Hydrogen Sulfide Regulates Autophagy Through TGF-?1/Smad2 Signaling Pathway To Ameliorate Myocardial Fibrosis In Hypothyroidism Rats Induced By Propylthiouracil

Objective:This study aimed to investigate whether hydrogen sulfide?H2S?could regulate autophagy through TGF-?1/Smad2 signaling pathway to ameliorate myocardial fibrosis in rats with hypothyroidism induced by propylthiouracil?PTU?.Methods:40 male SD rats of 8 weeks old were randomly divided into 4groups,including Control group,PTU group,PTU+H₂S group and H₂S roup?n=10?.Rats in PTU group and PTU+H2S group were treated with propylthiouracil gavage method for 10 weeks,the dose was 0.2g/kg/d,and the control group and H2S group were given the same volume of drinking water.After 6 weeks,the tail vein blood of each group was taken.The serum free thyroxine?FT4?and thyroid stimulating hormone?TSH?concentrations were detected by ELISA.After the model of hypothyroidism was successfully established,rats in PTU group and PTU+H2S group were still treated with propylthiouracil,while rats in the PTU+H2S group and H2S group were given intraperitoneal injection with NaHS?56umol/kg/d?.The control group and PTU group were injected intraperitoneally with the same dose of normal saline.A total of intervention time was 4 weeks.After the 10th week,color Doppler ultrasound was used to measure structure and function of heart,including the left ventricular posterior wall?LVPW?,the left ventricular end-systolic diameter?LVES?,the left ventricular end-diastolic dimension?LVED?,and the left ventricular fractional shortening?LVFS?.When the rats were sacrificed,the body weight?BW?and heart weight?HW?were weighed to calculate the heart weight index?HW/BW?;Masson staining was used to detect myocardial collagen fiber deposition;qRT-PCR method was adopted to detect COL1?2 and COL3?1expression.The expression levels of MMP13,TIMP2,BECLIN1,ATG4,ATG5,TGF-?1,SMAD2 and CSE were detected all by Western blotting.Results:1.After 6 weeks,compared with the Control group,the FT4 values of the PTU group and the PTU+H2S group were significantly decreased,the TSH values were significantly increased?P<0.05?,and the FT4 and TSH values of the H2S group were not significantly changed?P>0.05?.Compared with the Control group,the BW and HW values of the PTU group and the PTU+H2S group decreased,and the HW/BW values increased?P<0.05?.Compared with the PTU group,the BW value and the HW value of the PTU+H2S group decreased,there was no significant difference?P>0.05?,while the HW/BW ratio decreased statistically?P<0.05?.There were no significant differences in BW,HW and HW/BW values between the Control group and the H2S group?P>0.05?.3.The results of cardiac ultrasonography showed that the LVED and LVES values were both increased in the PTU group compared to the Control group,and the LVFS values were reduced?P<0.05?.Compared with the PTU group,the LVED and LVES values of the PTU+H2S group were decreased,the LVFS value was increased?P<0.05?,and the LVPW values were not statistically different?P>0.05?.4.Masson staining results showed that compared with the Control group,the interstitial fibrosis in the PTU group was obvious.Compared with the PTU group,the interstitial fibrosis of the PTU+H2S group was improved.The deposition of myocardial interstitial collagen fibers in H2S group was higher than that in Control group,which was less than that in PTU group.5.The results of qRT-PCR showed that the expression of COL1?2 and COL3?1 in the myocardium of PTU group and H2S group increased compared with Control group?P<0.05?.Compared with the PTU group,the expression of COL1?2 and COL3?1 in the PTU+H2S group was significantly decreased?P<0.05?.6.The results of Western Blotting showed that the expression of MMP13,TGF-?1 and SMAD2 protein in the myocardium of PTU group was significantly higher than that in Control group?P<0.05?,while the expression of BECLIN1,ATG4,ATG5,TIMP2 and CSE protein was significantly decreased?P<0.05?.Compared with the PTU group,the expression of MMP13,TGF-?1 and SMAD2 protein in the myocardial tissue of PTU+H2S group was significantly decreased?P<0.05?,and the expression of BECLIN1,ATG4,ATG5,TIMP2 and CSE protein was significantly increased?P<0.05?.The expressions of TGF-?1 protein in H2S group were different from those in Control group?P<0.05?,but there was no significant difference in MMP13,TIMP2,CSE,SMAD2,BECLIN1,ATG4 and ATG5 expression?P>0.05?.Conclusion:Myocardial fibrosis in hypothyroidism rats is associated with down-regulation of endogenous H2S levels and activation of TGF-?1/Smad2signaling pathway and autophagy.This study demonstrates that the gas signal molecule H2S can ameliorate propylthiouracil-induced myocardial fibrosis,which may be related to inhibiting TGF-?1/Smad2 signaling pathway and activating autophagy.