The Study Of Notopterol Alleviate Cardiac Dysfunction By Suppressing Cardiomyocyte Pyroptosis In Hyperuricemic Mice

Objective:Hyperuricemia is a prevalent metabolic disorder and is closely related to cardiovascular diseases.Pyroptosis and inflammation play vital roles in cardiac damage of hyperuricemic mice.Notopterol is a naturally occurring furanocoumarin compound found in the root of Notopterygium incisum and has been reported to exhibit anti-proliferative and anti-inflammatory properties.However,whether notopterol has cardioprotective potential in hyperuricemic mice and the possible molecular mechanism remain elusive.This study will investigate the effect of notopterol on cardiac dysfunction in hyperuricemic mice and explore its potential mechanism.Methods:Part 1:Western blot was applied to detect P2X7R,NLRP3,Cleaved caspasel,IL-1β,IL-18 expression levels in H9c2 cells induced by uric acid.To investigate the role of P2X7R in uric acid-stimulated pyroptosis and inflammation in H9c2 cells,the selective P2X7R inhibitor Brilliant blue G-250 was used to induce H9c2 cells.Moreover,P2X7R siRNA was applied to knock down P2X7R in H9c2 cells treatment with uric acid and then the pyroptosis and inflammation-related protein levels were examined by Western blot.The cytotoxicity of notopterol was detected by CCK8 assay.Western blot,RT-PCR or immunofluorescence were utilized to verify the effect of uric acid and notopterol on pyroptosis and inflammation cytokine production.P2X7R overexpression plasmid was transferred to H9c2 cells to overexpress P2X7R and pyroptosis and inflammation related protein levels were evaluated by Western blot.Part 2:The hyperuricemic mouse model was constructed by administration of potassium oxonate and adenine every other day.Notopterol(20 mg/kg/day)was orally administrated daily after 2 weeks of hyperuricemia induction,allopurinol(10 mg/kg/day)was applied as a positive control.The serum uric acid concentration was detected after 1 week and 6 weeks of hyperuricemia induction.Cardiac function and exercise capacity were examined by echocardiography and treadmill fatigue test.Immunohistochemistry,RT-PCR and Western blot were applied to evaluate P2X7R,pyroptosis and inflammation related protein expression levels in heart tissues.RNA-seq and JASPAR website were used to screen the possible transcription factor of P2X7R.Western blot was used to detect the effect of notopterol and uric acid on SOX4 expression levels.SOX4 siRNA was applied to knock down SOX4 in H9c2 cells then treatment with uric acid and P2X7R and pyroptosis-related protein expression levels were examined by Western blot.Results:Part 1:P2X7R,NLRP3,Cleaved caspase1,IL-1β and IL-18 expression levels were upregulated in uric acid-stimulated H9c2 cells.Additionally,it was verified that inhibition or knockdown P2X7R both alleviated expression levels of NLRP3,Cleaved caspase-1,IL1β and IL-18 in uric acid-treated H9c2 cells.Notopterol administration increased cell viability in H9c2 cells stimulated by uric acid.Moreover,notopterol treatment significantly suppressed expression levels of NLRP3,Cleaved caspasel,IL-1β and IL-18 in vitro.Meanwhile,P2X7R expression level induced by uric acid was suppressed by notopterol.Mechanistically,P2X7R overexpression abolished the inhibition effect of notopterol on pyroptosis and inflammation.Part 2:Hyperuricemia dampened heart function and reduced exercise capacity of mice.Allopurinol treatment significantly reduced serum uric acid level.Notopterol administration had no significant effects on serum uric acid levels.Allopurinol treatment increased running time and running distance of hyperuricemic mice but not significant.Notopterol treatment improved exercise capacity of hyperuricemic mice significantly.Both allopurinol and notopterol treatment alleviated cardiac dysfunction in hyperuricemic mice.The DNA motif of SOX4 and its binding site on P2X7R promoter were obtained from JASPAR website.RNA-seq and Western blot showed that the expression of transcription factor SOX4 increased in the heart tissue of hyperuricemic mice.Notopterol inhibited SOX4 expression level induced by uric acid in vivo and in vitro.SOX4 knockdown suppressed P2X7R expression level and pyroptosis signal in uric acid treated H9c2 cells.Conclusions:Collectively,our findings suggested that P2X7R played a critical role in uric acid-induced pyroptosis and inflammation cytokine production in H9c2 cells.Notopterol inhibited NLRP3,Cleaved caspasel,IL-1β and IL-18 expression via inhibiting the P2X7R signal under uric acid stimulation.Notopterol alleviated cardiac pyroptosis,cardiac inflammatory response,exercise intolerance and cardiac dysfunction induced by hyperuricemia.Moreover,we found that notopterol could inhibit pyroptosis partly via suppressing SOX4/P2X7R in H9c2 cells.Notopterol represent a potential therapeutic strategy against pyroptosis and improve cardiac function in hyperuricemic mice.