| Basing on the cardiovascular protective function of Shenmai injecta,Ophiopogonin D(OPD),as a steroidal glycoside isolated from Radix ophiopogonis,was focused on to exploring its pharmacological effect to alleviating the cardiac hypertrophy.According to the researches,OPD has ability of restoring the total anti-oxidant and anti-apoptotic capability,and inhibiting the release of inflammatory factors,but the pharmacological effect on hypertrophy is unknown.Cardiac hypertrophy is the major risk factors in cardiovascular diseases.We treated Ang II in vitro and infused Ang II and constricted the renal artery of Sprague Dawley rats to induce cardiac hypertrophy.The administration of Ang II resulted in a significant increase in the level of the NF-?B,which is a critical signaling molecule that is produced in response to a variety of stimuli and is required for the hypertrophic response of neonatal rat cardiomyocytes invitro.DNA-binding of NF-?B dimers facilitates the activation of gene transcription,such as inflammation and apoptosis.NF-?B binds with consensus sequences and subsequently stimulates the expression of various inflammatory genes.CYP2J3 is the predominant AA epoxygenase that metabolizes AA to EETs and are highly expressed in cardiomyocytes and vascular endothelial cells and plays a cardioprotective role in cardiac hypertrophy.In this study we investigated whether OPD is protective against inflammation to relieve cardiac hypertrophy via inhibiting the expression of NF-?B and examined whether CYP2J3 was involved in cardioprotective effects of OPD.AngII(10-6 mol/L)treated to induce the model of hypertrophy in vitro.Cardiomyocytes treated with different concentrations of OPD.The total protein content was detected by BCA method;Quantitative real-time PCR(qRT-PCR)technique was used to examine the expression of marker gene BNP and ?-MHC mRNA,represented the function of hear;Utilizing Western blot method to detect the expression of autophagy protein LC3 B and using high-throughput screening technology to corroborate it.In addition,we also examined the changes of mitochondrial membrane potential and the expression of F-actin in H9c2 myocardial cell.Overexpression and siRNA of CYP2J3 to detected the effect of CYP2J3 on the protective action of OPD in inflammatory pathway.On the overall level of animal,we created the hypertrophy by AngII and RAS,and utilized transthoracic echocardiography and histological analysis to identify the emergency of inflammation and hypertrophy.The expression of ET-1 was detected by ELISA.All analyses above purposed to determine whether OPD has a positive effect on inhibiting the NF-?B,inflammation and hypertrophy.The results of the study showed that high concentrations of OPD(50-100?M)can obviously inhibit the cell activity.In vitro,OPD cansignificantly reversed those pathological hypertrophical changes of cardiomyocytes induced by AngII.Such as: the growth level of specific hypertrophic gene mRNA expression,the remarkable increase of the total protein expression,the enhancement of autophagy expression,the reduction of mitochondrial membrane potential and so on.Our findings support that the overexpression of CYP2J3 results in the decreasing expression of NF-?B,and further evidenced CYP2J3 participates in the process of protective effect of OPD on cardiomyocytes.However,downregulation of CYP2J3 did not change the effect of OPD on blocking NF-?B or alleviating hypertrophy.In vivo,concentric hypertrophy induced by AngII and RAS can be relieied by OPD.Taken together,inflammation and NF-?B represent key events in cardiac hypertrophy.Our in vivo data serve as the first evidence that OPD prevents the pathogenesis and progression of cardiac hypertrophy,most likely by inhibiting the expression of NF-?B and inflammation in the heart.OPD exerts its effect also partially via upregulating the level of CYP2J3.Our finding that OPD imparts a positive effect on alleviating cardiac hypertrophy may be potentially applied to the development of shenmai injections. |
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