| Diabetic nephropathy(DN)is a common chronic complications caused by Diabetes mellitus(DM),and it is one of the major risk factors which leads to the end-stage renal disease.Micro RNAs(mi RNAs)are endogenous non-coding RNAs with regulatory levels of about 20-25 nucleotides in size.At present,the study of the specific mechanism of mi RNA in various diseases,especially the mechanism of mi RNA in kidney-related diseases,is not perfect.Herba artemisiae capillaris is a dry part of the Compositae plant Artemisia scoparia or Artemisia capillaries,and there are few studies on the related research of capillaris and DN,and most of the studies are found in the compound related to capillaris.The preliminary experiment showed that the herba artemisiae capillaris extracts had a protective effect on the kidney of DN rats,and discussed the mechanism from the perspective of extracellular matrix degradation.Based on the preliminary experiment,the current study extended the HACE intervention effect of kidney on the two time periods(8 weeks and 16weeks),and explored the possible mechanism of capillaris extracts in DN rats from mi RNAs level,which provided a new theoretical basis for the determination of DN in capillaris.In this study,DN rat model was injected by intraperitoneal injection of streptozotocin(STZ)(55 mg / kg)and treated with herba artemisiae capillaris extracts(HACE)(5 g / kg body weight).The study measured the indexes of blood and urine and the changes of renal function and renal histomorphology after 8 weeks and 16 weeks of administration.Mi RNAs chip were used to detect the expression of mi RNAs in the kidney of the rats.Seven high-expression mi RNAs were screened out in the high abundance(signal>800)mi RNAs with its differentially expressed levels higher than 1.74 times(log2 | Fold change |> 0.8),and verified using Realtime PCR.Combined with the results of the mi RNAs chip,Realtime PCR results and related literature,we selected mi RNA-21 for further functional studies.Mi RNA-21 target gene was predicted by mi RNAs target gene prediction database.Finally,Western Blot and immunohistochemistry were used to detect the expression of target gene in renal tissue of each group.The results showed that A number of renal function indicators were significantly changed and With varying degrees of renal lesions in DN group,after giving HACE treatment,some indicators were significantly reduced,Pathological changes have different degrees of improvement,and with the increase in administration time,its improvement also increased.The differentially expressed mi RNAs of normal and model groups were compared and analyzed,35 differentially expressed mi RNAs were screened out;the differentially expressed mi RNAs of HACE and model groups were compared and analyzed,17 differentially expressed mi RNAs were screened out.Realtime PCR was used to validate seven positive mi RNAs,after 8 weeks of administration,Compared with the normal group,the expression of mi R-1306-3p,mi R-672-5p and mi R-3550 were significantly down-regulated in the model group(P<0.01 or ?<0.05);After treatment with HACE,mi R-672-5p was significantly up-regulated(P<0.05).After 16 weeks of administration,compared with the normal group,the expression of mi R-21-5p was significantly up-regulated in the model group(P<0.01),and mi R-1306-3p,mi R-672-5p and mi R-466 d were significantly down-regulated(P<0.01 or P<0.05);After treatment with HACE,the expression of mi R-215 p and mi R-1306-3p was significantly down-regulated(P <0.05),and the expression of mi R-672-5p was significantly up-regulated(P<0.05).The results of Realtime PCR were analyzed after 8 weeks and 16 weeks of comprehensive analysis,mi R-21-5p and mi R-672-5p were found in the mi RNAs which differences changes occur after modeling and become normal after giving HACE.Mi RNA target gene prediction software(Targetscan,Pic Tar,micro RNA.org)predicts mi RNA-21 target gene,Smad7 found in three databases have appeared,analyze gene sequences found that the Smad7 gene has 7 base sites complementary to mi RNA-21.Prediction of binding site free energy analysis software(RNA Hybrid)validation revealed that mi RNA-21 had a greater probability of regulating the expression of Smad7.It was found that the TGF-?1 / Smad signaling pathway was enriched after using the KEGGdatabase to analyze all the predicted target genes.The expression of TGF-?1 / Smad pathway protein(TGF-?1,Smad2/3,Smad7)was detected by Western Blot and immunohistochemistry,the results showed that the expression of TGF-?1 and Smad2 /3 in model group was significantly increased(P <0.05),the expression of TGF-?1 and Smad2/3 was significantly decreased after HACE treatment(P <0.05).Compared with the normal group,the expression of Smad7 was significantly decreased(P<0.01),and the expression of Smad7 was significantly increased after HACE treatment(P<0.01).Based on the findings:1.Herba artemisiae capillaris extracts feeding can reduce kidney damage by blood lipid level reduce,inhibit renal pathological changes and renal function increase of diabetic nephropathy rats.2.There are several mi RNAs expressed in diabetic nephropathy rats,which with differential expression and participate in DN process.Among them,mi RNA-21 was found with obvious expression changing in this period.DN was affected by the TGF-?1/Smad pathway,and mi RNA-21 can modulate the expression of its specific proteins Smad7,TGF-?1 and Smad2/3.3.Herba artemisiae capillaris extracts effect the expression of multiple mi RNAs in different stages of diabetic nephropathy rats,especially mi RNA-21.Meanwhile,HACE can inhibit the expression of TGF-?1 and Smad2/3 and increase the expression of Smad7 in the DN rats,suggests that renal protective effects on diabetic nephropathy may be related on mi RNA-21-mediated TGF-?1/Smad pathway.Innovation:1.The results showed that the Herba artemisiae capillaris extracts could affect the expression of mi RNAs in diabetic nephropathy rats.2.Herba artemisiae capillaris extracts intervene diabetic in nephropathy rats may be related on mi RNA-21-mediated TGF-?1 / Smad pathway. |
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