| Objective:To investigate whether TDZD-8,an inhibitor of GSK3 beta,improves aldosterone induced heart inflammation and fibrosis by regulating autophagy.Methods:Adult male Wistar rats were raised in a sterile environment at room temperature and fed with normal diet and water.thirty-six adult male Wistar rats were divided into four groups for the following treatment.1)as the control group,eat and drink regularly.2)in the aldosterone group,Dissolve 0.2mg aldosterone salt in 800 ul sunflower seed oil and subcutaneously inject 1mg/kg per day.3)aldosterone salt+TDZD-8 group,aldosterone salt combined with TDZD-8(gsk-3 inhibitor): A mixture of0.2mg aldosterone salt and 0.3mg tdzd-8 was dissolved in 800 ul sunflower seed oil and injected subcutaneously into rats at 1.5mg/kg per day.4)aldosterone salt+TDZD-8+3MA group,aldosterone salt combined with TDZD-8(gsk-3 inhibitor)+3MA: Dissolve 0.2mg aldosterone salt,0.3mg tdzd-8 and0.2mg 3-ma in 800 ul sunflower seed oil and give subcutaneously to rats at 2mg/kg daily.All rats were treated and observed for 8 weeks.Hemodynamics and cardiac parameters of rats in each group were observed.Western blotting was used to detect the expression of GSK3,p-gsk3,VE-cad,alpha-SMA,LC3,P62 in cardiac tissue.The expression of il-1,ve-cad,alpha-sma,tnf-a,TGF-beta and Col I genes in cardiac tissue was detected by real-time quantitative PCR.LC3 expression in heart tissue was detected by immunofluorescence assay.The degree of fibrosis in cardiac tissue was observed by Masson staining.Results:1.TDZD-8 treatment cannot change the level of total GSK3 beta protein,but it can regulate phosphor-GSK3 beta S9 PSer9 at the level of heart tissue,indicating that TDZD-8 has the effect of inhibiting the activation of GSK3 beta.TDZD-8 can significantly inhibit the high expression of il-1beta and tnf-a mRNA caused by Aldo-salt.2.Aldosterone treatment resulted in decreased ve-cadherin mRNA and protein levels and increased a-sma expression levels,indicating that aldosterone induced EndoMT.Perivascular fibrosis and collagen deposition can be seen in the biopsy.This indicates that TDZD-8 can significantly inhibit the EndoMT induced by aldosterone salt.Aldosterone salt upregulated the expression of TGF-? and Col I in heart tissue,while TDZD-8 significantly inhibited the upregulation of TGF-? and Col I in heart tissue.3.Western blotting results showed that with or without aldosterone treatment,TDZD-8 significantly increased the protein level of LC3-II(a marker of autophagy activation)in myocardial tissue,indicating that autophagy was activated after TDZD-8 treatment.The decrease of p62/SQSTM1 protein level after TDZD-8 treatment was verified,indicating that the autophagy flux increased.Immunofluorescence results showed that LC3 green spots representing autophagy vesicles were significantlyincreased,and LC3-II was accumulated in heart tissues after TDZD-8treatment,indicating that TDZD-8 activated autophagy.4.The level of perivascular fibrosis increased after aldosterone treatment,and TDZD-8 could reverse this change.Autophagy inhibition of3-MAsignificantly inhibited the function of TDZD-8 in inhibiting perivascular fibrosis.It indicates that TDZD-8 protects the heart injury induced by aldosterone salt to some extent by activating autophagy.Conclusions:1.TDZD-8 inhibits GSK3 beta and relieves heart inflammation caused by aldosterone.2.TDZD-8 inhibited cardiac fibrosis caused by aldosterone.3.TDZD-8 activates autophagy in heart tissue.4.TDZD-8 inhibits aldosterone induced cardiac fibrosis through activation of autophagy. |
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