Intervention Effect And Mechanism Of Ophiopogonin D On Myocardial Injury In CYP2J Gene Knockout Rats Under Hypoxia Condition

Objective:The previous cell and animal experiments in this study showed that CYP2 J is a potential key target of Ophiopogonin D(OPD)in exerting its cardiovascular protective effect.It needs to be explored,which is also the scientific problem of this study.Combined with the previous findings that OPD can protect myocardial damage caused by hypoxia,and OPD can potently induce CYP2 J and have a direct interaction with it,but whether it can be verified at the whole animal level that the effect of OPD is directly related to the target of CYP2 J still needs to be explored.Therefore,this study aims to use CYP2 J gene knockout rats to simulate the myocardial injury model under hypoxic conditions,and compare the differences between wild type and gene knockout rats in the intervention of myocardial injury.The targets are directly related,which will help to further interpret the cardiovascular protective effect of the traditional Chinese medicine Ophiopogon japonicus and its important component OPD from the molecular target,clarify the new possible mechanism of the cardiovascular protective effect of the traditional Chinese medicine Ophiopogon japonicus,and screen more with CYP2J3 as the target Traditional Chinese medicine or natural active small molecules with cardiovascular protection.Methods:1.Study on the normoxic anti-fatigue effect of OPDThe mouse swimming fatigue model was constructed under normoxic conditions,and the administration method was as follows: mice were given OPD(20mg/kg,ig),OPD(10mg/kg,20mg/kg,40mg/kg,ig)for seven days,After the experiment,samples were taken to detect the anti-fatigue indicators of the mice(weight-bearing swimming time,CK,UREAL,liver glycogen,blood lactic acid).2.Study on the protective effect and mechanism of OPD on myocardial injury in simulated hypoxic wild-type rats and CYP2 J KO ratsSimulate hypoxia at an altitude of 6000 m for 72 hours to construct an acute hypobaric hypoxia stress model in rats.Two batches of rat experiments were conducted,with the first batch of OPD single dose prophylactic gavage administered at a concentration of OPD(20mg/kg,ig);The second batch of OPD multi dose prophylactic gavage was administered at a concentration of OPD(10mg/kg,20mg/kg,40mg/kg,ig),and a running load experiment was conducted in rats under hypoxic conditions to further investigate the anti fatigue effect under hypoxic conditions.After the experiment,the samples were collected to detect the blood routine(RBC),blood biochemical indicators(c Tn,MYO,CK,CKMB,AST,LDHI,HDLC,LDLC,etc.),oxidative stress level indicators(SOD,MDA,GSH),Western Blot was used to detect the protein expression of endoplasmic reticulum stress-related molecules HIF-1a,PERK,ATF4,ATF6,Bax,Bcl-2,and mitochondria-related molecules MTCO-1,SDHB,NDUFB8,UQCRC2,and q PCR was used to detect HIF-1a,ATF4,CHOP,and CYP2J3 gene expression,and then use immunohistochemistry to detect the expression of key molecules CYP2J3,CHOP,MTCO-1,SDHB,and use enzyme-linked immunosorbent assay to detect the activity of the mitochondrial respiratory chain enzyme complex and the production of ATP Finally,HE staining and Sirius red staining were used to analyze the pathological changes of myocardial tissue.3.Research on the mechanism of action of OPD based on metabolomics technology Further,through the non-targeted metabolomics technology,by comparing the differences in metabolites between wild-type and gene knockout rats and different dosages,the possible mechanism of CYP2 J gene action and OPD intervention under hypobaric and hypoxic conditions and Key metabolites and metabolic pathways.Results:1.The anti-fatigue experiment showed that OPD intervention could significantly improve the weight-bearing swimming time of the mice under normal oxygen condition,improve their blood biochemical indexes(CK and TRIGL),increase the liver glycogen reserve of non-weight-bearing swimming mice,and reduce the accumulation of blood lactic acid of non-weight-bearing swimming mice,indicating that OPD could improve the anti-fatigue ability of mice2.The experiment of simulating the myocardial injury caused by rapidly advancing to the plateau showed that OPD intervention can significantly increase the running displacement of hypoxic wild-type rats,increase the number of red blood cells,reduce the levels of myocardial injury markers and key myocardial enzymes in rat serum,and reduce the myocardial damage.Tissue oxidative stress level,increase the activity of mitochondrial respiratory chain enzyme complex,and can inhibit the expression of endoplasmic reticulum stress-related molecules,apoptosis protein and HIF-1a,increase the expression of mitochondria-related molecules,increase ATP content,pathology The results showed that OPD intervention could significantly reverse the myocardial injury in wild-type rats.However,the protective effect of OPD on CYP2 J KO rats was significantly weakened,which was reflected in the effect of OPD on the myocardial enzyme spectrum,the activity of mitochondrial respiratory chain enzyme complexes,and the expression of mitochondria-related molecules in rats,and the pathological results showed that OPD had a greater effect on CYP2 J KO rats.Mice have a partial protective effect,suggesting that OPD partly exerts cardioprotective effect through CYP2 J.3.The results of the metabolic group showed that there were significant differences between CYP2 J KO rats and wild-type rats in the pathways of amino acid metabolism and unsaturated fatty acid synthesis,suggesting that myocardial damage in CYP2 J KO rats was more serious than that of wild-type rats.After OPD intervention,the differential metabolites of hypoxic wild-type rats were significantly more than those of hypoxic CYP2 J KO rats,and the metabolite callback was better than that of CYP2 J KO rats,suggesting that CYP2 J targets play an important role in the myocardial protection of OPD.Conclusion:OPD intervention can significantly improve the myocardial injury caused by wild-type rats rushing to plateau,and the protective effect of OPD on myocardial injury in CYP2 J KO rats is weaker than that of wild-type rats.Therefore,by comparing the wild type and the knockout type,it can be seen that the initiation link may be closely related to the CYP2 J target,and its molecular events are related to the inhibition of myocardial oxidative stress and endoplasmic reticulum stress,the regulation of the activity of mitochondrial respiratory chain complex,and the maintenance of the expression level of related proteins.Metabolomics results showed that the protective effects of OPD intervention on myocardial injury in wild-type anoxic rats were mainly related to amino acid metabolism,lipid metabolism,purine metabolism,pentose phosphate pathway,etc,but had little effect on CYP2 J KO rats.It is suggested that CYP2 J target plays a key role in myocardial protection of OPD.