| Objective: Semen Sojae Preparatum, a traditional drug that fermented from ripe beans, can be used as both drug and food, but we usually believe that it worked in curing minor disease such as cold in the past. Researches nowadays show that SSP isoflavone(SSP), which is the main active component, has pharmacological effects. Our earlier researches show that SSP has a series of biological activities, incluing decreasing blood glucose, regulating lipid metabolism, protecting aorta tunica intima and inhibiting smooth muscle cell proliferation, and it has a visible protection on atherosclerosis too. For the purpose of elucidating the mechanism of SSP's anti-atherosclerosis effect, enlarging the clinical use of SSP and developing new drugs for AS, in our research we systematically approached the mechanism of SSP's anti-atherosclerosis effect utilizing the primary culture vascular smooth muscle cell in cellular and molecular levels.Methods:1 The effect of SSP Isoflavone on rats'VSMC Proliferation induced by AngⅡ1.1 Primary culture and identification of rat's aorta VSMCThe primary culture VSMC of rat by the tissue sticking mass method was used for the study; VSMC were identified byα-actin immunohistochemistry technology, cell morphology and SM-actin staining rate were detected by inverted phase contrast microscope.1.2 Detection of SSP Isoflavone on in vitro cultured VSMC cytotoxicityConcentration gradient of SSP was set to 1, 10, 100, 500, 1000, 5000, 10000, 50000μg/L, and the action time was 24h; Persistence of each hole was detected by MTT method, denoting by optical density.1.3 Interference of SSP Isoflavone to AngⅡ-induced VSMC proliferationInterference gradient concentrations was set as follows: 10, 50, 100, 200, 500μg/L; the incubation times were 1, 4, 12, 24 h;the final concentration of AngⅡwas 10-7mol/L which was co-cultured with SSP for 24h; the optimal concentration and action time that could inhibit proliferation were detected and determined by MTT method.1.4 Detection of VSMC cell cycleExperiment were divided into control group, AngⅡmodel group, SSP isoflavone (50, 100, 200μg/L)+AngⅡgroup, soybean isoflavone(200μg/L) + AngⅡgroup. Both SSP isoflavone and soybean isoflavone group were incubated for 24h; the final concentration of AngⅡwas 10-7mol/L and co-cultured with SSP for 24h; then cells were collected and cell cycles were detected by flow cytometer. 2 The Effect of SSP Isoflavone on JAK2/STAT3 signal transduction pathway in proliferated VSMC2.1 The interference of AG490, SSP Isoflavone and soybean Isoflavone to AngⅡ-induced VSMC ProliferationExperiment were divided into control group, AngⅡmodel group, AG490(50μmol/L)+AngⅡgroup, SSP isoflavone(50, 100, 200μg/L)+AngⅡgroup, soybean isoflavone (200μg/L) + AngⅡgroup; AG490 was incubated for 16h before AngⅡinterference, SSP isoflavone, soybean isoflavone were incubated for 24h before AngⅡinterference. Optical density of each hole was detected using MTT method.2.2 Detection of mRNA expression of AT1RExperimental groups and drug interference (AG490, SSPsoflavone, soybean isoflavone) were the same as 2.1. AngⅡstimulation lasted 30min. Whole RNA was extracted by Trizol, expression of AT1R in cell membrane was detected by RT-PCR method.2.3 Detection of related protein expressions of JAK2/STAT3 signal transduction pathwayExperimental groups and drug interference (AG490, SSP isoflavone, soybean isoflavone) were the same as 2.1. AngⅡstimulation lasted 2h. Whole protein was extracted by SDS lysate. JAK2, STAT3 and phosphorylation protein expressions were detected by Western Blot technology.Results:1 The effect of SSP Isoflavone on rats'VSMC Proliferation induced by AngⅡ1.1 Identification of rats'VSMCCells swam out from tissues after 72h culture of VSMC and were radiated in clostridial forms, triangles or irregular. The cytoplasm were plenty and the nucleus were oval and in the center. There were several nucleolus and they grown up in a characteristic"peak valley"VSMC way. Large amount of buffy filaments parallel to the long axis of the endochylema were observed afterα-actin antibody marking. More than 97% cells were stained by SM-actin.1.2 The cytotoxicity of SSP IsoflavoneOD value was decreased significantly(P<0.01) when the drug concentration exceeded 10μg/ml and obvious cytotoxicity emerged(P<0.01). Most cells were shrinked to round shape and few of dead cells were floating in the holes.1.3 Effect of SSP Isoflavone on the AngⅡ-induced VSMC proliferation10-7mol/L AngⅡcould obviously promote VSMC proliferation (P<0.05 or P<0.01); preincubation cells by SSP isoflavone could inhibit VSMC proliferation induced by AngⅡin different levels: compared with AngⅡgroup, 50μg/L preincubated 12, 24h; 100μg/L preincubated 1, 4, 12, 24h; 200μg/L preincubated 1, 4, 24h; 500μg/L preincubated 4h could decrease the OD value significantly(P<0.05 or P<0.01). 10μg/L preincubated 1, 4, 12, 24h; 50μg/L preincubated 1, 4h; 200μg/L preincubated 12h; 500μg/L preincubated 1, 12, 24h, that the OD value tended to decline, but had no significant difference (P>0.05). In conclusion, the optimal concentrations of SSP isoflavone that inhibitted VSMC proliferation were 50, 100, 200μg/L and the optimal time was 24h.1.4 Effect of SSP Isoflavone on VSMC cell cycleCompared with control group, the percentage of cells in phase G0/G1 in AngⅡgroup was significantly decreased (P<0.01), while the percentage of cells in phase S was obviously increased (P<0.01); compared with AngⅡgroup, all drugs could obviously increase the percentage of cells in phase G0/G1(P<0.01), and decreased the percentage of cells in phase S significantly (P<0.05 or P<0.01).2 The Effect of SSP Isoflavone on JAK2/STAT3 signal transduction pathway in proliferated VSMC2.1 Effect of AG490, SSP Isoflavone and Soybean Isoflavone on AngⅡ-induced VSMC ProliferationOD value of AngⅡgroup was increased significantly than that in control group (P<0.01); compared with the AngⅡgroup, OD values of AG490 group, SSP isoflavone 100μg/L group, 200μg/L group and the soybean isoflavone group were decreased significantly (P<0.05 or P<0.01); the OD value of 50μg/L group tended to decline, but had no significant difference (P>0.05).2.2 Effect on the expression of VSMC AT1R mRNAExpression of AT1R in AngⅡgroup was increased significantly than that in control group (P<0.01); compared with the AngⅡgroup, expressions of AG490 group, SSP isoflavone 100μg/L group, 200μg/L group, soybean isoflavone 200μg/L group were obviously decreased (P<0.01); expression of SSP isoflavone 50μg/L group tended to decline, but had no significant difference (P>0.05).2.3 Effect on protein expression of VSMC JAK2/STAT3 signal transduction pathwayThe whole protein of JAK2, STAT3 didn't show significant difference in all groups (P>0.05). Protein expressions of p-JAK2, p-STAT3 in AngⅡgroup were obviously increased than that in control group (P<0.01, P<0.05); Compared with the AngⅡgroup, protein expressions of p-JAK2, p-STAT3 in AG490 group, SSP isoflavone 200μg/L group and soybean isoflavone group were obviously decreased (P<0.01 or P<0.05); protein expression of p-STAT3 in SSP isoflavone 50μg/L group was decreased significantly (P<0.01); protein expression of p-JAK2 tended to decline, but had no significant difference (P>0.05); protein expressions of p-JAK2, p-STAT3 in SSP isoflavone 100μg/L group were lower, but had no significant difference (P>0.05).Conclusions: SSP isoflavone (10~500μg/L) could obviously inhibit rats'VSMC proliferation induced by AngⅡ, among the total, SSP isoflavone of 50, 100, 200μg/L that effected 24h best inhibitted VSMC proliferation. The effect might be implemented by down regulating the expression of AT1R, thus arresting the phosphorylation of JAK2/STAT3 signal transduction pathway, and consequently marching the regulation of VSMC cell cycle. |
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