| BACKGROUNDDiabetic nephropathy(DN)is the most common microvascular complication of diabetes,which occurs in 30%-40%of diabetic patients.It is an important cause of the end-stage renal disease(ESRD)and an independent risk factor for predicting the death of patients with chronic kidney disease(CKD).Although the control of blood glucose,blood pressure,and inhibition of the renin-angiotensin system(RAS)can help to reduce the incidence and delay the progress of DN,it does not improve the present situation of diagnosis and treatment in DN.It is urgent to strengthen the mechanism research and search for new prevention and treatment strategies.Recent studies have found that chronic inflammation played an important role in DN,may be caused by innate immune cells recognizing the endogenous damage signals and exogenous antigen molecules.In animal experiments,pathogenic microorganisms with abnormal proliferation can be recognized directly by intestinal innate immune cells,reducing the inflammatory response of the lung through interleukin 22(IL22).It has also been confirmed that IL22 can alleviate renal tubulointerstitial injury in DN.Is it possible that the changes of intestinal flora may also participate in the occurrence and development of DN through IL22?The interaction between intestinal flora and drugs has attracted much attention.Drugs may lead to dysbiosis of intestinal flora,which in turn affects the efficacy and side effects of antidiabetic drugs.Cinnamaldehyde(CIN)is a new antidiabetic drug,which can reduce DN proteinuria.At the same time,it has an antibacterial effect.Is it possible that the protective effect on DN is realized by regulating intestinal flora and affecting IL22 related inflammatory response?Therefore,based on the DN rat model,this study will explore the changes of intestinal flora structure and function in the process of DN,and search for possible key microorganisms.And we will observe the effect of cinnamaldehyde on DN and intestinal flora,and initially explore its possible mechanism.OBJECTIVE1)To establish DN rat model and observe the changes of intestinal microflora during the progression of DN,also its correlation with renal injury(proteinuria and pathological changes),trying to find the key microorganisms that may participate in the pathogenesis;2)To observe the effect of cinnamaldehyde on renal injury and intestinal flora in DN rats and its possible mechanism.METHODS1)Establishment of DN rat model and intervention of CinnamaldehydeRats with the same age,blood glucose,and body weight were randomly divided into 4 groups(16 rats/group):(1)Diabetic nephropathy(DN)group:DN rat model was established by injection of streptozotocin(STZ).After 72 hours,the rats with fasting blood glucose?16.7 mmol/L were included in DN group;(2)Normal control(NC)group:72 hours after injection of 1%sodium citrate buffer,those with fasting blood glucose less than 7 mmol/L were included in NC group;(3)DN+cinnamaldehyde(DNC)group and(4)NC+cinnamaldehyde(NCC)group:the modeling was the same as above,and cinnamaldehyde was given by gavage every day from the 4th day.Blood glucose,body weight,24-hour urinary protein(24hUP),24-hour urinary volume(24hUV),and fresh feces were monitored at Od,4W,8W,and 12W.At 8th weeks(NC8,DN8,NCC8,DNC8)or 12th weeks(NC12,DN12,NCC12,DNC12)after modeling,rats were sacrificed(8 rats/group).The blood and kidney tissue samples were collected.HE,PAS,Masson staining,and electron microscope were used to evaluate the renal pathological injury.2)16S rDNA sequencing of intestinal floraAfter clustering operational taxonomic units(OTUs),adiversity was analyzed using Chaol and ACE for richness,Shannon and Simpson for diversity.And?diversity was analyzed by Weighted Unifrac distance with principal co-ordinates analysis(PCoA).Metastats and linear discriminant analysis(LDA)were used to analyze the difference in intestinal flora among groups.Spearman was used to analyze the correlation between the abundance of intestinal flora and renal injury indicators.The function of intestinal flora was predicted by Tax4fun.3)Mechanism of cinnamaldehyde alleviating DNThe expression of the megalin protein in the kidney was detected by immunohistochemistry and semi-quantitative analysis.Western blot was used to detect the innate immune pathway IL22/IL17A,inflammation pathway caspase-1/IL18,and fibrosis pathway fibronectin and TGF-? protein expression.The differences in protein expression between NC8 and DN8 group,DN8 and DNC8 group,NC8 and NCC8 were analyzed.4)Statistical analysisKolmogorov Smirnov test was used to test the normality of measurement data.The continuous variable with normal distribution was represented by mean value±standard deviation(mean ? SD);the non-normal distribution variable was represented by median(Q25,Q75).For the continuous variable with normal distribution,a t-test was used to compare the differences between the two groups;For those with non-normal distribution,the differences between the two groups were compared by non-parametric Mann Whitney test.The difference was statistically significant if bilateral test p<0.05.RESULTS1.Characteristics of intestinal flora changes in diabetic nephropathy1)Establishment of DN rat modelCompared with the control group,72 hours after STZ injection,the blood glucose of the DN group was significantly increased,showing polydipsia,polyuria and polydipsia in DN rats.From the 4th week after modeling,the bodyweight of the DN group was significantly lower than that of the NC group(p<0.001),and the blood glucose,24hUV of the DN group were significantly higher than that of NC group(p<0.001).From the 8th week,the 24hUP of the DN group was significantly higher than that of the NC group(p<0.001),indicating that the DN rat model was successfully constructed.There was pathological damage of the kidney in the DN group after 8 weeks of modeling,which showed glomerular enlargement,accompanied by mesangial cells and matrix proliferation;renal tubular swelling,and lumen stenosis.Electron microscope showed glomerular basement membrane thickening,podocytes fusion;tubular basement membrane thickening,mitochondrial swelling,and brush border partial falling off.2)Characteristics of intestinal flora in DN ratsStructure of intestinal flora:OTU analysis showed that DN8/DN12 shared 764 OTUs,with 159(17.2%)and 218(22.2%)unique OTUs respectively.Compared with the control group,the diversity and richness of DN8 had no significant change;but the Simpson index was decreased significantly(0.95±0.01 vs 0.90±0.01,p<0.05)in DN12.? diversity analysis showed that the community structure difference between NC12/DN12 group was larger than that of the NC8/DN 8 group.Species Difference:There was little difference between DN8 and NC8,but there was a great difference between DN12 and NC12.Metastats analysis showed that there were significant differences between NC8 and DN8 groups in 1 phylum,1 class,0 order,0 family,0 genus,and 9 species(q<0.05).There were significant differences between NC12 and DN12 groups in 3 phyla,7 classes,10 orders,18 families,19 genera,and 14 species.There were significant differences between DN8 and DN12 groups in 8 phyla,10 classes,10 orders,16 families,39 genera,and 30 species.It indicated that the structure of intestinal flora changed significantly with the development of DN.Compared with the same age control group,s__Bacteroides_Cellosilyticus and s__Bacteroides_Thetaiotaomicron were significantly decreased in DN8 and DN 12 groups.Correlation analysis:The relative abundance of 13 genera and 12 species were significantly correlated with 24hUP(p<0.05),among which,s__Bacteroides_Cellosilyticus(-0.39,p<0.01)and s__Bacteroides_Thetaiotaomicron(-0.42,p<0.01)were significantly negatively correlated with 24hUP.Intestinal flora function:With the development of DN,intestinal flora function changed significantly.DNA_repair_and_recombination_Protein,transfer_RNA_Purine metabolism,purine_Metabolism were significantly enriched in DN12.2.Effect of cinnamaldehyde on early DN and its mechanism1)The effect of cinnamaldehyde on renal injury in DNAfter 8 weeks of intragastric administration of cinnamaldehyde,compared with DN8,the 24hUP(86.65±10.08 vs 52.53±7.03 mg,p=0.02),and 24hUV of DNC8 were decreased significantly,but blood glucose and body weight showed no significant difference.After 12 weeks of gavage,there were no significant differences in 24hUP,24hUV,blood glucose,and body weight between DNC12 and DN12.Under the light microscope,there was no significant difference in renal pathological changes between DN8 and DNC8 groups.Under the electron microscope,compared with DN8,the thickness of the renal tubular basement membrane(TBM)of DNC8 was significantly decreased(276.30±10.16 vs 239.50±12.43 nm,p=0.02).There was no significant difference in glomerular basement membrane(GBM)thickness between the two groups.2)Effect of cinnamaldehyde on intestinal flora in DN ratsStructure of intestinal flora:Cinnamaldehyde intervention changed the intestinal flora of DN rats.DN8 and DNC8 groups shared 774 OTUs,and the unique OTUs were 149(16.1%)and 247(24.2%)respectively.Compared with the DN8 group,the Simpson index of the DNC8 group was significantly decreased(0.96±0.01 vs 0.94±0.01,p<0.01),suggesting that cinnamaldehyde can reduce species diversity,but does not affect species richness.Compared with NC8/DN8 group,the distance between NC8/DNC8 group was closer,suggesting that cinnamaldehyde may partially restore the community structure of DN rats.Species difference:Compared with DN8,there were significant differences in the relative abundance of 1 phylum,2 classes,0 order,5 families,13 genera,and 10 species in DNC8.At genera,g__Lactobacillus,g__Alloprevotella,g__Oscillospira,g__Weissella,and some other probiotics increased significantly in DNC8.At species,the abundance of 3 species decreased in DN8 and recovered after cinnamaldehyde intervention,including s Bacteroides massiliensis,s__Oscillibacter_sp_ER4,and s__Lachnospirace ae_bacterium_A2.The abundance of 2 species increased in DN8,and decreased after cinnamaldehyde intervention,including s__Ruminococcus_sp_ID1 and s__Comamonas_denitrificans?Function prediction:Cinnamaldehyde can significantly change the intestinal flora function of DN rats.Compared with the DN8 group,transporters,purine metabolism,and the ABC transporter pathway were significantly enriched in the DNC8 group.3.Mechanism of cinnamaldehyde improving early DNImmunohistochemistry and semiquantitative analysis showed that compared with NC8,the expression of megalin in the brush border of the proximal tubule in the DN8 group was significantly decreased(0.017±0.001 vs 0.006±0.001,P<0.01).Compared with DN8,megalin in DNC8 was significantly increased(0.006±0.001 vs 0.011±0.001,P<0.01).Compared with NC8,the expression of fibronectin(FN)in the DN8 group was significantly increased(1.00±0.28 vs 4.57±1.20,p=0.04),and the expression of IL22 in the kidney was significantly decreased(1.00±0.12 vs 0.61±0.11,p=0.04),but there was no significant difference of TGF-?.Compared with DN8,FN(4.57±1.20 vs 0.68±0.06,p=0.03)and TGF-p(1.85±0.37 vs 0.57±0.15,p=0.03)in DNC8 was significantly decreased.There was no significant difference in IL-22 and IL-17A protein expression between DN8 and DNC8.CONCLUSION1.There was intestinal flora dysbiosis in DN rats,related to the severity of DN.The diversity of intestinal flora decreased significantly at 12th weeks,with more significant difference species correlated with 24hUP.2.Cinnamaldehyde can partially restore the intestinal flora dysbiosis of DN rats,and selectively increase the abundance of anti-inflammatory probiotics,such as g__Lactobacillus and g__Alloprevotella.3.Cinnamaldehyde can not only reduce the early proteinuria and decrease the thickness of TBM in DN rats but also increase the expression of Megalin in the kidney and inhibit FN and TGF-?.But it did not affect the expression of IL22 and IL17A. |
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