| Objective :To analyze the effect and injury mechanism of R5 HIV-1 gp120 on cardiomyocyte and Salvianolic acid B(SAB) protection effect.Methods:The cardiomyocytes isolated from SD rat Newborn 1-4 days cultured for4 days were used for the following experiments.1 To observe the R5 HIV-1 gp120 binding to CCR5 receptor on cardiomyocyte: The c?ltured cardiomyocytes were divided into 3 groups: control, gp120, gp120 +DAPTA group. gp120 + DAPTA group was added 10?M DAPTA and incubated for 0.5h; then added Alexa Flour 532 HIV-1 gp120(JRFL) 0.1?g in gp120 and gp120 + DAPTA group and c?ltured for 4h.2 To observe the injury effect of different concentration of R5 HIV-1 gp120 on cardiomyocytes: The cultured cardiomyocytes were divided into 6 groups(n = 5), added different doses of R5 HIV-1gp120(0,0.2,0.4,0.8,1.0, 2.0 ?g/ml) respectively. Incubated for 4h, mediums were collected and assayed LDH and CK content.3 To observe the phosphorylation effects of different concentrations of R5 HIV-1gp120 on the P38 MAPK and c Tn I of cardiomyocytes: The cultured cardiomyocytes were divided into 4 groups(n = 3), added different doses of R5 HIV-1gp120(0, 0.2, 0.4,0.8 ?g/ml) respectively, and incubated for 4h. Cardiomyocytes protein was extracted and assayed P38 MAPK and c Tn I phosphorylation levels by Western Blot.4 To observe the effects of P38 MAPK inhibitor on the injury of cardiomyocytes induced by R5 HIV-1gp120: The cultured cardiomyocytes were divided into 4 groups(n=5): Control, gp120, gp120 + SB203580, SB203580 group. Added 0.8?g/ml R5HIV-1gp120 in gp120 group, the same amount of gp120 and 10?M SB203580 in gp120+ SB203580 group, and 10?M SB203580 in SB203580 group. Incubated for 4h,mediums were collected and assayed LDH and CK content.5 To observe the effects of CCR5 blockers DAPTA on cardiomyocytes p38 MAPKphosphorylation induced by R5 HIV-1 gp120: The cultured cardiomyocytes were divided into 4 groups(n = 3): control, gp120, gp120 + DAPTA, DAPTA. Added 20?M DAPTA in gp120 + DAPTA and DAPTA groups and incubated for 0.5h; then except fo Control group, added 0.4?g/ml R5 HIV-1gp120 in other groups and incubated for 4h;cardiomyocytes protein was extracted, and detected P38 MAPK and c Tn I phosphorylation levels by Western Blot.6 To observe the effects of NMDAR blockers MK801 on cardiomyocytes P38 MAPK phosphorylation induced by R5 HIV-1gp120 : The cultured cardiomyocytes were divided into 5 groups(n = 3):Control, gp120, gp120 + MK801(2?20?200?M)group. Added 2, 20,200?M DAPTA in different doses of MK801 groups respectively and incubated for 0.5h; then except for Control group, added 0.4?g/ml R5 HIV-1gp120 in other groups, and incubated for 4h in incubator; Cardiomyocytes protein was extracted, and P38 MAPK and c Tn I phosphorylation levels detected by Western Blot.7 To observe the protective effects of SAB on the injury of cardiomyocytes induced by R5 HIV-1gp120: The cultured cardiomyocytes were divided into 7 groups(n = 3):Control, gp120, gp120+SAB(20,40,80,100?M) and gp120+SB203580 group. Added0.8?g/ml R5 HIV-1gp120 in gp120 group, 0.8?g/ml R5 HIV-1gp120 and SAB(20,40,80,100?M) in different doses of SAB groups respectively, and 10?M SB203580 with 0.8?g/ml R5 HIV-1gp120 in gp120+SB203580 group, and incubated for 24 h. The medi?Ms were collected and measured LDH and CK content, and Western Blot detected P38 MAPK phosphorylation levels.Res?lts:1 Compared with gp120, the fluorescence on cardiomyocytes significantly reduced(p <0.05), revealing R5 HIV-1 gp120 binding to CCR5 receptor on cardiomyocyte.2 The R5 HIV-1 gp120 was concentration dependently increased medium LDH, CK content, especially 0.8,1.0 and 2.0 ?g/ml groups significantly increased(p<0.05),indicating R5 HIV- 1gp120 induced cardiomyocytes injury.3 R5 HIV-1gp120 increased P38 MAPK and c Tn I phosphorylation, especially 0.4and 0.8?g/ml groups increased significantly(p<0.05), indicating R5 HIV-1gp120 induced P38MAPK and c Tn I phosphorylation.4 SB203580 significantly reduced LDH and CK content(p<0.05) in medium,indicating P38 MAPK inhibitors protected cardiomyocytes from R5 HIV-1gp120 injury.5 Compared with the control group, the levels of P38 MAPK and c Tn I phos-phorylation increased(p<0.05); compared with the gp120 group, DAPTA significantly inhibited P38 MAPK and c Tn I phosphorylation induced by R5 HIV-1gp120(p<0.05),revealing CCR5 blockers DAPTA inhibited P38 MAPK and c Tn I phosphorylation induced by R5 HIV-1gp120.6 Compared with the control group, the levels of P38 MAPK and c Tn I pho-sphorylation increased(p<0.05); compared with the gp120 group, P38 MAPK and c Tn I phosphorylation levels did not significantly change in different doses MK801groups(p>0.05), indicating NMDAR blockers MK801 did not inhibit cardiomyocytes P38 MAPK and c Tn I phosphorylation induced by R5 HIV-1gp120.7 Compared with the control group, the levels of LDH and CK increased in gp120group(p<0.05); compared with the gp120 group, the levels of LDH and CK obviously decreased in 80 and 100?M SAB groups(p<0.05).Western Blot detected P38 MAPK phosphorylation levels showed that, compared with the control group, the level of P38 MAPK phosphorylation in gp120 group increased(p<0.05); compared with the gp120 group, P38 MAPK phosphorylation reduced in 20, 40, 80 and 100?M SAB groups(p<0.05), especially in 80?M SAB group, indicating that appropriate dose of SAB protected the cardiomyocytes from P38 MAPK phosphorylation injury induced by R5 HIV-1gp120.Conclusions:R5 HIV-1gp120 induced cardiomyocytes P38 MAPK phosphorylation through the CCR5 and mediated cardiomyocytes injury, and appropriate dose of salvianolic acid B protect cardiomyocytes from p38 MAPK phosphorylation injury induced by R5 HIV-1 gp120. |
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