LncRNA MHRT Protects Cardiac Cell Damage Induced By Angiotensin Ⅱ

Background and objective: Pathological activation of the renin-angiotensin system(RAS)is a key pathogenic factor in various cardiovascular diseases.Angiotensin II(Ang II)is the main effector peptide of RAS,and is involved in the occurrence and development of various cardiomyopathy,and oxidative stress and inflammatory response are the main pathogenic factors.Nrf2 is an important regulator of maintaining redox balance in the body.By combining with the antioxidant response element(ARE),Nrf2 up-regulates the expression of multiple phase II detoxifying enzymes,antioxidant genes and cytoprotective proteins to resist oxidative stress damage and clear excess generated ROS.Studies have shown that NLRP3 inflammatory bodies can be activated by ROS and that blockling the activation of NLRP3 inflammatory bodies,can inhibit the occurrence of myocardial inflammation,myocardial fibrosis and cardiac remodeling caused by Ang II.The Nrf2 agonist resveratrol protects myocardial damage caused by chronic intermittent hypoxia by blocking the activation of NLRP3 inflammatory bodies.It can be seen that Nrf2 is at the center of myocardial oxidative stress and NLRP3-mediated inflammatory response.The expression and function of Nrf2 are regulated by many factors,including the kinase pathways such as AMPK,PI3 K,and Lnc RNA.Lnc RNA is a type of non-coding RNA with a length of more than 200 nt.Among them,MHRT is an lnc RNA produced by the gene encoding myosin heavy chain 7 through variable splicing.It is highly conserved in humans and mice.Studies have shown that MHRT can inhibit the occurrence of myocardial hypertrophy through Brg1;MHRT can up-regulate Nrf2 when it protects adriamycin-induced cardiomyocyte apoptosis.Combined with our previous experiments,we found that in the late stage of myocardial injury caused by Ang II,the expression and activity of Nrf2 were down-regulated,and accompanied by the decrease of MHRT expression level,suggesting that MHRT may be involved in the regulation of Ang II-induced myocardial oxidative damage and activation of NLRP3 inflammatory bodies and it is a potential target for preventing myocardial damage caused by Ang II.Therefore,We investigated the role of MHRT in regulating Ang II-induced myocardial injury and inflammatory response in vivo and in vitro experiments,overexpression of MHRT may inhibit the activation of NLRP3 inflammatory bodies and oxidative stress in cardiomyocytes by activating Nrf2,thereby inhibiting Ang IIinduced cardiomyocyte injury,to provide molecular targets for early diagnosis and treatment of Ang II-related myocardial injury and prevention of cardiomyopathy in the clinicMethods: In vivo experiments,refer to the method reported by the research group to establish an Ang II-induced chronic myocardial injury model[23].8-week-old male C57 / BL mice were subcutaneously injected with Ang II(0.5mg / kg)every other day for 2 consecutive months(2M),and the observation was stopped until 6 months(6M);the control group was given the same dose of physiological saline,and cardiac function was detected by echocardiography at 2 months(2M),4 months(4M),and 6 months(6M).CODA Monitor was used to detect changes in blood pressure in mice;cardiac tissue was collected,and relevant indicators were detected using q RT-PCR and Western Blot methods,including: oxidative damage indicators(3-NT,4HNE),NLRP3 inflammation body-related indicators(NLRP3,caspase-1,IL-1β),Lnc RNA MHRT and Nrf2.In vitro experiments,lentiviral transfection was used to overexpress MHRT in AC16 cardiomyocytes,and 100 nmol / L Ang II was given to the cells for 24 h.The cells were collected and tested as follows:(1)q RT-PCR and Western Blot were used to detect the oxidative damage of AC16 cardiomyocytes and the activation of NLRP3 inflammatory bodies;(2)FISH test to detect the localization of MHRT in cell;(3)Flow cytometry to detect intracellular ROS content;(4)q RT-PCR test to detect the expression of Nrf2 and its downstream antioxidant genes CAT,HO-1;(5)Coimmunoprecipitation,q RT-PCR and Western Blot experiments were used to detect the expression of TXNIP m RNA and protein,and the binding of TXNIP and NLRP3.Results:In vivo experiments found that long-term low-dose Ang II stimulation can cause ventricular dilatation and reduce cardiac function in mice;the occurrence of myocardial oxidative damage(3-NT,4-HNE),the activation of NLRP3 inflammatory bodies(NLRP3,caspase-1,IL-1β),accompanied by the down-regulation of Nrf2 expression and activity,and the decreased expression levels of MHRT,indicating that Ang IIinduced myocardial damage is closely related to the down-regulation of Nrf2 activity and down-regulation of MHRT expression.In vitro experiments found that MHRT is distributed in the nucleus and cytoplasm of AC16 cardiomyocytes.Overexpression of MHRT can inhibit the oxidative damage of AC16 cardiomyocytes(3-NT,4HNE)induced by Ang II stimulation,and the expression of NLRP3 inflammatory body-associated marker genes(NLRP3,caspase-1,IL-1β);and overexpression of MHRT up-regulates the expression of Nrf2 and its downstream antioxidant genes CAT,HO-1 m RNA in AC16 cardiomyocytes;inhibiting the production of ROS induced by Ang II stimulation;at the m RNA and protein levels,the expression of TXNIP and the direct binding between TXNIP and NLRP3 protein were inhibited.Conclusion: Chronic Ang II stimulation activates myocardial NLRP3 inflammatory bodies and myocardial oxidative damage,leading to myocardial hypertrophy and cardiac dysfunction.However,overexpression of MHRT may inhibit Ang II induced oxidative damage of AC16 cardiomyocytes and activation of NLRP3 inflammatory bodies by activating Nrf2,thus preventing Ang II induced cardiomyocyte damage.