Gaseous Signal Molecule SO₂ Up-regulates Autophagy Through The PI3K/AKT1 Signaling Pathway To Improve Myocardial Fibrosis In Rats With Hyperthyroidism

Objective:This study aimed to establish a rat model of hyperthyroidism by levothyroxine,and to explore whether SO₂ can ameliorate myocardial fibrosis in rats with hyperthyroid cardiomyopathy by regulating autophagy via the PI3K/AKT1 signaling pathway.Methods:According to the random principle,40 male SD rats were divided into 4 groups with the same number in each group:normal group（Control）,L-Thy model group（L-Thy）,SO₂ intervention group（L-Thy+SO₂）,HDX intervention Group（L-Thy+SO₂+HDX）.On the basis of standard feed feeding,levothyroxine（L-Thy,dissolved in physiological saline at a concentration of 250ug/ml,a dose of 2ml/100g）was injected intraperitoneally for 5 consecutive weeks.L-Thy+SO₂ group and L-Thy+SO₂+HDX group were added with sulfur dioxide donor Na₂SO₃/NaHSO₃ 85mg/Kg（0.54:0.18mmol/kg freshly dissolved in distilled water before use）;In the HDX group,an additional SO₂generating enzyme inhibitor（HDX）was given at 25 mg/kg/week.Five weeks after the first L-Thy injection,cardiac ultrasound was performed to detect the cardiac structure of rats,then the rats were sacrificed and the rat body weight（BW）and heart weight（HW）were weighed,the rat heart weight index（HW/BW）were calculated;Elisa method was used to measure the serum TT3 and TT4 values and the myocardial tissue SO₂content;Masson staining was used to detect the deposition of collagen fibers in rats;Transmission electron microscope was used to observe the myocardial fibers and some ultrastructure changes;Western blotting method was used to detect relative protein expression levels.Results:1.ELISA detection of SO₂ content showed that compared with the Control group,the SO₂ content in the myocardium of the L-Thy group rats was significantly reduced;compared with the L-Thy group,the SO₂ content in the myocardium of the L-Thy+SO₂ group rats was significantly increased;and the myocardial SO₂ content in L-Thy+SO₂+HDX group was lower than that in L-Thy+SO₂ group.2.Compared with the Control group,the TT3 and TT4 values in the L-Thy group were significantly increased（P<0.05）;compared with the L-Thy group,the TT3 and TT4 levels were slightly lower in the L-Thy+SO₂ group,and compared with the L-Thy+SO₂ group,the TT3and TT4 values of the L-Thy+SO₂+HDX group were increased（P>0.05）.3.Compared with the Control group,the BW in the L-Thy group was significantly reduced,and the HW/BW value was significantly increased（P<0.05）;the BW value in the L-Thy+SO₂ group was significantly higher than that in L-Thy group（P<0.05）,while the HW/BW value was slightly lower（P>0.05）;compared with the L-Thy+SO₂ group,there was no significant difference in HW/BW value and BW value in the L-Thy+SO₂+HDX group.4.Echocardiography showed that compared with the Control group,rats in the L-Thy group had impared cardiac function,the LVFS in the L-Thy group decreased significantly,and the LVEDD and LVESD values showed a significant increase（P<0.05）;Compared with the L-Thy group,the cardiac function of the L-Thy+SO₂group was improved,the LVFS increased compared with the L-Thy group;the LVEDD value and the LVESD value decreased obviously（P<0.05）;Compared with the L-Thy+SO₂ group,the LVFS in the L-Thy+SO₂+HDX group decreased significantly,and the LVEDD and LVESD values increased significantly（P<0.05）.5.Masson results showed that the area of blue staining in the L-Thy group was significantly larger than that in the Control group,the myocardial interstitial tissue was rich in collagen fibers,and the myocardial cells were arranged disorderly;compared with the L-Thy group,the area of blue staining in the L-Thy+SO₂ group was lessened,the myocardial interstitial collagen fibers were relatively small,and the cardiomyocytes were arranged relatively regularly;while the degree of myocardial fibrosis in the L-Thy+SO₂+HDX group was significantly worse than that in the L-Thy+SO₂ group.6.TEM results showed that compared with the Control group,the myocardial fibers in the L-Thy group were significantly disordered,mitochondria and endoplasmic reticulum were damaged,and autophagosomes were visible;compared with the L-Thy group,the cardiomyocytes in the L-Thy+SO₂ group were relatively ordered,and the damage of mitochondria and endoplasmic reticulum was better than before,and the autophagosomes were significantly reduced;the degree of myocardial damage in the L-Thy+SO₂+HDX group was significantly worse than that in the L-Thy+SO₂ group,and autophagosomes increased.7.Western Blotting results showed that compared with the Control group,MMP2,MMP3,CollagenIII,CollagenI,PI3K,AKT1,ERP72,CHOP,and GRP78/BIP had enhanced expression in the L-Thy group,LC3II,Beclin-1,ATG5,ATG12,ATG16,and TIMP2expressions were weakened（P<0.05）;compared with the L-Thy group,MMP2,MMP3,Collagen III,CollagenI,PI3K,AKT1,ERP72,CHOP,and GRP78/BIP in the myocardium of the L-Thy+SO₂ group expression levels were significantly reduced,LC3II,Beclin-1,ATG5,ATG12,ATG16 and TIMP2 expressions were significantly increased（P<0.05）;while in the L-Thy+SO₂+HDX group,the expression levels of MMP2,MMP3,CollagenIII,CollagenI,PI3K,AKT1,ERP72,CHOP,and GRP78/BIP were higher than those in the L-Thy+SO₂ group（P<0.05）.Conclusion:The gaseous signal molecule SO₂ could ameliorate myocardial fibrosis in rats with hyperthyroidism,the mechanism of which may involve the activating of autophagy via the PI3K/AKT1 signaling pathway and the inhibition of endoplasmic reticulum stress.