endothelial(e NOS),inducible(i NOS),and neural(n NOS),and the expression levels of PDE5 m RNA and protein in the kidney of each group of rats were detected by RT-PCR and WB method.3 Results:3.1 Chaga ameliorates disorders of glucolipid metabolism in T2 DM rats(1)At weeks 8,13 and 17,the BW in the DN group was significantly higher than that in the NOR group(P < 0.01).After 8 weeks of chaga intervention,BW was significantly less in the DN+I and DN+II groups than in the DN group(P < 0.05).KW and renal index(RI)were significantly higher in the DN group than in the NOR group(P < 0.01).Compared with the DN group,KW and RI were significantly reduced in the DN+I and DN+II groups(P < 0.001).(2)FBG was significantly higher in the DN group at week 17 compared to the NOR group(P < 0.001).FBG levels in the DN+I and DN+II groups at week 17 were significantly lower compared to the DN group(P < 0.001).TC and TG levels in the DN group were significantly higher than those in the NOR group at weeks 8,13,and 17,respectively(P < 0.05).Both TG and TC levels were significantly increased in the DN group at week 13 compared to week 8(P < 0.01)and further significantly increased from week 13 to week 17(P < 0.01).Compared to the DN group,the DN+I group had lower TC and TG levels at weeks 13 and 17,but significant differences could be seen only in TG levels(P < 0.05),and TC and TG levels were significantly lower in the DN+II group at weeks 13 and 17(P < 0.05).(3)At week 8,FINS and IRI in the DN,DN+I and DN+II groups,were significantly higher than those in the NOR group(P < 0.05).At week 17,FINS was significantly lower in the DN group compared to the NOR group(P = 0.002).FINS levels at week 17 were significantly increased only in the DN+II group compared to the DN group(P = 0.021).At week 17,IRI was significantly higher in the DN group than in the DN group at week 8 and the NOR group at week 17(P < 0.001).IRI was significantly lower in both the DN+I and DN+II groups than in the DN group(P < 0.01).3.2 Chaga improves renal function in T2 DM rats(1)At week 17,the levels of SCr,BUN and 24h-UP were significantly higher in the DN group than in the NOR group and in the DN group at week 13(P < 0.05).The levels of UCr and the calculated creatinine clearance(Cr Cl)were significantly lower in the DN group than in the NOR group(P < 0.001 and P = 0.015).After 8 weeks of chaga intervention,SCr,BUN,24h-UP and UCr were significantly improved in the DN+I and DN+II groups compared with the DN group(P < 0.05),and Cr Cl levels were significantly increased only in the DN+II group(P = 0.005).(2)Glomerular diameter and relative area of the thylakoid matrix were significantly increased in the DN group compared with the NOR group(P < 0.001),and the above renal pathological changes were significantly improved in the DN+I and DN+II groups compared with the DN group(P < 0.05).3.3 Chaga ameliorates podocyte injury in T2 DM rats(1)The GBM and FPW were significantly increased in the DN group compared to the NOR group(P < 0.001).There was a significant effect of both chaga treatment groups on reducing GBM thickness and FPW compared to the DN group(P < 0.001).(2)PCR and WB results showed that the levels of Nephrin and WT-1 m RNA and protein were significantly reduced in the DN group compared with the NOR group(P < 0.01),and the levels of Nephrin and WT-1 expression were significantly increased in the DN+I and DN+II groups,respectively,compared with the DN group after 8 weeks of intervention with chaga(P < 0.01).3.4 Chaga elevated c GMP levels in kidney tissue and serum of T2 DM rats Elisa results showed that serum c GMP levels were significantly lower in the DN group than in the NOR group at weeks 13 and 17(P < 0.01),renal c GMP levels were lower in the DN group than in the NOR group,but there was no significant difference between the two groups.Compared with the DN group,the DN+I and DN+II groups had significantly increased serum c GMP levels at weeks 13 and 17(P < 0.001),and increased renal c GMP levels,but only the DN+II group was statistically different(P < 0.05).3.5 Chaga elevated e NOS,n NOS and decreased i NOS and PDE5 m RNA and protein levels in kidney tissues of T2 DM rats(1)RT-PCR results showed that e NOS m RNA was significantly decreased(P < 0.01)and i NOS and PDE5 m RNA levels were significantly increased(P < 0.01)in the DN group compared with the NOR group,and n NOS m RNA levels were not significantly different compared with the NOR group.(2)WB results showed no significant difference in e NOS and n NOS protein expression levels and a significant increase in i NOS and PDE5 protein expression levels in the DN group compared to the NOR group(P < 0.01).Compared with the DN group,the m RNA and protein expression levels of e NOS and n NOS were significantly increased(P < 0.05),the m RNA of i NOS and PDE5 and the protein expression of i NOS were significantly decreased(P < 0.05)in both the DN+I and DN+II groups,while the protein expression level of PDE5 was significantly decreased only in the DN+II group(P < 0.05).4 Conclusions:(1)Chaga improved hyperglycemia,hyperlipidemia and insulin resistance in HFD/STZ-induced T2 DM rats.(2)Chaga improved proteinuria,GBM thickening and podocyte fusion in T2 DM rats,and increased the m RNA and protein expression of podocyte-specific markers Nephrin and WT-1.(3)Chaga increased c GMP levels in blood and kidney tissues of T2 DM rats.The effect of chaga in increasing c GMP levels in the kidney tissues of T2 DM rats was associated with increased e NOS and n NOS and decreased i NOS and PDE5 expression.In conclusion,we suggest that chaga can protect the renal podocytes of T2 DM rats by improving the disorder of glucolipid metabolism and increasing the c GMP level,thus reducing proteinuria. |
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