The Hepatic And Cardiac Protective Effects And Underlying Mechanisms Of DSS Based On Iron Metabolism-related Proteins And Nrf2 Pathway

Part one Protective effects and mechanisms of Danshensu against liver injury induced by iron overload.Objective:To determine the hepatic protective effects and possible underlying mechanisms of Danshensu（DSS）on iron overload mice.Methods:Fifty male KM mice（six to eight weeks old）were randomly divided into five groups（n=10 per group）:control（10 m L/kg/d saline）,iron overload（Fe,50 mg/kg/d iron dextran）,low dose Danshensu（L-DSS,Fe+50mg/kg/d DSS）,high dose Danshensu（H-DSS,Fe+100 mg/kg/d DSS）,and deferoxamine positive control（DFO,Fe+100 mg/kg/d DFO）groups.The entire experimental period lasted for 14 days.When the animals were anesthetized（sodium pentobarbital,50 mg/kg）,liver samples were quickly removed and fixed in 4%paraformaldehyde solution or frozen in liquid nitrogen for the next analysis.HE staining,Prussian blue staining,immunohistochemistry and Western blot were used to observe the regulatory effects of DSS on oxidative stress,inflammation,apoptosis and iron transportation-related proteins in iron overload hepatic tissues.Results:1.HE staining results showed that DSS treatment significantly improved the disordered liver structure and inflammatory infiltration caused by iron overload.2.Biochemical results showed that DSS treatment significantly increased serum levels of AST and ALT,alleviated iron overloaded-liver injury.3.Prussian blue staining results showed that DSS treatment significantly reduced hepatic iron deposition in iron overloaded mice.4.Western blot results showed that DSS significantly down-regulated the expression level of DMT-1,Tf R and LTCCα1C in iron overloaded liver,that may be one of the mechanisms of DSS reduced hepatic iron deposition.5.Biochemical results showed DSS significantly increased the serum activities of SOD and GSH-Px and decreased the concentration of MDA,antagonized oxidative stress damage caused by iron overload.6.Immunohistochemical results showed that DSS significantly reduced the expression of TGF-βand IL-6,antagonized iron overloaded inflammation.7.TUNEL and Western blot results showed that DSS significantly reduced expression of Bax and Caspase-3,increased the expression of Bcl-2,and inhibited hepatocyte apoptosis.Conclusion:Iron overload can lead to hepatic structural and functional damage in mice.DSS plays a protective role in the iron oberloaded liver by anti-oxidation,anti-inflammation,anti-apoptosis and regulating the expression of iron transport associated proteins.Part two Mechanisms of Danshensu combined with Verapamil on heart injury in iron overloaded mice.Objective:To determine the cardiac protective effects and possible underlying mechanisms of Danshensu combined with Verapamil（DSS+VER）on iron overloaded mice.Methods:Fifty male KM mice（six to eight weeks old）were randomly divided into five groups（n=10 per group）:control（10 m L/kg/d saline）,iron overload（Fe,50 mg/kg/d iron dextran）,DSS+VER（Fe+50 mg/kg/d DSS+2mg/kg/d VER）,DSS（Fe+50 mg/kg/d DSS）,and VER（Fe+2 mg/kg/d VER）groups.The entire experimental period lasted for 14 days.When the animals were anesthetized（sodium pentobarbital,50 mg/kg）,heart samples were quickly removed and fixed in 4%paraformaldehyde solution or frozen in liquid nitrogen for the next analysis.HE staining,Prussian blue staining,immunohistochemistry and Western blot were used to observe the regulatory effects of DSS on oxidative stress,inflammation,apoptosis and iron transportation-related proteins in iron overload cardiac tissues.Results:1.DSS+VER treatment significantly improved the disordered myocardial structure caused by iron overload.2.DSS+VER treatment significantly increased serum levels of CK and LDH,and improved cardiac function injury caused by iron overload.3.Prussian blue staining results showed that DSS+VER treatment significantly reduced cardiac iron deposition.4.DSS+VER significantly increased the activities of SOD and GSH-Px and decreased the concentration of MDA,antagonized oxidative stress damage caused by iron overload.5.DSS+VER significantly reduced the expression of TNF-α（11）TGF-βand IL-6（11）antagonized iron overload inflammation.6.DSS+VER significantly reduced expression of Bax and Caspase-3 and increased the expression of Bcl-2,inhibited cardiomyocyte apoptosis.7.DSS+VER significantly down-regulated the expression level of DMT-1,Tf R and LTCCα1C in iron overloaded heart,which may be one of the mechanisms of DSS reduced cardiac iron deposition.Conclusion:Iron overload can lead to cardiac structural and functional damage in mice.DSS and VER plays a protective role in the iron oberloaded heart by anti-oxidation,anti-inflammation,anti-apoptosis and regulating the expression of iron transport associated proteins.The effect of DSS combined with VER on iron overloaded myocardial injury was better than that of DSS or VER.Part three Protective effects and mechanisms of Danshensu against iron overload in H9c2 cellsObjective:To determine the protective effects and possible underlying mechanisms of Danshensu（DSS）on Fe SO₄-induced iron overload in H9c2cells.Methods:H9c2 cells were assigned into six groups:control;DSS control（30μM DSS,24 h）;iron overload group（Fe,3μMFe SO₄,24 h）;low-dose DSS group（L-DSS,10μMDSS 4h+3μMFe SO₄ 24 h）;high-dose DSS group（H-DSS,30μMDSS 4h+3μMFe SO₄,24 h）;DFO group（1μMDFO4h+3μMFe SO₄,24 h）.Colorimetric method,ELISA,Flow cytometry,q RT-PCR and Western blot were used to observe the protective effects of DSS on Fe SO₄-induced iron overload in H9c2 cells.Results:1.The potential cytotoxic results on H9c2 showed that DSS treatment had little effect on cell viability at the concentrations of 10μM and 30μM DSS.The cell viability of H9c2 cells were significantly decreased after treatment of 3μMFe SO₄.Therefore,3μMFe SO₄ was selected to induce iron overload injury in the H9c2 cells.2.The results of colorimetry and ELISA showed that DSS significantly reduced the activites of LDH,CK and the concentration CK-MB in cell supernatant.3.The results of inductively coupled plasma emission spectrometry（ICP-OES）and Prussian blue staining showed that DSS significantly reduced the iron content in H9c2 cells.4.The results of colorimetry showed that DSS significantly reduced the concentration of MDA and increased the activities of SOD and GSH-Px in cell supernatant.5.DCFH-DA reactive oxygen species assay resultes showed that DSS significantly reduced the ROS level in iron overloaded H9c2 cells.6.JC-1 assay results showed that DSS could significantly inhibited the mitochondrial membrane potential decreasing in iron overloaded H9c2 cells.7.Annexin V-PI test results showed that the rate of apoptosis was significantly decreased after treatment of DSS.8.The q RT-PCR and Western blot results showed that DSS significantly increased the m RNA levels of Nrf2 and up-regulated the protein expression levels of Nrf2,HO-1 and NQO1 in iron overloaded H9c2 cells.9.The q RT-PCR and Western blot results showed that DSS significantly decreased the m RNA levels of hepcidin and down-regulated the protein expression levels of hepcidin,DMT-1,Tf R and Ft L in iron overloaded H9c2cells.Conclusion:The protective mechanisms of DSS on iron-overloaded cardiomyocytes include antioxidant stress and reduction of myocardial iron deposition.The mechanism of antioxidant stress is related to the activation of Nrf2 pathway,and the mechanism of reduction of iron deposition is related to down-regulation of the expressions of iron regulatory proteins such as hepcidin,DMT-1,Tf R and Ft L.Conclusions:1.The protective mechanisms of DSS on liver injury in iron overloaded mice might be related to the inhibition of oxidative stress,inflammation,apoptosis and reduction iron deposition,and the mechanism of decreasing iron deposition is related to the down-regulation of iron-uptake related proteins,such as DMT-1,Tf R and LTCCα1C.2.The mechanism of DSS and VER against iron overload myocardial injury is related to the inhibition of oxidative stress,inflammation,apoptosis,reduction of iron deposition and down-regulation of iron uptake related proteins DMT-1,Tf R and LTCCα1C.The effect of DSS combined with VER on iron overloaded myocardial injury was better than that of DSS or VER.Combined the effective component of traditional Chinese medicine with calcium channel blocker may be a potential new therapy to treat iron overload cardiomyopathy.3.The protective mechanisms of DSS on iron overload induced oxidative damage in H9c2 cells may be related to activating Nrf2 pathway,including inhibition of ROS production,up-regulation of Nrf2,NQO1 and HO-1expression,increase of SOD and GSH-Px activities,inhibition of mitochondrial membrane potential decline,and inhibition of cell apoptosis.4.The mechanism of DSS reducing iron deposition in iron-overloaded H9c2 cells is related to decreasing the expression level of hepcidin,DMT-1,Tf R and Ft L.