The Effects Of Naringin On Myocardial Fibrosis In Type 1 Diabetic Mice And It's Possible Mechanism

Objective: To investigate the effect of naringin on myocardial fibrosis in type 1 diabetic mice and it's possible mechanism.Methods: C57BL/6 mice(n=32)were randomly divided into normal group(NS group,n=8),diabetes group(DM group,n=8),diabetes with low dose of Naringenin group(D+LN group,n=8),and diabetes with high dose of Naringenin group(D+HN group,n=8).Mice were intraperitoneally injected with streptozotocin(STZ)daily for 5 consecutive days.After 2 weeks,the tail was cut off for blood collection to test the random blood glucose.It was considered that type 1 diabetes mouse model was successfully established when the level of random blood glucose was greater than 16.7 mmol/L.After type 1 diabetes mouse model established,Naringin suspension was given to D+LN group and D+HN group mice by intragastric administration at the dose of 25 mg/kg/d and 75 mg/kg/d once a day for 8 weeks,respectively.Simultaneously,the normal saline of equal volume was given to the NS group and DM group for 8 weeks.At the end of the study,all mice were sacrificed.The effects of Naringin on body weight and blood glucose of mice were observed.Hematoxylin eosin(HE)and Masson staining to observe the changes of heart morphological structure and measure the myocardial collagen volume fraction(CVF).The protein expression of RhoA,ROCK1 and ROCK2 in heart tissues was detected by immunohistochemistry(IHC).Western blot analysis was applied to determinate the protein expression of RhoA,ROCK1,ROCK2,type IV collagen and Fibronectin(FN)in heart tissue.Result: 1.Compared with the NS group,the body weight decreased and blood glucose increased in DM group mice(P<0.05).And there was no significant difference among DM group,D+LN group and D+HN group(P>0.05).2.HE staining: The myocardial cells in NS group had clear contour,neat arrangement and uniform cytoplasmic staining.However,the myocardial cells in the DM group were not clear in contour,disordered structure in arrangement,uneven in cytoplasm coloring,and the myocardial cells in the middle layer showed wave-like degeneration.In the D+LN group and the D+HN group,the myocardial cells were generally clear in contour,orderly in structure,uniform in cytoplasmic staining,and the wave-like degeneration of the mesenchymal myocardial cells was significantly alleviated compared with that in the DM group.3.The distribution of collagen fibers around myocardial cells and small vessels was observed by MASSON staining: there was a little collagen fiber distribution around myocardial cells and small vessels in NS group.However,the collagen fiber was significantly increased in DM group.And,the collagen fiber in D+LN and D+HN group was significantly decreased than that in DM group.Moerover,the CVF analysis showed that CVF in DM group was significantly increased than that in NS group,and decreased than D+LN and D+HN group(All P<0.05).Compared with the D+LN group,CVF was more decreased in the D+HN group(P<0.05).4.IHC was performed to observe the protein levels of RhoA,ROCK1 and ROCK2: The presence of brown-yellow granules indicates a positive expression and RhoA,ROCK1 and ROCK2 proteins were mainly expressed in the cytoplasm.Compared with NS group,RhoA,ROCK1 and ROCK2 protein expression were increased in DM group(P<0.05).Compared with the DM group,the protein expression of RhoA,ROCK1 and ROCK2 protein decreased in the D+LN and D+HN group(P<0.05).And their expressions more decreased in D+HN group than that in D+LN group(P<0.05),which indicated that the degree of decreased protein showed a dose-dependent relationship with naringin.5.The levels of RhoA,ROCK1,ROCK2,type IV collagen and FN protein in the heart tissue of mice was test by Western blot: Compared with the NS group,the protein levels of RhoA,ROCK1,ROCK2,type IV collagen and FN in the DM group were markedly augmented(P<0.05).Compared to the DM group,the levels of RhoA,ROCK1,ROCK2,type IV collagen and FN in the D+LN and D+HN group decreased(P<0.05),And their expressions more decreased in D+HN group than that in D+LN group(P<0.05),showing that the degree of decreased protein showed a dose-dependent relationship with naringin.Conclusion: 1.Naringin can improve myocardial fibrosis in type 1 diabetic mice without affecting the body weight and blood glucose.There is a dose-dependent effect of naringin on the anti-fibrosis in type 1 diabetes mice,and with the increase dose of naringin,the anti-fibrosis effect becomes more stronger.2.The over-activation of RhoA/ROCK signaling pathway was involved in myocardial fibrosis in type 1 diabetic mice.And one of the mechanisms of naringin to improve myocardial fibrosis in type 1 diabetic mice may be achieved by inhibiting the over-activation of RhoA/ROCK signaling pathway.