Effect Of High Salt On The Proliferation Of Rat Vascular Smooth Muscle Cells And The Intervention Of Drugs

Objective: To study the influence of high salt on the proliferation of rat vascular smoothmuscle cells (VSMCs) and the intervention of telmisartan and capsaicin in vivo and in vitroand to explore its possible mechanisms.Methods:1.Experiment in vivo Male Wistar rats were randomly divided into Fourgroups: control group (0.5%NaCl), high salt group (4%NaCl), high salt+telmisartan group(4%NaCl+telmisartan), high salt+and capsaicin group (4%NaCl+0.01%capsaicin), ratswere feed for24weeks. At the end of the experiment, the aorta was fixed, embedded, slicedand stained with hematoxylin and eosin(HE), the protein expression of α-smooth muscularactin （α-SMA） and proliferating cell nuclear antigen(PCNA) were determined byimmunohistochemistry, and proliferation index(PI) was calculated according toimmunohistochemistry of PCNA.2. Experiment in vitro (1) To determine optimal actiontime and concentration of salt, telmisartan and capsaicin VSMCs were obtained fromthe rat’s thoracic aorta by tissue adherent method and divided into four groups: control group（Na+139mmol/L）, high-salt group（Na+149mmol/L、159mmol/L、169mmol/L、179mmol/L）, high salt+telmisartan group （Na+159mmol/L+telmisartan10﹣8mol/L、10﹣7mol/L、10﹣6mol/L、10﹣5mol/L、10﹣4mol/L）and capsaicin group（Na+159mmol/L+capsaicin10﹣8mol/L、10﹣7mol/L、10﹣6mol/L、10﹣5mol/L、10﹣4mol/L）; DMSO solvent group (equalvolume DMSO corresponding to telmisartan group and capsaicin group), MTT colorimetricassay was used to measure vitality of VSMCs, the cell proliferation rate (PR) and inhibitionrate (IR) were calculated according to result of MTT.(2) Effects and mechanisms theoptimal action time and concentration of salt and telmisartan and capsaicin were confirmedby protocol (1) After synchronization for24h, VSMCs were randomly divided into controlgroup（Na+139mmol/L）, high-salt group（Na+159mmol/L） and capsaicin group（Na+159mmol/L+capsaicin10-5mol/L）, then continued to culture for72h. Flow cytometrytechnology was used to analyze cell cycle. The cell area, cytoplasm area and nuclear area, the ratio of nuclear area to cell area and the ratio of nuclear area to cytoplasm area wererespectivrely calculated for VSMCs dyed by hematoxylin and eosin (HE). Theimmunofluorescence methods were applied to analyze the protein expression levels oftransforming growth factor beta-1(TGF-β1), pSmad2/3and Smad7in VSMCs. Theimmunocytochemical method were applied to analyze the protein expression levels ofangiotensin Ⅱ type1receptor (AT1R), angiotensin Ⅱ type2receptor (AT2R),angiotensin converting enzyme1(ACE), angiotensin converting enzyme2(ACE2),angiotensin Ⅱ (AngII). Meanwhile, the expressions of TGF-β1, Smad2, Smad3, Smad7,AT1R, AT2R, and ACE mRNA in rat VSMCs were measured by the real time RT-PCR.Results:1. Experiment in vivo HE staining showed that arterial intima-media thickness,are obvious in high salt group compared with the control group, telmisartan attenuated aorticpathological damage (P<0.05), but capsaicin have no similar effect with telmisartan.Telmisartan and capsaicin alleviated down-regulating of protein expression of α-SMA andup-regulating protein expression of PCNA.2. Experiment in vitro (1) To explore optimalaction time and concentration of salt and telmisartan and capsaicin MTT assay showedthat Na+159mmol/L at72h promoted VSMCs proliferation on a dose and time dependentmanner. The optimal concentration of anti-proliferative effect of telmisartan and capsaicinare10-5mol/L.(2) Effects and mechanisms At Na+159mmol/L for72h, the OD and PRwere remarkably higher, the expression of PCNA increased, telmisartan and capsaicinantagonized it significantly, α-SMA expression reduced, telmisartan and capsaicin had noeffects. HE dyeing showed that cell area, cytoplasm area and nuclear area of the VSMCs inthe high salt group were evidently increased, while the ratio of nuclear area to cell area andthe ratio of nuclear area to cytoplasm area decreased (P<0.05), G0/G1phase was decreased(P<0.05), while the percentage of cells in S stage increased (P<0.05), telmisartan andcapsaicin improved these changes. The protein and mRNA expressions of TGF-β1, Smad2and Smad3were upregulated in high-salt group (P<0.05), however, Smad7downregulatedcompared with control group (P<0.05), telmisartan and capsaicin significantly decreased theprotein and mRNA expressions of TGF-β1, Smad2and Smad3expression and increased theexpression of Smad7(P<0.05). In the high-salt group, the gene expression of AT1, AT2, ACE and protein expression of AT1, AT2, ACE, AngII were significantly increased (P <0.05),whereas ACE2protein expression was decreased (P <0.05), telmisartan significantlyimproved AT1, AT2, ACE mRNA and protein expression levels, but no significant effect ofcapsaicin.Conclusion:I. Long-term high salt diet can lead to aortic remodeling, characterized by the arterialmedia thickening, vascular smooth muscle proliferation and phenotypictransformation.II. VSMCs proliferation and hypertrophy can be induced by high salt through promotingG0/G1phase into S phase of cell cycle.III. The mechanisms of the VSMCs proliferation induced by high-salt may be related toabnormal expressions of TGF-β1/Smads signaling pathway and RAS components.IV. Telmisartan and capsaicin can inhibit the VSMCs proliferation induced by high-saltvia regulating TGF-β1/Smads pathway and RAS.