Effect Of DFMG On The Phenotype Transformation Of Vascular Smooth Muscle In Atherosclerotic Mice

Objective:To verify whether 7-difluoromethoxy-5 chrysoflavin(7-difnuoromethoxy-5)-dimethoxyoxygenisteine(DFMG)could inhibit the contractile to synthetic phenotype transformation of vascular smooth muscle cell(VSMC).Methods:1.Thirty healthy male wild type and ApoE-/-mice were randomly divided into five groups(6 mice per group):wild type mice normal diet group,wild type mouse high-fat vehicle group,wild type mouse high-fat DFMG group,ApoE-/-mouse high-fat vehicle group,and ApoE-/-mouse high-fat DFMG group.PCR and agarose gel electrophoresis were used to identify the phenotype of mice in each group.At the same time,DMSO 10 mg/(kg·d)was added to the vehicle group,and DFMG 10 mg/(kg·d)was added to the DFMG group for 12 weeks.2.The weight of each group was measured at the end of the experiment.The supernatants were used to measure total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),and high-density lipoprotein(HDL).3.Oil red O staining was used to detect the percentage of thoracic aortic intimal plaques in the aortic area;HE staining was used to observe the morphological changes of the aorta in each group;Above comprehensive judgment of C57,ApoE-/-mice can successfully establish atherosclerosis model.4.Masson staining was used to determine the collagen fibers of the thoracic aortic plaques in each group.5.Western-blot and immunohistochemistry were used to observe the expression of contraction-type a-SMA and synthetic marker OPN in vascular smooth muscle cells.Results:1.The amplified fragments of ApoE and wild type mouse gene were 245bp and 155bp respectively.It was confirmed to be characteristic fragments of ApoE gene knockout and wild mouse.Identification of mouse bases due to ApoE gene deletion homozygote and wild type.2.Blood lipid results showed that there was no significant difference in serum lipids between the C57 vehicle group and the C57 DFMG group,compared with C57 control group(P ＞ 0.05).Serum TC,TG,and LDL levels in ApoE vehicle group and ApoE-/-DFMG group were significantly increased(P ＜ 0.05).Compared with the ApoE-/-vehicle group,the serum HDL level in the ApoE-/-DFMG group increased,and the TC,TG,and LDL decreased.(P ＜ 0.05).At body weight level,compared with C57 control group,the weight of the other four groups of mice was significantly increased(P＜ 0.05).There was no significant difference between the body weight of C57 vehicle group and C57 DFMG group(P ＞ 0.05),there was no significant difference in body weight between the ApoE-/-DFMG group and the ApoE-/-vehicle group(P＞ 0.05).3.HE staining and oil red O staining showed that there were obvious lipid plaques in the thoracic aorta of ApoE-/-vehicle and ApoE-/-DFMG mice,and there was no formation of lipid plaques in the thoracic aorta of C57 mice.It has been demonstrated that ApoE-/-mice can successfully build an atherosclerotic model.Compared with the ApoE-/-vehicle group,the percentage of plaque area in the aortic intima of the ApoE-/-DFMG group was significantly reduced(P ＜0.05).4.Masson staining showed that smooth muscle collagen fibers in the other four groups of mouse vascular smooth muscle cells were significantly increased(P ＜ 0.05),compared with the C57 control group,C57 DFMG group compared with the C57 vehicle group had no significant difference in collagen content(P ＞0.05).Compared with ApoE-/-vehicle group,the collagen fiber content of mice in ApoE-/-DFMG group was significantly increased(P ＜ 0.001).5.Western blot and immunohistochemistry results indicated that there was no significant difference in a-SMA protein expression between C57 vehicle group and C57 DFMG group(P＞0.05),compared to C57 control group.The expression of OPN in C57 DFMG group was significantly lower than that in C57 vehicle group(P ＜ 0.05).Compared with ApoE-/-vehicle group,ApoE-/-DFMG group increased the expression of contraction phenotype α-SMA in vascular smooth muscle cells(P ＜ 0.05),and decreased the expression of synthetic phenotype OPN(P ＜ 0.05).Conclusions:1.ApoE-/-mice which fed with a high-fat diet were successfully established AS mouse model.DFMG could improve the metabolism of blood lipids and reduce the formation of atherosclerotic plaque.2.DFMG could inhibit the conversion of a contraction phenotype to a synthetic phenotype in atherosclerotic mouse vascular smooth muscle cells.