| BackgroundCoronary heart disease ?CHD? threatening human health seriously, is called "the first killer of human". The 2012 Chinese cardiovascular disease report pointed out:29 million of cardiovascular disease patient in china, about one person died of cardiovascular disease every 10 seconds, including coronary heart disease mortality for cardiovascular diseases 10-20%. Diabetes mellitus is the world's fastest-growing disease, and at present, approximately 250 million people worldwide and by 2025 this is expected to increase to over 380 million, with type 2 diabetes mellitus ?T2DM? accounting for 90-95% of them. Diabetes mellitus is concerned because of its high incidence and mortality of cardiovascular complications,55% of adult diabetics combined coronary heart disease ?CHD?.Coronary reperfusion therapy can quickly restore coronary artery, then the following "myocardial ischemia reperfusion injury ?MIRI?" improve myocardial blood flow but increase the injury at the same time. And the role of T2DM in myocardial ischemia reperfusion is controversial; some scholars think that diabetes mellitus increase myocardial ischemia-reperfusion injury. We found that the level of ischemia-reperfusion injury is different in CHD patients with T2DM after Percutaneous coronary intervention ?PCI?. So we are going to further investigate the correlation between them and the underlying mechanisms.High glucose can cause oxidative stress reaction, reactive oxygen species ?ROS? excessively generation. Nicotinamide adenine dinucleotide phosphate oxidase ?NADPH? is the main source of ROS in the myocardial cells, and Nox2 is the main subtype in myocardial cells. Studies have shown that high glucose increase ROS, can improve transient receptor potential ?TRP? expression in human mononuclear cells. TRP channels are an important calcium cation channels which located in cell membrane. The canonical transient receptor potential 6 ?TRPC6? is widely expressed in the cardiovascular system, participating in a variety of cardiovascular disorders, results suggested that TRPC6 expression can be improved to increase the flow of calcium ions, accelerated the deterioration of various heart diseases. Studies show that intracellular Ca2+overload which starts the apoptosis plays a central role in the pathogenesis of MIRI. As a calcium ion, whether TRPC6 involved in MIRI still unknown. Nuclear factor-kappa B ?NF-?B? is a transcription factor with multidirectional regulating role of a ubiquitous intracellular, studies show that the NF-?B associated with MIRI, and TRPC6 promoter binding site region with NF-?B. C-Jun N-terminal kinase ?JNK? cell signal transduction pathway is an important signaling pathway in apoptosis of myocardial cells, and JNK mediated apoptosis in MIRI, including three subtypes ?JNK1, JNK2 and JNK3?, JNK land JNK2 widely expressed in organization. The present research confirmed that JNK/NF-?B signaling pathway participated in apoptosis of myocardial cells and MIRI. There may be a positive feedback effect between ROS and JNK, ROS can promote the activation of JNK, the activation of JNK in turn stimulated more ROS, finally sustained activation JNK. The role of NADPH oxidase activation, ROS increase and JNK/NF-?B in MIRI in high glucose concentration, whether related to TRPC6, and Nox2 expression in it, remains to be further discussed.Chinese medicine for prevention and treatment of MIRI is confirmed in basic and clinical experiment. Danshen ?Salvia miltiorrhiza? is a traditional Chinese medicine, the current Danshen injection ?DSI? has been widely used in clinical treatment of cardiovascular diseases, including Danshensu ?DSS? as the content of effective components of recognized the highest, and on their biological activity level plays a main role. Research shows that Danshen could decrease the intracellular calcium overload, reduce MIRI damage, but the specific role in which calcium channel, specific signal transduction mechanisms known yet.Therefore, the purpose of this study was to investigate whether the protective effect of Danshensu and Danshen Injection on MIRI in high glucose concentration is related to TRPC6, the influx of Ca2+, and the role of Nox2/ROS/JNK2/NF-?B signaling pathway, to provide more sufficient theoretical basis for MIRI in high glucose concentration in injury mechanism and clinical treatment.ObjectivesClinical observation on the relationship between diabetes mellitus and myocardial ischemia-reperfusion injury and the role of TRPC6 in it and the protective effects of Danshen injection; Further to investigate the relationship between glucose concentration and H9c2 cells hypoxia-reoxygenation injury in basic research, and detect the level of TRPC6 expression, calcium influx and apoptosis; explore the regulating effect of Nox2/ROS/JNK2/NF-?B signaling pathways; inspect the protection and mechanism of Danshensu on myocardial hypoxia-reoxygenation in high glucose environment; Provide a more adequate theoretical basis for diabetic myocardial ischemia-reperfusion injury mechanism and clinical treatment.Methods1. Clinical grouping for coronary heart disease ?CHD? ?136 cases? and normal Control group ?Control? ?95 cases?, all objects were performed coronary angiography ?CAG?, CHD group was stable angina with? 75% stenosis degree and treated by coronary artery intervention ?PCI?. Compared the creatine kinase isoenzyme MB ?CKMB? content between before and after CAG or PCI1d. CKMB< 32 u/L in all selected objects. Record the age, gender, calculate body mass index ?BMI?, detect:Total cholesterol?TC?, Low Density Lipoprotein cholesterol ?LDL?, Triglycerides ?TG?, Glycosylated hemoglobin ?HbAlc?, Serum Creatinine ?SCr?, Uric acid ?UA?, Random blood glucose ?RBG?, records of coronary arterial stenosis, number of stent placement,24 hours of postoperative arrhythmia score, etc. Analysis CKMB content between before and after CAG or PCI1d. Analysis CKMB content after PCI1d with the above indicators.2. According to the presence of diabetes and HbA1c content. Divided into the following five groups ?25 cases?:1) Control:coronary angiography to rule out coronary heart disease; 2) CHD:coronary angiography to rule in coronary heart disease patients without diabetes mellitus; 3) CHD+H1:coronary heart disease ?CHD? combined with HbAlc< 8%; 4) CHD+H2:coronary heart disease ?CHD? combined with HbA1c between 8-10%; 5) CHD+H3:coronary heart disease ?CHD? with HbA1c> 10%. Western Blot detection research TRPC6 protein expression in mononuclear cells in all objects.3.20 cases of coronary heart disease ?CHD? with HbAlc> 10%,10 cases of coronary heart disease without T2DM. According use of DSI and before or after PCI, compared the following six groups:1) CHD:coronary heart disease without T2DM; 2) CHD+DSI:coronary heart disease patients without T2DM, injected DSI; 3) CHD ?H3?:coronary heart disease with T2DM, HbA1c> 10%; 4) CHD ?H3?+ Insulin:coronary heart disease with T2DM, HbA1c> 10%, insulin injection;5) CHD ?H3?+Insulin+DSI:injected DSI except for insulin; 6) CHD ?H3?+ Insulin+DSI+PCI 1d:1 day after PCI. TRPC6 protein expression was detected before and after PCI 1 day.4. CHD Patients with T2DM, HbA1c? 7%, were divided into two groups ?60 cases?:1) injected DSI for 7 days before PCI; 2) didin't injected DSI for 7 days before PCI. Detected the content of myocardial injury factor CKMB, endothelin ?ET? and malondialdehyde ?MDA?.5. H9c2 cells were taken as target cells and divided into 4 group:1) NG group ?5 mmol/L D-glucose?; 2) LG group?15 mmol/L D-glucose?; 3) HG group ?25 mmol/L D-glucose?; 4)MA group, mannitol osmotic control group ?20 mmol/L mannitol+5 mmol/L D-glucose?. MTT detected 36 h,24 h,48 h,72 h proliferation.6. H9c2 cells were taken as target cells and divided into 6 group:1) HG 3h group:25 mmol/L D-glucose, cultivated 3 hours; 2) HG 6 h groups:HG,6 hours; 3) HG 12 h groups:HG,12 hours; 4) HG 24h groups:HG,24 hours; 5) HG 48 h groups:HG,48 hours; 6) NG 48 h groups:5 mmol/L D-glucose,48 hours. Using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity; using Western Blot method to detect TRPC6 protein expression.7. H9c2 cells were taken as target cells and divided into 4 group:1) NG group; 2) LG group; 3) HG group; 4) MA group. Above groups incubated for 48 hours in each glucose concentration. Using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity; using the fluorescent probe DCFH-DA to detect the intracellular ROS levels; using Real time PCR to detect TRPC6 mRNA expression; using Western Blot method to detect TRPC6 protein expression.8. H9c2 cells were taken as target cells and divided into 4 group:1)NG group; 2) LG group; 3) HG group; 4) MA group. Above each glucose concentration in the incubation for 48 hours then following 2 h of hypoxia then 3 h of reoxygenation, detected the levels of CKMB, cTnl and LDH; using ELISA to detect the levels of BCL-2 and BAX; using Fluo-3AM to observe intracellular calcium fluorescence intensity by flow cytometry; using Annexin V-FITC/PI double staining method to detect H9c2 cell apoptosis by flow cytometry.9. H9c2 cells were taken as target cells and divided into 7 group:1) NG group; 2) MA group; 3) NG+DSS 2 group:pretreated by DSS 2 mg/L; 4) NG+DSS 10 group: pretreated by DSS 10 mg/L; 5) NG+DSS 50 group:pretreated by DSS 50mg/L; 6) NG+DSS 100 group:pretreated by DSS 100mg/L; 7) NG+DMSO:pretreated by 0.5% DMSO. DSS incubated with H9c2 cells for 2 hours in advance, and then incubation in the NG or MA for 48 hours. Using MTT assay to examine the OD value of each group.10. H9c2 cells were taken as target cells and divided into 6 group:1) HG; 2) DSS5+ HG:pretreated by DSS 2mg/L; 3)DSS25+HG:pretreated by DSS 25 mg/L; 4) DSS50+HG:pretreated by DSS 50 mg/L; 5) DSS 100+HG:pretreated by DSS 100 mg/L; 6) Tempol+HG:pretreated by Tempol ?100 ?M? ?ROS inhibitor?. Using the fluorescent probe DCFH-DA to detect the intracellular ROS levels; using Real time PCR to detect TRPC6 mRNA expression; using Western Blot method to detect TRPC6 protein expression. Then carried out 2 h of hypoxia then 3 h of reoxygenation. Using Fluo-3AM to observe intracellular calcium fluorescence intensity by flow cytometry; using Annexin V-FITC/PI double staining method to detect H9c2 cell apoptosis by flow cytometry.11. H9c2 cells were taken as target cells and divided into 6 group:1) NG 48 h; 2) HG 1 h; 3) HG 6 h; 4) HG 12 h; 5) HG 24 h; 6) HG 48 h:pretreated by Tempol. Using Western Blot method to detect p-JNK2, NF-?B in cytoplasm and nucleus protein expression respectively.12. H9c2 cells were taken as target cells and divided into 4 group:1) NG group; 2) LG group; 3) HG group; 4) MA group. Using Western Blot method to detect p-JNK2, NF-?B in cytoplasm and nucleus protein expression respectively.13. H9c2 cells were taken as target cells and divided into 4 group:1) NG group; 2) HG group; 3) HG+PDTC ?10?M? ?NF-?B inhibitor? group; 4) HG+SP600125 ?10?M? ?JNK inhibitor? group. Using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity; using Western Blot method to detect Nox2, p-JNK2, NF-?B ?nucleus? and TRPC6 protein expression respectively.14. H9c2 cells were taken as target cells and divided into 4 group, using JNK2 siRNA specificity JNK2 gene silence:1) NG group; 2) HG group; 3) HG+FAM siRNA group; 4) HG+ JNK2 siRNA group. Continue to cultivate after transient transfection siRNA 48 h, using Western Blot method to detect Nox2, JNK2, p-JNK2, NF-?B ?nucleus? and TRPC6 protein expression respectively. Using the fluorescent probe DCFH-DA to detect the intracellular ROS levels. Then following hypoxia-reoxygenation, detected the level of CKMB and LDH.15. H9c2 cells were taken as target cells and divided into 6 group, using Nox2 siRNA specificity Nox2 gene silence:1) NG group; 2) NG+ FAM siRNA group; 3) NG+ Nox2 siRNA group; 4) HG group; 5) HG+ FAM siRNA group; 6) HG+Nox2 siRNA group. Using Western Blot method to detect Nox2, p-JNK2, NF-?B ?nucleus? and TRPC6 protein expression respectively. Using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity; using the fluorescent probe DCFH-DA to detect the intracellular ROS levels. Then following hypoxia-reoxygenation, using Annexin V-FITC/PI double staining method to detect H9c2 cell apoptosis by flow cytometry; using ELISA to detect the levels of BCL-2 andBAX.16. H9c2 cells were taken as target cells and divided into 7 groups:1) NG group; 2) HG group; 3) D10+HG group:pretreated by DSS 10mg/L; 4) D 50+HG group: pretreated by DSS 50mg/L; 5) D 100+HG group:pretreated by DSS 100mg/L; 6) PDTC+HG group:pretreated by PDTC:7) SP600125+HG group:Pretreated by SP600125. Using Western Blot method to detect p-JNK2, NF-?B ?nucleus? protein expression respectively.17. H9c2 cells were taken as target cells and divided into 6 group:1) NG group; 2) HG group; 3) D 10+HG group:pretreated by DSS 10mg/L; 4) D 50+HG group: pretreated by DSS 50mg/L; 5) D 100+HG group:pretreated by DSS 100mg/L; 6) DPI ?20?M? ?NADPH inhibitor?+HG group:pretreated by PDTC. Using the cell NADPH oxidase colorimetric assay kit to examine the NADPH oxidase activity; using Western Blot method to detect Nox2 protein expression. Then following hypoxia-reoxygenation, Using ELISA to detecting the levels of BCL-2 and BAX; detect the levels of CKMB?cTnI and LDH.Results1. CKMB content significantly increased in coronary heart disease ?CHD? group after PCI than the control group after CAG. There were no significant correlation between CKMB content in CHD group after PCI with age, gender, BMI, triglyceride, SCr, and uric acid etc. while there were significant correlation between CKMB content in CHD group after PCI with TC, LDL, RBG, HbA1c, records of coronary arterial stenosis and number of stent placement. Logistic regression analysis results show that CKMB content in CHD group after PCI have the closest relationship with the HbAlc content; Suggested that myocardial ischemia-reperfusion injury is associated with persistent increased blood glucose.2. TRPC6 protein content increased significantly in CHD patients with the increase of HbAlc content, while there was no significant difference before and after PCI in CHD group. TRPC6 protein content decreased obviously when injected DSJ before PCI. The content of CKMB, ET and MDA decreased obviously. Suggested that DSI could alleviate MIRI in CHD patients with high HbAlc content.3. NADPH oxidase activity and TRPC6 protein expression increased significantly in H9c2 cells when cultivated in HG concentration, showed a trend of time dependence, climbed the highest at 48 h; NADPH oxidase activity, ROS, TRPC6 mRNA and protein expression increased with the increase of the concentration of glucose in the culture medium, and climbed the highest at HG concentration. Prompted NADPH oxidase activity, ROS activity and TRPC6 expression increased when H9c2 cells cultivated in HG concentration for a long time.4. H9c2 cells cultivated in different glucose concentrations and hypoxia-reoxygenation after 48 h incubation, the content of CKMB, cTnI, LDH, BAX and cell apoptosis and intracellular calcium fluorescence intensity increased with the increase of concentration of glucose, and reached the highest in HG group; while the BCL-2 content showed an downward trend adversely.5. DSS had no significant effect on H9c2 cells vitality. ROS levels, TRPC6 mRNA and protein expression decreased in dose dependent when H9c2 cells pretreated by DSS; H9c2 cells apoptosis, intracellular calcium fluorescence intensity decreased when H9c2 cells pretreated by DSS following hypoxia-reoxygenation, and reached the lowest in the DSS 100 pretreatment group; Suggested that DSS pretreatment could reduce hypoxia-reoxygenation injury of H9c2 cells cultivating in high glucose, may be by reducing ROS, TRPC6 expression, and reduce the intracellular calcium fluorescence intensity, reduce apoptosis.6. P-JNK2 and NF-?B ?in nucleus? protein expression increased when H9c2 cells cultivated in HG in different periods, and reached the highest at 48 h; p-JNK2 and NF-?B ?in nucleus? protein expression increased in dose dependent when H9c2 cells cultivated in different glucose for 48 h, and reached the highest at HG group; while the NF-?B ?in cytoplasm? protein expression showed an downward trend adversely. Suggested JNK2 phosphorylation, NF-?B activation and inner transfer in the process of H9c2 cells cultivated in high glucose. NADPH oxidase activity and Nox2, p-JNK2 protein expression has no obvious change in H9c2 cells when pretreated by PDTC, while which decreased when pretreated by SP600125. NF-?B ?in nucleus? and TRPC6 protein expression when pretreated by PDTC or SP600125. Suggested that p-JNK2 is located in the upstream of NF-?B and TRPC6, and can affect the Nox2 expression.7. Nox2, p-JNK2, NF-?B ?in nucleus? and TRPC6 protein expression, ROS levels decreased obviously H9c2 cells cultivated in HG when pretreated by JNK2 siRNA transfection; CKMB and LDH level also obviously decrease when H9c2 cells accepted hypoxia-reoxygenation after JNK2 siRNA transfection; Nox2, JNK2, p-JNK2, NF-?B ?in nucleus? and TRPC6 protein expression, NADPH oxidase activity and ROS levels decreased obviously H9c2 cells cultivated in HG when pretreated by Nox2 siRNA transfection; cell apoptosis and BAX content also reduced obviously, while BAX content increased when H9c2 cells accepted hypoxia-reoxygenation after Nox2 siRNA transfection. Combined with the above 3, 4 and 6 results suggested high glucose cultivation aggravating H9c2 cells of hypoxia-reoxygenation injury, and Nox2, ROS and p-JNK2 cross each other, coexist in the upstream of NF-?B and TRPC6 in the process of H9c2 cells hypoxia-reoxygenation cultivating in HG; the specificity silence of Nox2 and JNK2, p-JNK2 can reduce H9c2 cells hypoxia-reoxygenation injury cultivating in HG.8. P-JNK2 and NF-?B ?in nucleus? protein expression presented a downward trend in H9c2 cells cultivated in HG when pretreated by DSS, and reach the minimum at D100 pretreatment group. p-JNK2 and NF-?B ?in nucleus? protein expression markedly reduced when H9c2 cells pretreated by SP600125. NF-?B ?in nucleus? protein expression reduced while p-JNK2 protein expression has no obvious drop when pretreated by PDTC. Suggested that JNK2 phosphorylation, NF-?B activation and internal transfer reduced when H9c2 cells cultivated in HG pretreated by DSS.9. NADPH oxidase, Nox2 protein expression presented a downward trend in DSS dose dependent trend in H9c2 cells cultivated in HG when pretreated by DSS or DPI. Myocardial injury factor CKMB, cTnI, LDH level and pro-apoptotic factor BAX, cell apoptosis also obviously decrease while antiapoptotic factor BCL-2 increased when H9c2 cells accepted hypoxia-reoxygenation pretreated by DSS or DPI. Suggested that DSS pretreatment could reduce H9c2 cells hypoxia-reoxygenation injury cultivated in HG, by reducing the NADPH oxidase and Nox2 expression.Conclusion1. Coronary heart disease combined with T2DM prone to myocardial ischemia reperfusion injury, and glycated hemoglobin levels ?HbAlc? were positively correlated, may is associated with persistent hyperglycemia promote TRPC6 protein expression increase; Danshen injection pretreatment can reduce TRPC6 protein expression, reduce myocardial ischemia-reperfusion injury in coronary heart disease patients with T2DM.2. H9c2 cells prone to myocardial hypoxia-reoxygenation injury in high glucose environment, maybe through regulating Nox2/ROS/JNK2/NF-?B pathway, promote TRPC6 protein expression, increase calcium influx in myocardial hypoxia-reoxygenation, lead to the calcium overload and increased cell apoptosis in the end. In the signal path, Nox2, ROS and p-JNK2 activation may be cross each other and coexist in the upstream of the NF-?B signaling pathway. Pretreatment by Danshensu could reduce H9c2 cells hypoxia-reoxygenation injury, may be through inhibiting Nox2/ROS/JNK2/NF-?B pathway, reduce TRPC6 expression, reduce calcium influx and calcium overload, thus to play a role of protection in H9c2 cells hypoxia-reoxygenation injury when cultivated in high glucose. Salvia miltiorrhiza maybe effective candidate drugs in clinical prevention and treatment of myocardial ischemia-reperfusion injury in high sugar environment. |
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