| Objective: To explore the influence of angiotensin-(1-7) [Ang-(1-7)]on proliferation and secretion in cultured Rat's Glomerular MesangialCells(GMC) induced by AngiotensinⅡ(AngⅡ). Methods: Experiments wereperformed after the mesangial cells had been cultured in serum-free medium for24 hours. The cells, all cultured in 96-well plates, were divided into 6 groups.①Control group: the messangial cells were cultured in serum-free RPMI1640for 48 hours without any stimulators or inhibitors;②AngⅡgroup: themesangial cells were stimulated with AngⅡof 10-7mol/L for 48 hours;③Ang-(1-7) group: the mesangial cells were stimulated with Ang-(1-7) of10-6mol/L for 48 hours;④ Ang-(1-7)+AngⅡgroup: the mesangial cells weretreated with AngⅡof 10-7mol/L,and then adding Ang-(1-7) of 10-6mol/L;Ang-(1-7)10-7mol/L;Ang-(1-7)10-8 mol/L;Ang-(1-7)10-9 mol/L;⑤AngⅡAT1 recepter antagonist +Ang-(1-7) group: the mesangial cells werepretreated with [Sar1,Ile8]-AngⅡ(10-6mol/L)for 30 minute and then stimulatedwith Ang-(1-7) of 10-6mol/L for 48 hours.⑥ AngⅡAT2 receptor antagonist+Ang-(1-7) group: the mesangial cells were pretreated with PD123319(10-5mol/L)for 30 minute and then stimulated with Ang-(1-7) of 10-6mol/L for48 hours. 3[H]thymidine and 3[H]Leucine incorporation: The mesangial cellsfrom each group were exposed to 3[H]thymidine and 3[H]Leucine at aconcentration of 1uCi/ml. Taking off the medium and then addingtrichloroacetic acid of 100g/L to fixation at 4℃ for 30min, Then washing for 2times by balance salt, at last, cracking the cells by 10g/L SDS 0.1mol/LNaOH,Placeing over night, Collecting the cells to mensuration cup tomorrow morning,after uniformly shakening and resolution, mesangial cells was quantified by aliquid scintillation counter. Cell numbers: Using crystal violet to dying cells andcounter the number: 0.1ml 5%(50g/L) glutaraldehyde 15 minute was used tofixation cells, then 1g/L Crystal violet to dying 20 minutes, 0.1ml TritonX-100was used to absorb crystal violet in cell nucleus, the absorbency (A) at 630nmof each well plate was measured by the spectrophotometer. Secretion of PcⅢand HA: The secretion of PcⅢ and HA was measured by radioimmunoassayin culture medium of rats Glomerular Mesangial Cells. Ang-(1-7) was used incultured rats GMC induced by AngⅡ, The synthesis of DNA and proteinmeasured by Incorporation of 3[H]thymidine and 3[H]leucine;cell numbers ofrat's GMC were detected by crystal violet, The secretion of PcⅢ and HA wasmeasured by radioimmunoassay in culture medium of rat's GMC . Statisticalanalysis: All data are reported as mean±standard deviation. Statisticalsignificance was determined by one-way ANOVA followed by Bonferroni's test.Significance was defined as a value of P<0.05. Results: Ang-(1-7) inhibitedsynthesis of DNA and protein as well as the secretion of cultured rat's GMCinduced by AngⅡ in a dose-dependent manner. Ang-(1-7) also reduced thenumbers of cultured rats GMC induced by AngⅡ. The effects of Ang-(1-7)could not be blocked by [Sar1,Ile8]-AngⅡ, and PD123319 have no effect on ityet. Ang-(1-7) can inhibit proliferation and secretion of cultured rats GMCinduced by AngⅡ, The effects of Ang-(1-7) can not be inhibited by[Sar1,Ile8]-AngⅡor PD123319. Incorporation of [3H]thymidine and [3H]leucinein rats mensangial cells: AngⅡ induced the synthesis of protein and DNA incultured mesangial cells, in contrast, Ang-(1-7) siginificantly inhibited thesynthesis, in addition, the inhibition effect of Ang-(1-7) appears a concentrationdependent matter. The inhibition effect of Ang-(1-7) was not attenuated by[Sar1,Ile8]-AngⅡ, an Ang II AT1 specific antagonist, no significance difference(p>0.05) as compared with Ang-(1-7), and administratrition of a specificantagonist to AT2 recepter(PD123319) had no effect yet, p>0.05 as comparedwith Ang-(1-7). Conclusions: The present findings demonstrate that AngⅡinduced the numbers , the synthesis of protein and DNA , The secretion of PcⅢand HA in cultured mesangiol cells, In contrast, Ang-(1-7) significantlyinhibited the numbers , the synthesis and the secretion, in addition, Ang-(1-7)inhibited the synthesis of protein and DNA , the secretion of mesangiol cells ina concentration dependent matter induced by AngⅡ. The inhibition effect ofAng-(1-7) was not attenuated by [Sar1,Ile8]-AngⅡ, a specific AngII AT1receptor antagonist , or a specific antagonist to AT2 recepter(PD123319), nosignificance difference. The result suggest that The inhibition effect ofAng-(1-7) was not through the AT1 and AT2 receptors.
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