The Protective Mechanism Of Carbon Monoxide Regulating IL-33/ST2L Axis In Diabetic Cardiomyopathy

Diabetic cardiomyopathy（DCM）is one of the most common complications of diabetes mellitus,and about three-quarters of diabetic patients die of DCM-induced cardiovascular events.The DCM has a complex and long course,and the pathogenesis has not been unraveled fully so far.The chronic and low-grade inflammatory response induces the inflammatory death-pyroptosis of cardiomyocytes and fibrosis.In DCM,the pyroptosis of cardiomyocytes leads to impaired cardiac contraction and relaxation,myocardial fibrosis stiffens the heart and reduces its compliance,while pyroptosis of cardiomyocytes and cardiac fibrosis promote each other,resulting in progressive impairment of cardiac function.Thus the chronic inflammation is considered to be the key pathophysiological factor in the development of DCM.Further elucidation of inflammatory mechanisms in DCM and exploration of effective interventions are urgent.The inflammatory cytokine interleukin 33（IL-33）is identified as an IL-1 family cytokine in 2005.Tumor suppressor protein 2（ST2）is the receptor of IL-33,which mainly includes two forms:soluble ST2（s ST2）and transmembrane ST2（ST2L）.The binding of IL-33 and ST2L on the cell membrane surface is known as the IL-33/ST2L axis,which can cause alterations of different intracellular signaling pathways such as intracellular proliferation,pyroptosis and inflammation.Studies have reported that the IL-33/ST2L axis could exert cardioprotective effects by improving pressure-overload cardiomyocyte hypertrophy and reducing the inflammatory response.In addition,IL-33/ST2L axis knockout promoted ischemia-reperfusion-induced heart failure.Nevertheless,there are still many opposite conclusions that the IL-33/ST2L axis was upregulated in cardiac infection and myocardial infarction and induced cardiomyocyte death,cardiac fibrosis and impaired cardiac function by promoting inflammatory response.It is one-sided if simply believed that the IL-33/ST2L axis plays a protective or damaging role in cardiac disease and how it is involved in the progression of DCM is not fully understood,which urgently needs to be elucidated by further studies.Gas messenger molecules carbon monoxide（CO）have good protective effects in a variety of diseases,especially cardiovascular diseases.Inducing endogenous CO or using exogenous CO-releasing molecules（CORMs）with good fitting effects can reduce myocardial injury caused by hypertensive cardiomyopathy,heart transplantation and myocardial infarction,and the mechanism is related to the inhibition of apoptosis,anxiety or necrosis,and the block of fibrosis.More importantly,recent studies found that induction of endogenous CO can reduce inflammatory response,alleviate lipopolysaccharide-induced lung injury,and reduce rheumatoid arthritis fibrosis by regulating IL-33.So,does CO also play protective role in DCM through anti-pyroptosis and anti-fibrosis?What’s the role of IL-33/ST2L axis in the process of CO attenuating myocardial injury of DCM?These issues still need further study and disclosure.Therefore,in this study,C57BL/6J mice with wild-type（WT）,IL-33^-/-,and ST2^-/-were used to establish DCM models,and HL-1 cardiomyocytes and mouse primary cardiac fibroblasts were treated with high glucose.Using CO releasing molecule 2（CORM-2）to intervene the DCM mice and high glucose treated cells to reveal the role of the IL-33/ST2L axis in DCM and the protective mechanism of CORM-2.Specifically,the study consists of the following three sections:Section 1:Diabetic cardiomyopathy and carbon monoxide intervention:preliminary exploration of the role of IL-33/ST2L axisObjective:The DCM model was established in the mice of WT,IL-33^-/-and ST2^-/-,and CORM-2 intervention was given to investigate the role of IL-33/ST2L axis in DCM and the intervention effect of CORM-2 at the overall animal level.Methods:1.Male C57BL/6J mice aged 7～8 weeks were fed with normal diet or high-fat diet（60%fat-derived calories）for 8 months.The high-fat diet mice were continuously intraperitoneally injected with streptozotocin（STZ,50 mg/kg·bw）in the middle stage（4 months）.One week later,mice with fasting plasma glucose（FPG）level≥16.9 mmol/L were intraperitoneally injected with CORM-2 or ineffective CORM-2（i CORM-2）for intervention.After 8 months,the cardiac function of mice in each group was measured by ultrasound,and serum samples were collected to determine the blood glucose and creatine kinase MB isoenzyme（CK-MB）levels of mice.The ratio of heart to tibia length was calculated.The heart histopathological changes were observed by H&E,Sirius Red staining and transmission electron microscopy.The levels of IL-33 and ST2 were detected by ELISA,immunohistochemistry and Western blot,and the co-localization of myocardial fibroblasts and IL-33 in myocardial tissue was detected by immunofluorescence.Spontaneously diabetic animals of ob/ob mice and ZDF rats were used to verify IL-33 and ST2L protein expression in myocardial tissue.Further,IL-33^-/-and ST2^-/-mice were used to establish DCM models according to the above methods and given CORM-2 intervention to compare cardiac histopathological and cardiac function changes.Results:1.1 Compared with WT mice,DCM mice had increased blood glucose and the CK-MB level ratio of heart weight to tibia length and（P<0.05）;disorganized myocardial tissue structure,increased collagen deposition,swelling mitochondrial structure and broken myofilament;decreased cardiac ejection fraction and fractional shortening（P<0.05）.CORM-2 intervention in DCM mice had no effect on blood glucose and the ratio of heart weight to tibia length but serum CK-MB,cardiac histopathology and cardiac function parameters were significantly improved（P<0.05）.However,the i CORM-2 intervention had no significant effect on DCM mice.1.2 Compared with WT mice,IL-33 and s ST2 levels in the serum of DCM mice increased by 21.9%and 22.6%,respectively（P<0.05）,and the protein expression of IL-33 and ST2L in myocardial tissue increased by 118.2%and 116.8%,respectively（P<0.05）;the protein expression of IL-33 and ST2L in myocardial tissue of ob/ob mice and ZDF rats was also significantly increased compared with its control group（P<0.05）.After CORM-2 intervention in DCM mice,IL-33 and s ST2 levels in serum and IL-33 and ST2L protein expression in the heart were decreased（P<0.05）.However,the i CORM-2intervention had no significant effect on IL-33 and ST2L expression in DCM mice.1.3 Compared with DCM mice,DCM^IL-33-/-and DCM^ST2-/-mice had decreased survival rate,increased CK-MB levels（P<0.05）,deteriorative cardiac histopathology and cardiac function parameters（P<0.05）.However,cardiac collagen deposition was reduced.1.4 Unlike the intervention in DCM mice,CORM-2 intervention in DCM^IL-33-/-and DCM^ST2-/-mice showed no improvement in serum CK-MB levels,myocardial histopathology and cardiac function parameters.Summary:The upregulation of IL-33/ST2L axis directly affected the development of DCM,but its absence also aggravated DCM myocardial injury;carbon monoxide intervention may alleviate DCM lesions by maintaining IL-33/ST2L axis homeostasis.Section 2:The regulation of IL-33/ST2L axis on cardiomyocyte pyroptosis in diabetic cardiomyopathy and the intervention of carbon monoxideObjective:Investigate the role of IL-33/ST2L axis in cardiomyocyte pyroptosis in DCM and the intervention effect of CORM-2.Methods:The animal treatment and grouping were the same as in the first section.The expression of pyroptosis-related proteins in myocardial tissue was detected by Western blot.In the high glucose incubated HL-1 cardiomyocytes,lactate dehydrogenase（LDH）release and CCK-8 were used to assess the damage of HL-1 cardiomyocytes and the protein expression of IL-33 and ST2L was detected by Western blot;recombinant IL-33（r IL-33）and IL-33 neutralizing antibody（αIL-33）were added to respectively to detect cardiomyocyte injury and the expression of pyroptosis related proteins;IL-33 si RNA was further used to reduce IL-33,and cardiomyocyte pyroptosis-related protein expression was measured after high glucose treatment.CORM-2（100 n M）was added to high glucose incubated HL-1 cardiomyocytes,and the cell injury and the expression of pyroptosis-related proteins,IL-33 and ST2L were detected.Results:2.1 Compared with the WT mice,the expression of pyroptosis-related proteins NLRP3,GSDMD-N,cleaved caspase-1 and cleaved IL-1βincreased in myocardial tissue of DCM mice（P<0.05）.Compared with DCM mice,the expression of pyroptosis-related proteins increased further in DCM^IL-33-/-and DCM^ST2-/-mice（P<0.05）.2.2 After CORM-2 intervention in DCM mice,the expression of pyroptosis-related proteins was decreased（P<0.05）.Unlike the intervention in DCM mice,CORM-2intervention in DCM^IL-33-/-and DCM^ST2-/-mice showed no effect on the expression of pyroptosis-related proteins.2.3 Compared with the control group（5.5 m M glucose）,LDH release was increased（P<0.05）,cell activity was decreased（P<0.05）and the expression of pyroptosis related proteins,IL-33 and ST2L was increased（P<0.05）in HL-1 cardiomyocytes treated with high glucose（33 m M,HG）for 48 h.2.4 In the HG incubated HL-1 cells,r IL-33（1–10 ng/m L）treatment increased the expression of pyroptosis-related proteins（P<0.05）.2.5 In the HG incubated HL-1 cells,1 ng/m LαIL-33 treatment showed no effect on HL-1 cardiomyocytes;10 ng/m LαIL-33 treatment decreased the LDH release（P<0.05）,improved the activity of cells（P<0.05）and decreased the expression of GSDMD-N and cleaved caspase-1（P<0.05）;100 ng/m LαIL-33 treatment increased the expression of cleaved caspase-1（P<0.05）;1000 ng/m LαIL-33 treatment increased the LDH release,decreased the activity of the cells and increased the expression of NLRP3,GSDMD-N and cleaved caspase-1（P<0.05）.2.6 HL-1 cardiomyocytes treated with different IL-33 si RNA and then incubated with high glucose,compared with the HG group,when the interference efficiency of IL-33 si RNA was about 90%,the expression of pyroptosis related proteins increased（P<0.05）;when the interference efficiency of IL-33 si RNA was about 70%,the expression of pyroptosis related protein showed no change.2.7 In the HG incubated HL-1 cells,the protein expression of GSDMD-N,cleaved IL-1β,IL-33 and ST2L were decreased（P<0.05）and cell injury was alleviated（P<0.05）after CORM-2（100 n M）intervention for 48 h.Summary:Both upregulation or deletion of IL-33/ST2L axis promoted cardiomyocyte pyroptosis in DCM;carbon monoxide reduced cardiomyocyte pyroptosis and alleviated DCM lesions by maintaining IL-33/ST2L axis homeostasis.Section 3:The mediating of IL-33/ST2L axis on activation of cardiac fibroblasts and the intervention of carbon monoxide in diabetic cardiomyopathyObjective:Investigate the role of IL-33/ST2L axis in the activation of myocardial fibroblasts and the intervention effect of CORM-2 in DCM.Methods:The animal treatment and grouping were the same as in the first section.Western blot was used to detect the expression of fibrosis-related proteins in myocardial tissue and immunofluorescence was used to determine the co-staining of IL-33 and fibroblasts.In the high glucose incubated WT mouse primary cardiac fibroblasts,α-SMA immunofluorescence and Ed U staining were used to detect the activation of fibroblasts,and immunofluorescence was used to detect IL-33 and ST2L expression;r IL-33 andαIL-33 were added respectively,andα-SMA immunofluorescence,Ed U staining,collagen I and CTGF m RNA levels were used to evaluate the degree of fibroblast activation.Added the r IL-33 to L-33^-/-and ST2^-/-fibroblasts incubated with high glucose to detect the degree of fibroblast activation.In addition,CORM-2 was added to the WT fibroblasts incubated with high glucose,the degree of fibroblasts activation and IL-33 and ST2L expression were detected.Results:3.1 Compared with WT mice,the expression of fibrosis-related proteinsα-SMA,Collagen I,TGF-βand CTGF were increased in myocardial tissue of DCM mice（P<0.05）.Compared with DCM mice,DCM^IL-33-/-and DCM^ST2-/-mice had decreased expression of fibrosis-related proteins（P<0.05）.3.2 After CORM-2 intervention in DCM mice,the expression of fibrosis-related proteins was decreased（P<0.05）.Unlike the intervention in DCM mice,CORM-2intervention in DCM^IL-33-/-and DCM^ST2-/-mice showed no effect on the expression of fibrosis-related proteins.3.3 Compared with the control group（5.5 m M glucose）,α-SMA expression and Ed U positive cells were increased（P<0.05）and the protein expression of IL-33 and ST2L was also increased（P<0.05）in the cardiac fibroblasts treated with high glucose（HG）.3.4 In the HG incubated fibroblasts,r IL-33（10 ng/m L）treatment increasedα-SMA expression,the Ed U positive cells（P<0.05）and the m RNA levels of collagen I and CTGF（P<0.05）;αIL-33（1000 ng/m L）treatment alleviated the activation of fibroblasts（P<0.05）.In addition,under the HG incubated condition,compared with the WT fibroblasts,the activation of IL-33^-/-and ST2^-/-fibroblast was decreased（P<0.05）,and r IL-33（10 ng/m L）treatment increased the activation of IL-33^-/-fibroblast（P<0.05）while had no effect on ST2^-/-fibroblast.3.5 In the HG incubated WT fibroblasts,CORM-2（30 n M）treatment alleviated the activation of fibroblasts and decreased the fluorescence expression of IL-33 and ST2L（P<0.05）.Summary:The IL-33/ST2L axis directly induced cardiac fibroblast activation;carbon monoxide reduced cardiac fibroblast activation and alleviated DCM fibrosis by inhibiting the IL-33/ST2L axis.Conclusions:Under the pathological conditions of DCM,the upregulation of IL-33/ST2L axis induced cardiomyocyte pyroptosis and promoted myocardial fibroblast activation;and the deletion of IL-33/ST2L axis also promoted cardiomyocyte pyroptosis and mediated cardiac dysfunction.Carbon monoxide mitigated cardiomyocyte pyroptosis,inhibited myocardial fibroblast activation and alleviated DCM lesions by maintaining IL-33/ST2L axis homeostasis.