The Cardioprotective Effects And Underlying Mechanisms Of 6-gingerol Based On TLR4/MAPKs/NF-?B Signaling Pathway And Calcium Channel

Oxidative stress,immune inflammatory reaction,apoptosis and calcium ion overload are the main biological events that induce myocardial damage.The secretion of these cytokines is regulated by toll-like receptor-4?TLR4?/NF-?B signaling pathway activity,and the MAPKs signaling pathway is activated,aggravating inflammatory responses,inducing apoptosis,and causing cell necrosis.There is ample evidence that excess Ca²⁺can produce pathological changes in cardiac tissue,such as increased contractility,hypertrophy,and apoptosis.Ca²⁺mainly enters through L-type Ca²⁺channels?LTCCs?,which are essential to cardiac excitability and excitation-contraction coupling.LTCC blockers generally protect against myocardial ischemic injury via the inhibition of calcium channels.Hence,drugs that can weaken the L-type Ca²⁺current(I_Ca-L)are promising for myocardial protection.The major bioactive constituent of ginger is 6-gingerol?6-Gin?.6-Gin has diverse and interesting pharmacological effects,including antipyretic,anti-inflammatory,anti-angiogenic,anti-cancer,cardiotonic,and anti-aging effects.However,the protective effects and underlying mechanisms 6-Gin remain largely unexplored.In this study,the myocardial protective effect and the mechanism of 6-Gin based on the TLR4/MAPKs/NF-?B signaling pathway and calcium channel were observed.By this approach,we investigate the protective effect and mechanism of 6-Gin against ISO-induced myocardial fibrosis?MF?in mice,the protective effect and mechanism of 6-Gin against CoCl₂-induced hypoxia in H9c2 cells,and the effects of 6-Gin on I_Ca-L,contractility,and Ca²⁺transients in isolated rat ventricular myocytes.Part 1 Protective effect and mechanism of 6-Gin against myocardial fibrosis in miceObjective:To determine the cardioprotective effects and possible underlying mechanisms of 6-Gin on myocardial fibrosis mice.Methods:Sixty male KM mice were randomly assigned into five groups?n=10 per group?:control group?Con?,ISO alone group?ISO?,low-dose6-Gin group?L-6-Gin?,high-dose 6-Gin group?H-6-Gin?,and propranolol group?Pro?.Animals in the Con group were gavaged and injected subcutaneously with normal saline.Animals in the ISO group were gavaged with saline and injected subcutaneously with ISO?10 mg/kg/day?.Mice in L-6-Gin and H-6-Gin groups were gavaged with 6-Gin?10,20 mg/kg/day?and injected subcutaneously with ISO?10 mg/kg/day?.Mice in Pro group were injected intraperitoneally with Pro?40 mg/kg/day?and injected subcutaneously with ISO?10 mg/kg/day?.This experimental procedure was carried out for 14 days in all groups.Colorimetric method,immunohistochemistry,TUNEL staining,and Western blot were used to observe the effects of 6-Gin on oxidative stress,inflammation,apoptosis and TLR4/MAPKs/NF-?B signaling pathway.Results:1.6-Gin treatment significantly decreased the J-point and heart rate in ISO-induced MF rats.2.6-Gin treatment significantly decreased CWI and LVWI in ISO-induced MF rats.3.6-Gin treatment significantly decreased levels of CK and LDH in ISO-induced MF rats.4.6-Gin improved ISO-induced morphological pathologies.5.6-Gin significantly decreased levels of calcium and MDA and increased levels of SOD,CAT,and GSH.6.6-Gin significantly reduced the expression of TNF-a,IL-6,c-fos,and c-jun.7.6-Gin significantly reduced expression of Bax and Caspase-3,increased the expression of Bcl-2 and reduced the rate of apoptosis.8.6-Gin significantly reduced expression of TLR4,p38,p-p38,ERK1/2,p-ERK1/2,JNK,p-JNK,NF-?B.Part 2 Protective effect and mechanism of 6-Gin against hypoxia in H9c2cellsObjective:To determine the protective effects and possible underlying mechanisms of 6-Gin on CoCl₂-induced hypoxia in H9c2 cells.Methods:H9c2 cells were assigned into four groups:control group?Con?,CoCl₂-induced hypoxia group?CoCl₂,400?M?,CoCl₂+low-dose6-Gin group?L-6-Gin,20?M?,and CoCl₂+high-dose 6-Gin group?H-6-Gin,40?M?.Colorimetric method,Elisa,Flow cytometry,and Western blot were used to observe the protective effects of 6-Gin on CoCl₂-induced hypoxia in H9c2 cells.Results:1.The cell viability of H9c2 cells were significantly decreased after treatment of CoCl₂?200,300,400,500,600?M?for 22 hours.The concentration of CoCl₂?400?M?was selected to induce hypoxia injury in the H9c2 cells.2.The activity of H9c2 cells was not affected by 6-Gin of 10,20,30,40,50,and 60?M?13?Concentration of 6-Gin?20 and 40?M?were selected for the prevention in the H9c2 cells.3.Levels of CK and LDH were significantly decreased after treatment of6-Gin.4.Levels of ROS,calcium,and MDA were decreased,while levels of SOD,CAT,and GSH were increased after treatment of 6-Gin.5.Levels of TNF-aand IL-6 were significantly decreased after treatment of 6-Gin.6.The rate of apoptosis was significantly decreased after treatment of6-Gin.7.The expression of p38 and NF-?B were significantly decreased,while the expression of Nrf2,HIF-1a,and HO-1 were significantly increased after treatment of 6-Gin.Part 3 Effects of 6-Gin on I_Ca-L,contractility,and Ca²⁺transients in isolated rat ventricular myocytesObjective:To investigate the effects of 6-Gin on I_Ca-L,contractility,and Ca²⁺transients of rat cardiomyocytes.Methods:The whole patch-clamp technique and the Ion Optix system were used to investigate the effects of 6-Gin on I_Ca-L,contractility,and the Ca²⁺transients of rat cardiomyocytes.Results:1.The 6-Gin?300?M?decreased the I_Ca-La-L of normal and ischemic ventricular myocytes by 58.17±1.05%and 55.22±1.34%respectively.2.The inhibition rates of 6-Gin at 3,10,30,100 and 300?M were 8.71±0.60%,16.2±0.80%,32.67±0.76%,54.33±1.89%,and 58.17±1.04%respectively3.The I-V relationship curve was up-regulated after treatment of 6-Gin?3,30,and 300?M?.4.The steady-state activation and inactivation of I_Ca-La-L were not significantly changed.5.At 300?M,6-Gin reduced the cell shortening by 48.87±5.44%and the transients by 42.5±9.79%.6.6-Gin at 300?M decreased the Tp and Tr.Also,6-Gin at 300?M decreased the maximum velocity of contraction-relaxation?±dL/dt?.Conclusion:1.The protective effect of 6-Gin in mice with ISO-induced myocardial fibrosis might be related to the inhibition of oxidative stress,inflammation,and apoptosis,potentially through the TLR4/MAPKs/NF-?B signaling pathway.2.The protective effect of 6-Gin on CoCl₂-induced hypoxia in H9c2 cells might be related to the inhibition of oxidative stress,inflammation,and apoptosis,potentially via inhibition on p38/NF-?B pathway and activation on the expression of Nrf2,HIF-1a,and HO-1.3.The molecular mechanisms underlying the cardioprotective effects of6-Gin might be because of a decreasing of intracellular Ca²⁺via the inhibition of I_Ca-La-L and contractility in rat cardiomyocytes.