| Objective:To observe the method of “treating phlegm and blood stasis together” on the mechanism of renal fibrosis in diabetic rats,Study the JAK/STAT signaling pathway and its downstream indexes regulated by AGEs by using“treating phlegm and blood stasis together” method,and to give new ideas to treat diabetic nephropathy.Method:100 clean-grade Rats with the weight of 240±20g were adaptively fed for 7 days that were divided into two groups randomly,15 rats were taken as blank group,the remaining 85 rats were fasted but not water for 24 h,and STZ solution was injected intraperitoneally at a dose of 55mg/kg,and the blank group was injected with the same amount of buffer.After 3 days,the random blood glucose > of 16.7mmol/L was measured by tail vein method,which was considered to be successful replication of the DM model.The DN model was prepared by continuing feeding for 4 weeks,In the end.The successfully modeled rats were randomly divided into 5 groups: model group,phlegm and stasis co-treatment group,ALT-711(ALT-711)group,phlegm-reducing group,and stasis-reducing group.All rats were administered by gavage,blank group and model group: gavage with distilled water at a dose of 1.0 ml/(100 g·d);,the corresponding decoction was given gavage according to the doses of 4.05/(kg·d),8.10g/(kg·d)and 4.05/(kg·d)in the phlegm-reducing group,phlegm and stasis co-treatment group,stasis-reducing group,respectively.ALT-711 group: dissolve ALT-711 in distilled water and then gavage at a dose of 0.3 mg/(100 g·d).Gastric gavage was performed at a fixed time every morning,and the rats were closely monitored daily,and after 8 weeks of administration,the material was taken after anesthesia.Observe the general condition of rats every day during gavage,including rat hair gloss,mental condition,eating water,activity and the number of rat deaths,and record them in detail.HE and Masson staining to observe the renal tissue fibrosis of DN rats with“treating phlegm and blood stasis together”.Real-time PCR and Western blot method were used to detect DN rat kidney tissue AGEs,JAK2,STAT3,TGFβ1,FN,COLIV gene and protein and RAGE protein expression levels.Outcome:(1)Rats group had normal response in the blank,rapid movement,shiny hair,normal water intake and stool,while each module rat had different degrees of dull and yellow hair,mental malaise,slow action,increased dietary water and weight loss.(2)In HE-stain,The phlegm and stasis co-treatment group had the least pathological changes and is closest to the blank group.In Masson-stained,the model group had a large number of blue-stained collagen fibers in the glomeruli and renal interstitium,while the Phlegm and stasis co-treatment group had less collagen fiber deposition than in the other groups.(3)By comparing the model group and the blank group,The expression of AGEs,JAK2,STAT3,FN,Col IV,RAGE and TGFβ1 in the model group increased(P<0.01).Compared with the model group,the expression of genes and proteins of AGEs,JAK2,STAT3,FN,Col IV and TGFβ1 and protein of RAGE in renal tissues increased(P<0.05,P<0.01).Compared between the drug groups,the downregulation of AGEs,JAK2,STAT3,FN,Col IV,TGFβ1 proteins and genes and protein of RAGE was more obvious in the sputum and stasis treatment group(P<0.01).Conclusion:The “ treating phlegm and blood stasis together" method can improve DN fibrosis,and its mechanism may be related to inhibition of JAK/STAT signaling pathway regulated by AGEs-RAGE and the expression of FN,Col IV and TGFβ1 downstream of it,thereby improving DN renal fibrosis lesions and protecting the kidneys.The effect of "treating phlegm and blood stasis together " on DN renal fibrosis is more obvious than that of " phlegm-reducing " and "stasis-reducing ". |
