Effect Of Active Vitamin D On Intestine And Kidney Of Diabetic Kidney Disease Through TLR4/NF-κB Pathway Based On Intestinal Flora

Objective: Changes of intestinal flora,the expression of VDR,TLR4/NF-κB in intestinal and renal tissues and the concentration related cytokines in mice with diabetes mellitus and diabetic kidney disease were observed.Methods: 8-week old and 14-week old male KKay mice were fed with high-fat and high-sugar diet to establish DM and DKD models,while 8-week old male C57BL/6 mice were fed with normal diet as Control group.During the feeding period,blood glucose and body weight were monitored every 3 days,diet and water intake were observed every week,and urine volume was monitored every two weeks.At the 6th week,24-hour urine was collected from metabolic cage to detect urinary protein excretion rate,urinary microalbumin and urinary creatinine.and serum levels of 25(OH)D,C-peptide,insulin,endotoxin,IL-6,TNF-α,serum calcium and serum phosphorus were detected.At the age of14 weeks,the blood glucose in tail vein of Kkay mice was more than 16.7 mmol/L for 3consecutive times,which indicated that the DM model was constructed successfully.At the age of 20 weeks,the 24-hour urinary protein excretion rate>30mg and/or urine ACR>30mg/g judged that the DKD model was constructed successfully.After 6 weeks of feeding,the mice were killed,and the kidney and intestinal tissues were removed by laparotomy for follow-up experiments.the mRNA and protein expression of VDR,TLR4 and NF-κB in kidney and intestinal tissue were detected by quantitative realtime-polymerase chain reaction and western blotting.and the changes of intestinal tissue were observed by hematoxylin-eosin staining.The genomes of intestinal bacteria were extracted from fresh fecal samples from colon to rectum,and the taxonomic composition,diversity and species differences were analyzed by sequencing the V3-V4 region of 16s rRNA gene.Results:①When Kkay mice were 14 weeks old,the DM model of tail vein blood glucose greater than 16.7mmol/kg was successfully established for three consecutive times.At 20 weeks of age,24hUP > 30mg and or ACR > 30mg/g,DKD model was successfully constructed.②Compared with Control group,serum 25(OH)D decreased and endotoxin,IL-6,TNF-α levels increased in DM and DKD groups(P<0.05).Urine UP and Urine ACR levels increased significantly in DKD group(P<0.05).③Compared with Control group,q PCR and WB showed that the expression of m RNA and protein of VDR in kidney of DM and DKD mice decreased(P<0.05),and the expression of VDR in intestinal tissue decreased in DKD group(P < 0.05).The m RNA and protein expression of TLR4 and NF-κB were increased in intestinal and renal tissues,and the expression of DKD was higher than that in DM group(P < 0.05).④A total of 2502996 high quality sequences were obtained by Nova Seq sequencing platform,including 661250 sequences in Control group,901888 sequences in DM group and 939858 sequences in DKD group.The number of ASV of the three groups of samples was calculated by R software,and the petal chart was drawn to show the common and unique number of ASV in each group,including 16176 in the unique ASV Control group,3996 in the DM group and 2574 in the DKD group.The Good’s coverage index showed that the current sequencing coverage depth of the three groups was more than 98%,including 98.4% in the Control group,99.3% in the DM group and 99.4% in the DKD group(P<0.05).Chao1 index and Observed species index showed that the richness of DM and DKD decreased among the three groups,and the difference was statistically significant(P<0.05).PCo A analysis in Bray-Curtis distance matrix shows that all samples are representative.At the phylum level,the samples of the three groups were mainly composed of Firmicutes,Bacteroidetes and Proteobacteria,and their relative abundance accounted for more than 99%.Among them,the abundance of Proteobacteria increased gradually,showing a trend of Control group < DM group < DKD group(P < 0.05).Metagenome Seq difference analysis showed that the abundance of horizontal Bacteroides,Parabacteroides,Acinetobacter,Oscillospira,Pseudomonas,Sediminibacterium and other G-bacteria in DM and DKD groups were significantly higher than those in DM group(P < 0.05),while the abundance of Lactobacillus in DKD group was significantly lower than that in DM group(P < 0.05).LDA analysis showed the distribution of Bifidobacterium family and Allobaculum relative abundance in Control group.In DM group,G-bacteria such as Bacteroides,Clostridium,Oscillospira,Brevundimonas,Mycoplana,Burkholderia,Helicobacter and Acinetobacter were relatively enriched.In DKD group,Gram negative bacteria such as Odoribacter,Prevotella,Elizabethkingia,Clostridium,Ochrobactrum,Agrobacterium,Delftia,Desulfovibrio,Morganella,Proteus,Shigella,Pseudomonas and Stenotrophomonas were relatively enriched(LDA>2,P<0.05).⑤HE showed that DM and DKD showed different degrees of inflammatory cell infiltration and loss of crypt structure.Conclusion: The diversity of intestinal flora of DM and DKD mice decreased and the level of phylum and genus changed,intestinal bacterial disorders may be involved in the pathogenesis of DM and DKD,and chronic inflammatory state and VD/VDR disorders may be the key factors in the pathogenesis.Objective: To explore the changes of intestinal flora,VDR,TLR4/NF-κB and related inflammatory indexes in intestinal and renal tissues of DKD model rats treated with different concentrations of 1,25-(OH)2D3.Methods: Forty 20-week old DKD model mice weighing 40-50g were randomly divided into DKD group,LVD group,MVD group and HVD group.The mice were treated with25-(OH)2D3 for 14 days.The changes of body weight and blood glucose were observed every 3 days during the intervention.24-hour urine UP,urine m Alb and urine Cr were collected 14 days later.25(OH)D,C peptide,insulin,endotoxin,IL-6 and TNF-α were detected in serum.16s rRNA from colon to rectum was taken to determine the species,number and function of intestinal flora.QPCR and WB were used to detect the mRNA and protein expression of VDR,TLR4 and NF-κ B in intestinal and renal tissues.Intestinal pathological changes were observed by HE staining.Results:①With the increase of the concentration of 1p25-(OH)2D3,the blood glucose decreased gradually,and the trend of body weight increase tended to be smooth(P>0.05).②Compared with DKD group,the concentrations of endotoxin,IL-6,TNF-αand urinary UP and ACR levels decreased in 25-(OH)2D3 intervention group,and the differences were statistically significant(all P<0.05).③qPCR and WB confirmed that the expression of mRNA and protein of VDR in kidney and intestinal tissue of mice treated with 25-(OH)2D3 was significantly up-regulated,while the expression of m RNA and protein of TLR4 and NF-κ B was significantly down-regulated(P < 0.05).④A total of 2600515 high quality sequences were obtained by Nova Seq sequencing platform,including 936680 in DKD group,540594 in LVD group,557720 in MVD group and 565521 in HVD group.The number of ASV in four groups of samples was calculated by R software.the unique number of ASV was 3235 in DKD group,4990 in LVD group,4776 in MVD group and3673 in HVD group.The Good’s coverage index showed that the current sequencing coverage depths of the four groups of samples were all greater than 99%,including 99.4%in DKD group,99.0% in LVD group,99.0% in MVD group and 99.2% in HVD group(P <0.05).Bray-Curtis distance matrix and PCo A analysis showed that the four groups of samples were representative.Taxonomic composition analysis and difference analysis showed that 1the abundance of Proteus decreased and the abundance of horizontal lactic acid bacteria increased gradually in the DKD group.The expression of high abundance G-bacteria Shigella,Pseudomonas,Desulfovibrio,Oscillospira,Acinetobacter,Helicobacter and Odoribacter decreased in the DKD group(all P<0.05).LDA difference analysis showed that Sediminibacterium,Odoribacter,Elizabethkingia,Wautersiella,Vagococcus,Streptococcus,Thermotalea,Oscillospira,Brevundimonas,Ochrobactrum,Agrobacterium,Burkholderia,Delftia,Bilophila,Helicobacter,Morganella,Proteus,Shigella,Acinetobacter,Pseudomonas and Stenotrophomonas)were relatively enriched in DKD group,while Alistipes,Nitrosomonas and Dok59 were relatively enriched in LVD group.The MVD group was relatively enriched by S2407,while the HVD group was relatively enriched by Lactobacillus and Pediococcus(P < 0.05).⑤HE showed that inflammatory cell infiltration was improved after intervention with 25-(OH)2D3.Conclusion: 1,25-(OH)2D3 could improve the imbalance of intestinal flora in DKD model mice,such as the decrease of relative abundance of Proteobacteria,the increase of relative abundance of lactobacillus,the decrease of the proportion and relative abundance of G-bacteria at genus level,the up-regulation of VDR expression in intestine and kidney,the down-regulation of m RNA and protein expression of TLR4/NF-κB,the decrease of serum endotoxin level and the concentration of inflammatory cytokines IL-6 and TNF-α.the protective effect of 1,25-(OH)2D3 on DKD mice may be related to increasing the expression of VDR in intestinal and renal tissues and inhibiting the activation of TLR4/NF-κB signal pathway after improving intestinal flora imbalance.