Gaseous Signalling Molecule Sulfur Dioxide Alleviates Myocardial Fibrosis In Type 2 Diabetic Rats Through The Regulation Of Autophagy By Regulating TGF β-Smad2/3 Signaling Pathway

Objective:In this study,Type 2 diabetic myocardial fibrosis rat model was established by high-sugar and high-fat diet combined with intraperitoneal injection of streptozotocin.SO₂ donor sodium sulfite/sodium bisulfite（Na₂SO₃/NaHSO₃）was used to intervene to investigate the effect of SO₂ on myocardial fibrosis in type 2 diabetic rats,and to explore whether its mechanism of action is related to the regulation of TGFβ-Smad2/3 pathway to up-regulate autophagy.Methods:Forty male SD rats were raised in cages in a cleaner-grade laboratory and randomly divided into four groups:Control group（control group）,model group（DC group）,SO₂ intervention group（DC+SO₂ group）and SO₂control group（SO₂ group）.The Control group and SO₂ group were fed with standard feed,DC group and DC+SO₂ group were fed with high-sugar and high-fat feed for 1 month.after 1 month,the Control group and SO₂ group were intraperitoneally injected with citric acid-sodium citrate buffer of equal volume.DC group and DC+SO₂ group received intraperitoneal injection of 1%streptozotocin at a time of 35mg/kg（fasting and drinking for 12 hours before injection）.Blood glucose was detected on the 3rd and 7th day after intraperitoneal injection of STZ.Random blood glucose of each rat was more than 16.7mmol/l,indicating successful modeling of type 2 diabetic rats.After successful modeling,the rats were fed with high-sugar and high-fat feed for 8 weeks.SO₂ group and DC+SO₂ group received intraperitoneal injection of 85 mg/kg of SO₂ donor sodium sulfite/sodium bisulfite every day for intervention.Control group and DC group received intraperitoneal injection of the same amount of sterile water every day.After 8 weeks,glucose tolerance test and 24-hour urine protein quantification of rats were carried out,The rats were weighed（BW）and anesthetized at 400mg/kg by intraperitoneal injection of 4%chloral hydrate and cardiac color Doppler ultrasound examination,serum urea nitrogen（BUN）,creatinine（Cr）,β2-microglobulin（β2-MG）,fasting insulin level and synchronous fasting blood glucose value were detected,insulin resistance index was calculated,rats were executed,hearts of rats were collected,cardiac weight（HW）was weighed,and cardiac weight index（HW/BW）was calculated.Masson staining was used to observe the degree of myocardial interstitial fibrosis,Tunel staining was used to observe the apoptosis of myocardial cells,Transmission electron microscope was used to observe the autophagy of myocardial cells and Western Blotting was used to detect the expression of myocardial tissue fibrosis-related proteins,cell autophagy,cell apoptosis-related proteins and TGFβ-Smad2/3 signal pathway.Results:1.The results of 24-hour urine protein quantity show that,Compared with Control group,urine protein quantity and microalbumin quantification were significantly increased（P<0.05）.Compared with DC group,urinary protein and microalbumin in DC+SO₂ group showed a decreasing trend,with no statistically significant difference（P>0.05）;2.glucose tolerance test:Compared with Control group,DC group indicated diabetic glucose tolerance;Compared with DC group,indicating diabetic glucose tolerance in both groups,but the blood glucose value in the DC+SO₂ group showed a downward trend（P>0.05）.3.blood urea nitrogen,creatinine,β2-microglobulin:urea nitrogen,creatinine,β2-microglobulin in DC group were significantly higher than those in Control group（P<0.05）.Compared with DC group,urea nitrogen,creatinine,β2microglobulin in DC+SO₂ group decreased significantly（P<0.05）.4.fasting blood glucose,insulin level and insulin resistance index value:fasting blood glucose,insulin level and HOMA-IR value in DC group was significantly higher than that in Control group（P<0.05）;Compared with the DC group,fasting blood glucose,insulin level and HOMA-IR value in the DC+SO₂ group decreased no significantly（P>0.05）.5.GOT2 protein expression in rats:compared with the Control group,GOT2 protein expression in the myocardium of the DC group was slightly decreased,with no statistically significant difference（P>0.05）.Compared with the DC group,the expression of GOT2 protein in rat myocardial tissue of the DC+SO₂ group was significantly increased（P<0.05）.6.BW,HW,BW/HW:Compared with the Control group,the body weight of the DC group decreased significantly（P<0.05）,while the heart mass index increased significantly（P<0.05）.Compared with the DC group,the weight of rats in the DC+SO₂ group increased significantly（P<0.05）,while the heart mass index decreased,but there was no significant difference between the two groups（P>0.05）.7.cardiac color Doppler ultrasound:Compared with the Control group,the LVFS value of the DC group showed a decreasing trend（P<0.05）.Compared with the DC group,the LVFS value of the DC+SO₂ group decreased significantly（P>0.05）.8.Masson staining:Collagen deposition in myocardial interstitium in DC group was significantly higher than that in Control group.Compared with DC group,myocardial fibrosis degree in DC+SO₂ group was significantly reduced.9.Tunel staining:The number of apoptotic cells in myocardial tissue of DC group was significantly higher than that of Control group.Compared with DC group,the number of apoptotic cardiomyocytes in DC+SO₂ group decreased significantly.10.The results of transmission electron microscopy showed that vacuolization of autophagosomes and disordered arrangement of muscle fibers in the DC group,Compared with the DC group,the rats in the DC+SO₂ group showed autophagosomes,with no significant vacuolization and relatively neat arrangement of muscle fibers.11.WB:1)fibrosis related protein expression:compared with Control group rats,DC group rats myocardial tissue MMP3,collagen I,collagen III expression was significantly up-regulated,TIMP2 expression was significantly down-regulated（P<0.05）;Compared with DC group,these above-mentioned changes were obviously reversed in DC+SO₂ group（P<0.05）.2)autophagy related protein expression:compared with Control group,the expressions of Beclin-1,LC3,Atg16L1,Atg4b,P62 in myocardial tissue of DC group were significantly down-regulated（P<0.05）.compared with DC group,the expressions of Beclin-1,LC3,Atg16L1,Atg4b,P62 in myocardial tissue of DC+SO₂ group were significantly up-regulated（P<0.05）.3)expression of apoptosis-related proteins:compared with Control group,the expressions of Caspase3 and Caspase6 proteins in myocardial tissue of DC group were significantly up-regulated（P<0.05）.Compared with DC group,the expressions of Caspase3 and Caspase6 in myocardium of DC+SO₂ group were significantly down-regulated（P<0.05）.4)expression of TGFβ-Smad2/3 signal pathway:compared with Control group,the expression of TGFβ,smad2,Smad3 protein in myocardium of DC group was significantly up-regulated（P<0.05）.Compared with DC group,the expression of TGFβ,Smad2 and Smad3 protein in myocardium of DC+SO₂ group was significantly decreased（P<0.05）.Conclusion:SO₂ can alleviate myocardial fibrosis in type 2 diabetic rats,and i t s m e c h a n i s m m a y b e r e l a t e d t o d o w n-r e g u l a t i n g t h e e x p r e s s i o n o f TGFβ-Smad2/3 signaling pathway to regulate autophagy thereby reduce apoptosis.