Study On Intervention Effect Of Danhuang Wuwu Tang On Diabetic Nephropathy Model Rats Based On Gut Microbiota

Objective: In this study,streptozocin(STZ)-induced diabetic kidney disease(DKD or diabetic nephropathy,DN)rats were used as an animal model to intervene with Danhuang Wuwu Tang(DHWWT)and observe the intervention effect of DHWWT on DN.Through16 S r DNA gene sequencing,targeted metabolomics determination and fecal microbiota transplantation(FMT)technology,the aim was to deeply investigate the regulatory effect of DHWWT on the levels of gut microbioa and their metabolites short-chain fatty acids(SCFAs)in DN model rats,providing a reference for the treatment of DN by traditional Chinese medicine by regulating gut microbioa.Methods:1.Twenty-one healthy male Wistar rats were divided into normal model group after adaptive feeding.In the model group,the DN model was constructed by intraperitoneal injection of STZ 60 mg/kg.72 hours after STZ injection,blood samples were collected from the tail vein to determine fasting blood glucose(FBG)> 15 mmol/L(280mg/dl),that is,the diabetic model was judged successful.They were observed continuously for 14 weeks.Body weight was measured weekly to calculate water intake and food intake.24-hour urinary protein(24h-UP),glucose metabolism,lipid metabolism,renal function and serum albumin levels were measured every 2 weeks.At the end of the experiment,the eyeballs,kidneys,and pancreas were taken for pathological staining to comprehensively evaluate the pathological injury and provide the basis for the next pharmacodynamic experiment;2.After 100 Wistar rats were adaptively fed,10 rats were randomly selected as the normal control group,and the other rats were used to duplicate the DN model according to the same method,and the 24h-UP levels of rats in each group were dynamically monitored every 2 weeks.After the 24h-UP of model rats was significantly higher than that of the normal control group,they were included in this experiment,and divided into 6 groups according to the principle of randomization,that is,the model group,the low,medium,and high dose groups of DHWWT,the losartan group(LOS),and the Huangkui Capsule group(HKC)During the intervention period,body weight was weighed weekly and water intake,food intake,and urine volume were counted.24h-UP levels were measured every 2 weeks.At the end of the intervention,three stools were collected from rats in each group at the same time point,and microbial 16 S r DNA gene sequencing,SCFAs determination and preparation of fecal bacterial solution were performed,respectively.At the end point,the experiment was anesthetized and killed,and serum was obtained to measure biochemical parameters such as glucose metabolism,lipid metabolism,and renal function and kidney tissues were taken for pathological staining and transmission electron microscopy;3.After adaptive feeding of 60 Wistar rats in a single cage,the diabetic model was replicated by the same method,and 48 rats were randomly selected and divided into 4groups,namely,FMT control group(DM + PBS),FMT model group(DM + FMT DN),FMT DHWWT group(DM + FMT DHWWT),and FMT HKC group(DM + FMT HKC).Gut microbiota were removed by mixed antibiotic method,except for the control group transplanted with the same amount of PBS solution,the rest each group was transplanted with the fecal microbiota liquid of the corresponding experimental group.After the FMT,the observation was conducted for 4 weeks.Body weight was measured weekly,and water intake,food intake,and urine volume were counted.At the end of the 4th week,the feces and 24 h urine volume of rats in each group were collected for the determination of SCFAs and 24h-UP,the end point experiment was anesthetized and killed,the serum was obtained to measure biochemical indicators such as glucose metabolism,lipid metabolism,and renal function and the kidney tissues were taken for pathological staining;4.The chemical components of DHWWT were qualitatively and quantitatively analyzed using liquid chromatography-mass spectrometry(LC-MS).DHWWT sample solution was prepared by water extraction,and internal standard(propranolol,tolbutamide)was added,injected into LC-MS for detection under the same conditions,and the optimized chromatographic and mass spectrometric conditions were as follows: Chromatographic column: Thermo Hypersil Gold C18 3 μm,2.1 × 100 mm;mobile phase: A: water(2.5 m M ammonium formate),B: acetonitrile;gradient elution: 0 – 1.5 min,30 – 60% B,1.5 – 4 min,60 – 95% B,4 – 5.5 min,95% B,5.5 – 6 min,95 – 30% B,6 – 8 min,30% B;sample volume 5 μL,flow rate 0.3 m L/min.Mass spectrometry parameters: ESI(+): spray voltage,3500V;ESI(–): spray voltage,3200V;evaporation temperature,350oC;sheath gas,40Arb;auxiliary gas,10Arb;capillary temperature,320oC;S-lens RF,50;NCE,30.Each compound in DHWWT was identified by comparison with the standard,and quantitative analysis of each compound was performed.Results:1.Compared with the normal group,the rats in the model group showed hyperglycemia,polydipsia,polyphagia,polyuria and decreased body weight.At the beginning of 6 weeks,there was a continuous increase in 24h-UP,and serum tests revealed an increase in Scr,BUN,TC,TG,LDL-C,and HDL-C and a decrease in blood ALB.Pathological staining suggested that the glomerular volume increased and the intraglomerular capillaries expanded in the DN group from the 8th week.By the 10 th week of the experiment,the typical pathological manifestations of DN such as diffuse glomerulosclerosis occurred accompanied by pancreatic atrophy and retinopathy;2.After the intervention of DHWWT,the renal pathological damage was alleviated,24h-UP,Scr,BUN,FBG,GSP,TC,TG,LDL-C decreased,and the blood ALB,HDL-C and pancreatic index increased,of which the high-dose group of DHWWT had the most significant effect,and had a certain dose-dependent effect,superior to the HKC and LOS;sequencing analysis of the collected 56 stool samples showed that the gut microbial ACE and Shannon index decreased significantly and the Simpson index increased in the DN rats.Species composition phylum-level analysis showed that rats in the DN model group were significantly enriched with Actinobacteriota,and association analysis indicated a positive correlation with various metabolic parameters such as 24h-UP,FBG,and TG.At the genus level,rats in the DN model group were significantly enriched with Bifidobacterium,Turicibacter,and Phascolarctobacterium,and association analysis showed a significant positive correlation with 24-UP,FBG,GSP,TG,TC,and BUN parameters,and a significant inhibition of Lactobacillus,which was negatively correlated with 24-UP and TG and positively correlated with HDL-C.After the intervention of DHWWT,the abundance of Actinobacteriota,Bifidobacterium,Turicibacter,and Phascolarctobacterium was significantly reduced,and the abundance of Lactobacillus was significantly increased,with DHWWT having the best effect at high doses and a dose-dependent effect.Lefse multi-level species difference discriminant analysis(LDA)showed that Actinobacteriota and Bifidobacterium,Turicibacter,Phascolarctobacterium were the unique core bacteria of DN model rats;the results of quantitative detection of SCFAs showed that compared with the normal group,the content of total SCFAs in the feces of DN model group was decreased,and the content of total SCFAs in the feces of DHWWT group was increased,especially acetic acid,propanoic acid,and butanoic acid were increased,of which DHWWT had the best high-dose effect,and had a dose-dependent effect,superior to HKC and LOS;3.Fecal transplantation experiments confirmed that compared with DM + PBS group,DM + FMT DN group had increased 24h-UP,Scr,BUN,renal index,hyperplasia index,renal fibrosis index and severe renal pathological injury,DM + FMT DHWWT group had decreased above indicators,reduced renal pathological injury,and the effect was superior to DM + FMT HKC group;the quantitative results of fecal SCFAs showed that the content of fecal SCFAs in the DM+FMT DHWWT group increased,which was better than that of the DM+FMT HKC group;4.The results of LC-MS qualitative analysis showed that DHWWT contained phloridzin,salvianolic acid B,hyperoside,isoquercitrin,myricetin,rutin,astragaloside Ⅳ,tanshinone IIA and other chemical components,and the quantitative analysis results showed that DHWWT contained the highest contents of chemical constituents,including phloridzin,salvianolic acid B,hyperoside,and isoquercitrin.Conclusions: 1.The DN rat model of STZ-injury has good stability and high repeatability,which can provide an animal model for the pharmacological study of DN;2.DHWWT can reduce the urinary protein of DN model rats,and reduce glucose and lipid metabolism indicators,improve renal function and pathological injury,and can promote pancreatic weight gain,with a protective effect on the kidney and pancreas;3.DN rats show gut microbiota disorders,of which Actinobacteriota and Bifidobacterium,Turicibacter,Phascolarctobacterium can be used as potential core marker bacteria of DN.DHWWT can improve gut microbiota imbalance,reduce core bacteria abundance,and significantly enrich probiotic Lactobacillus abundance in DN rats.DHWWT can increase the content of total SCFAs in feces,of which acetic acid,propanoic acid,and butanoic acid are the main ones;the bacterial groups enriched in DHWWT can produce a large number of SCFAs and play a role in anti-DN using FMT techniques;4.The contents of chemical components such as phloridzin,salvianolic acid B,hyperoside,and isoquercitrin in DHWWT are high,and these chemical components may be the key components of DHWWT in exerting efficacy and responding to gut microbiota.