Effects Of Simvastatin On LPS-induced Angiotensin â…¡ Type 2 Receptor Protein Expression And Apoptosis Change Of Rat Cardiac Fibroblasts

Cardiac fibrosis is a major facet of a variety of cardiovascular diseases, including hypertensive cardiac disease and inflammation of the heart tissue. Fibrosis is the results of over-production of collagen and extracellular matrix (ECM) component proteins. Increased deposition of collagen is a hallmark of the remodeling process, whereas rebuilding of cardiac ECM results mainly from overproliferation of cardiac fibroblasts (CFs), which increases collagen synthesis and ECM component proteins expression. Two major types of AngⅡr eceptor are present in CFs, the type 1 receptor (AT1R) and the type 2 receptor (AT2R). AT2R is generally regarded to counteract the function of AT1R in various contexts, which leads to the inhibition of CFs proliferation and the induction of cell differentiation and apoptosis. Recently, substantial evidences have demonstrated that statins, inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, have anti-growth effects by inhibiting cell proliferation and collagen synthesis and inducing cell apoptosis in-dependent of the cholesterol-lowing mechanism. But so far no studies have addressed whether AT2R is involved in the above effects. It has been illustrated that mitogen-activated protein kinase (MAPK) is a convergence point for many signal pathways. It can transduce extracellular stimuli to the nucleus, regulate transcription factors, and drive expression of related genes. Extra-cellular signal-regulated kinase (ERK) is one subfamily of MAPK, which plays a pivotal role in modulating cellular processes such as proliferation, differentiation, transformation and cell death. Lipopolysaccharide (LPS) is a well recognized factor in inducing cardiac fibrosis through the induction of nitric oxide (NO), yet so far the mechanism by which statins affect CFs apoptosis and AT2R protein expression and the possible involvement of ERK1/2 have not been addressed. In the present study, we observed the effects of LPS on the production of NO and AT2R protein expression, and those of simvastatin (Sim). What's more, the modulatory effects of Sim and LPS on the phosphorylation of ERK1/2 is also addressed, in the hope of further elucidating the effects of statins in reversing CM and the possible mechanism involved in such a modulating effect.In the present study, cultured CFs from neonatal Sprague-Dawley (SD) rats were used as experiment model. Western blot is adopted to determine AT2R and ERK1/2 and phosphorylated-ERK1/2 protein expression, and Hoechst 33258 staining is employed to assess CFs apoptotic rate. Dynamic observation is made to identify: (1) the effects of LPS on NO production and AT2R protein expression, and those of Sim on such a modulation. (2) the effects of LPS on CFs apoptosis and those of Sim on such a modulation. (3) the modulatory effects of Sim on LPS-induced ERK1/2 phosphorylation chage. (4) the role of AT2R in Sim modulating LPS-induced ERK1/2 phosphorylation and CFs apoptosis change.The results of the study were as follows: (1) The AT2R/β-actin value was 0.18±0.02 when CFs were incubated with 0.1μg/ml LPS for 24 h, not being significantly different from control(0.18±0.02, P > 0.05); AT2R/β-actin values were 0.14±0.02, 0.10±0.01 and 0.15±0.01, respectively, when CFs were treated with LPS at concentrations of 1μg/ml, 10μg/ml and 100μg/ml for 24h, all being significantly lower than control(P < 0.05 ~ 0.01).(2)Treating CFs with LPS at concentrations of 0.1μg/ml, 1μg/ml, 10μg/ml and 100μg/ml for 24 h induced NO production in the culture media to 35.51±3.74μM, 49.72±4.6μM, 59.25±3.78μM and 76.45±3.93μM, respectively; NO levels in all but 0.1μg/ml LPS group were significantly higher than control(33.83±2.23μM)(P < 0.01).(3)AT2R/β-actin value was 0.17±0.02 when pretreating cells with 200μM L-NAME,a NO synthetase inhibitor, for 30 min before 10μg/ml LPS treatment; the value was significantly higher than that in 10μg/ml LPS single-drug group(P < 0.01)while no significant difference was detected when compared with control(P > 0.05). The value was not significantly different between L-NAME single-drug group and control (P > 0.05). (4)NO levels in the culture media were 60.19±5.74μM and 55.89±4.87μM when CFs were co-incubated with 0.01μM or 0.1μM Sim and 10μg/ml LPS, and no significant difference was evidenced when compared with that in 10μg/ml LPS single-drug group(59.25±3.77μM, P > 0.05). NO levels in the culture media were 48.97±2.25μM and 42.24±3.88μM when CFs were co-incubated with 1μM or 10μM Sim and 10μg/ml LPS for 24 h; both were significantly lower than that in 10μg/ml LPS single-drug group(P < 0.01). (5)AT2R/β-actin values were 0.10±0.02 and 0.12±0.02,respectively, when CFs were co-incubated with 0.01μM or 0.1μM Sim and 10μg/ml LPS for 24 h; no significant difference was observed when compared with that in LPS single-drug group(0.10±0.02, P > 0.05). AT2R/β-actin values were 0.15±0.01 and 0.18±0.02 when CFs were co-incubated with 1μM or 10μM Sim and 10μg/ml LPS for 24 h; both were significantly lower than that in 10μg/ml LPS single-drug treated group (P < 0.05 and P < 0.01, respectively). (6) Hoechst 33258 staining showed that the proportions of apoptotic CFs were (7.56±0.39)% and (9.23±0.86)%, respectively, when CFs were treated with 10μM Sim or 1μg/ml LPS for 24 h, and there was no difference when compared with control group's (8.67±0.59)% (P >0.05). After treating cells with 1μg/ml LPS plus 10μM Sim for 24 h,the proportion of apoptotic CFs was (29.56±2.53)%, being significantly different when compared with 1μg/ml LPS or 10μM Sim groups'(P<0.05). (7) Treating cells with 1μg/ml LPS combined with 2μM or 5μM or 10μM Sim for 24 h induced CFs apoptosis proportions of (19.37±1.79)% and (24.94±2.21)% and (29.56±2.53)%, respectively, and significant difference was evidenced when compared with 1μg/ml LPS single-drug treated group (9.23±0.86)% (P<0.01). After treating cells with 1μg/ml LPS plus 10μM Sim plus 1mM L-MVA for 24 h,the proportion of apoptotic CFs was (13.06±0.98)%,which was significantly different when compared with 1μg/ml LPS plus 10μM Sim group (P<0.05). (8) After treating cells with 10μM Sim for 4hs, phosphorylated ERK1/2(p-ERK1/2) in CFs was (56±5)% when compared with that in control group(100±6)% (P<0.05). After treating cells with 1μg/ml LPS for 4 hs, p-ERK1/2 proten level was (97±8)% when compared with that in control group, and no significant difference was detected between those two groups (P>0.05). After Treating cells with 1μg/ml LPS combined with 10μM Sim for 4hs, p-ERK1/2 proten level was (35±3)% when compared with control, and significant difference was detected between the two (P<0.05). But after 1μM PD123319(specific AT2R blocker) was added in this group, p-ERK1/2 proten level was restored to (50±6)%, being significantly higher than 1μg/ml LPS plus 10μM Sim group (P<0.05). (9) 2μM or 5μM PD123319 combined with1μg/ml LPS and with 10μM Sim induced CFs apoptotic proportion at (26.12±2.10 )% and (19.64±2.06 )%, which were significantly different from 1μg/ml LPS plus 10μM Sim group (P<0.05). 10μM PD123319 combined with1μg/ml LPS and with 10μM Sim induced CFs apoptotic proportion at (15.64±1.60) %, which was significantly different from 1μg/ml LPS plus 10μM Sim group (P<0.01). While 10μM PD123319 single-drug treated group, a proportion of (7.54±0.70) % was witnessed, not being significant different from control group (P > 0.05).The results above indicate that: (1) LPS can inhibit the expression of AT2R in CFs in a concentration-dependent manner via the induction of NO production. Sim can antagonize the effects of LPS in NO production and AT2R protein espression. (2) Sim can sensitize CFs to LPS-induced apoptosis by inhibiting the L-MVA pathway, and the effect follows a concentration-dependent pattern. (3) Sim can synergize with LPS to induce CFs apoptosis via inhibiting ERK1/2 phosphorylation. (4) The effects of Sim on CFs LPS-induced apoptosis and ERK1/2 phosphorylation are exerted via the induction of AT2R protein espression increasement.From the results above, it can be shown that Sim can antagonize LPS in NO production and AT2R protein espression, and enhanced CFs apoptosis via the inhibitory effects of AT2R on ERK1/2 phosphorylation, thus exerting its effects in damping cardiac fibrosis. Our present study furthered the knowledge of statins about their modulating effects on cardiac remodeling triggerd by proinflammatory factors and the possible mechanism involved, and added to the theoretical base and clinical applications for the prevention of cardiac remodeling in inflammatory state.