| Purpose:To explore the mechanism of spleen-invigorating and phlegm-eliminating prescription(JPQT)on VSMCs in mice with spleen deficiency and phlegm-turbid syndrome of AS,to provide more theoretical and experimental basis for JPQT prescription in the prevention and treatment of AS lesions.Material and method:The mice were divided into groups after 1 week of adaptive feeding.Eight 8-week-old SPF male C57BL/6J served as normal control group,24 8-week-old SPF male Apo E-/-mice were randomly divided into 3 groups with 8 mice in each group,the 3 groups name are model group,positive control group and JPQT group,The mice in the normal control group were fed with normal diet,and the mice in the other three groups were fed with high fat diet.At the same time,and were given single cage and small cage to restrict activities,and each group was free to eat and drink,and at the same time,the drug was given by intragastric administration,and the model group and normal group were perfused with the same amount of normal saline,the JPQT group was given 11.88g/(kg · d)JPQT prescription,and the positive control group was given 2.4mg/(kg ·d)Atto vastatin calcium solution,the gastric perfusion of mice in each group was calculated according to their body weight,intragastric administration of 0.2ml every 10 g,twice a day for 12 weeks,the samples were drawn in the13 th week.The general condition of the mice was observed every day,the body weight of the mice was measured every Wednesday,and the grasping force,fecal score and fecal water content were measured every two weeks.The content of PCNA in aorta was detected by Elisa,the content of α-SMA,MMP-2 and MMP-9 in aorta was detected by Western-blot,and the expression of α-SMAm RNA,MMP-2m RNA and MMP-9m RNA was detected by Real-time PCR.Results:1.General condition of mice1.1 observation of general stateThe mice in the normal control group had normal diet,sensitive response,agile action,bright coat color,no hair removal and so on.The mice in the model group were fed for 5-6weeks,there are some phenomena,such as reduced eating,tiredness,lethargy,slow movement,dark hair,local hair removal,loose stools and so on.The mice in the positive control group were fed for about 6 weeks,there were some phenomena,such as slightly slow response,less food intake,less active degree,slightly dark hair,thinning and softening of stool and so on.In the later stage of the experiment,the coat color was slightly dark,a small amount of hair removal,loose stool and so on.The mice in JPQT group were fed for about 6 weeks,in some cases,the reaction is slightly slow,the degree of activity is slightly reduced,eating is slightly less than before,the hair is slightly dark,and a few mice have sparse stools.In the later stage of the experiment,the coat color of mice returned to normal,and their stools basically returned to normal.1.2 Results of grip measurement of mice in each groupAt the 4th week,there was no statistical significance in the grip of mice in each group.At the 7th week,compared with the normal control group,the grip of mice in model group,positive control group and JPQT prescription group decreased significantly(P < 0.01).the difference is statistically significant.At the 10 th and 13 th weeks,compared with the normal control group,the grip of mice in the model group and positive control group decreased significantly(P < 0.01).,the difference was statistically significant.Compared with the model group,the grip of the JPQT group increased significantly(P < 0.01).,and the difference was statistically significant,but there was no significant difference in the positive control group.1.3 Body weight of mice in each groupAt the 1st and 4th week,the body weight of the mice in each group was not statistically significant,at the 7th and 13 th week,the body weight of the model group was significantly higher than that of the normal control group(P < 0.01),the difference was statistically significant,Compared with the model group,the body weight of mice in the positive control group and JPQT prescription group decreased significantly(P < 0.01),the difference was statistically significant,and at the 10 th week,the body weight of the model group was significantly higher than that of the normal control group(P < 0.05),the difference was statistically significant,compared with the model group,the body weight of mice in JPQT prescription group decreased significantly(P < 0.05),the difference was statistically significant,but the body weight of the positive control group was not statistically significant.1.4 Fecal water content and Bristol fecal scale of mice in each group1.4.1Fecal water content of mice in each groupAt the 4th,7th,10 th and 13 th weeks,compared with the normal control group,the fecal water content of mice in the model group increased significantly(P < 0.01),the difference was statistically significant,and the fecal water content in the positive control group increased significantly(P < 0.01),the difference was statistically significant,and the fecal water content in the JPQT prescription group was significantly lower than that in the model group(P <0.01),the difference was statistically significant,There was no significant difference in fecal water content in the positive control group.1.4.2 Bristol stool scale of mice in each groupAt the 4th week,compared with the normal control group,the stool score of the model group was significantly higher than that of the normal control group(P < 0.05),but there was no significant difference among the other groups.At the 7th week,compared with the normal control group,the stool scores of mice in the model group,positive control group and JPQT prescription group were significantly higher than those in the normal control group(P <0.01).At the 10 th and 13 th week,compared with the normal control group,the fecal scores of the model group and the positive control group were significantly higher than those of the normal control group(P < 0.01),the difference is statistically significant,Compared with the model group,the stool score of the JPQT prescription group was significantly lower than that of the model group(P < 0.01),the difference is statistically significant,The stool score of the positive control group was not statistically significant.2.HE stainingIn the normal control group,the tissue structure of aorta was normal and there was no AS plaque formation.Compared with the normal control group,there was obvious AS atherosclerotic plaque formation in the aorta of the model group.Compared with the model group,the structure of the aorta in the positive control group and JPQT group was basically normal,the intima was relatively smooth,the wall of the aorta was not significantly thickened,the epithelial cells were arranged relatively neatly,and the proliferation,degeneration and necrosis and plaque formation were significantly improved.The results showed that the model of AS mice was successful,and JPQT prescription could significantly improve the pathological changes of AS in mice.3.The PCNA content in the aorta was determined by ElisaCompared with the normal group,the PCNA content in the model group was significantly increased(P <0.01),the difference is statistically significant;Compared with the model group,the content of PCNA in the positive control group and JPQT prescription group decreased significantly(P < 0.01),the difference is statistically significant.4.The expression levels of α-SMA,MMP-2,and MMP-9 in aortic tissues were determined by Western-blot analysisCompared with the normal control group,the expression of α-SMA,MMP-2 and MMP-9protein in the model group increased significantly(P < 0.01),the difference is statistically significant.Compared with the model group,the expression of α-SMA,MMP-2 and MMP-9protein in JPQT prescription group and positive control group decreased significantly(P <0.01),the difference is statistically significant.5.Real-time PCR measured the m RNA expression levels of α-SMA,MMP-2,and MMP-9 in the aortic tissuesCompared with the normal control group,the expression of α-SMAm RNA,MMP-2m RNA and MMP-9m RNA in the aorta of the model group increased significantly(P <0.01),the difference is statistically significant.Compared with the model group,the expression of α-SMAm RNA,MMP-2m RNA and MMP-9m RNA in JPQT group and positive control group decreased significantly(P < 0.01),the difference is statistically significant.Conclusion:1.JPQT prescription can improve the general condition of AS mice,increase grip,reduce body weight,fecal water content and Bristol stool score,and it also can improve the formation of aortic plaque in AS mice.2.JPQT prescription can inhibit the expression of PCNA,α-SMA,MMP-2 and MMP-9 proteins related to abnormal proliferation and migration of VSMCs,the mechanism of therapeutic effect on AS may be related to its regulation of abnormal proliferation and migration of VSMCs. |
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