| ObjectiveTo observe the change of aortasinus plaque morphology and expression of LDLR and PPARy in atheromatous plaque,the change of hepatic morphology and expression of LDLR in hepatic cell and differences of oxidized low density lipoprotein and apolipoprotein B of mice serum among groups after acupuncture intervention in ApoE-/-mice and study mechanism of acupuncture on aortasinus plaque of ApoE-/-mice.Methods Experiment one:38ApoE-/-male mice at6weeks of age were divided randomly into34high fat and cholesterol diet mice group(HF group),which were feed on food containing21%fat and1.25%cholesterol,and4middle fat diet mice group(MF group),which were feed on food containing10%mice fat.One mouse in HF group died for improper feeding.After14weeks of feeding high fat forage,3mice selected randomly from HF group and4mice from MF group were all put to death.Plasma was collected to detect the content of blood lipid.Aortic sinus were taken,which were dyed by routine hematoxylin-eosin (HE) staining, frozen HE staining and oil red O staining methodes.Abdominal aorta were taken,which were dyed by routine HE staining.Then observe the change of aortasinus plaque morphology and judge whether atherosclerosis model were built successfully, and compare the change of Lee’s index, blood lipid, liver-to-body ratio, fat-to-body ratio and arterial pathological sections between HF group and MF group. Experiment two:The rest ApoE-/-30mice of HF group at20weeks of age were divided randomly into model control group(n=10,M group),pharmacy control group(n=10,F group) and acupuncture group(n=10,A group).Moreover,5C57BL/6J male mice were fed ordinary feed as blank control group (BC group).Pharmacy group were administered with simvastatin(40mg/kg,10ml/kg per day) by oral gavage. Acupuncture group are treated by low-frequency Electro-acupuncture at "neiguan","shenmen","housanli" and "sanyinjiao" point.After11weeks treatment, blood sampling were applied to detect the contents of blood lipid, oxidized low density lipoprotein and apolipoprotein B to compare the differences of blood lipid,oxidized low density lipoprotein and apolipoprotein B of serum.Besides that,their arterial aortas were separated and atherosclerosis lesions tissues were observed with HE and immunohistochemical staining to compare the differences of morphology change of aortasinus plaque and expression of PPARy and LDLR in atheromatous plaque among groups.And their hepar were separated,the hepatic tissues were observed with HE and immunohistochemical staining to compare the differences of morphology change of hepatic cell and expression of LDLR in hepatic cell among groups.The expression of LDLR protein in hepar were detected by western blotting among groups to compare the differences among groups.ResultsExperiment one:1. Testing result of Lee’s index, liver-to-body ratio, fat-to-body ratio of HF group and MF group: The Lee’s index, liver-to-body ratio, fat-to-body ratio of MF group were slightly higher than the HF group, but which had no significant difference.2. Testing result of blood lipid of HF group and MF group: The blood lipid level (TC, TQHDL-C,LDL-C) of MF group were slightly higher than the HF group, but which had no significant difference.3. Testing result of aortasinus sections of HF group and MF group: There were obvious aorta lipid plaque in aortic sinus of MF and HF group.There were a tremendous amount of lipid and foam cell accumulation,a large piece of necrotic core, willow leaf shape cholesterol crystals and the clear arrangement of elastic fiber of middle membrane in plaque.So the plaque of MF and HF group were early stage of fibrous plaque.4.Testing result of abdominal aorta’s slices of HF group and MF group: There were no lipid deposition in vascular intima in abdominal aorta of MF and HF group’ HE staining sections. MF group formed abdominal aneurysm.Experiment two:1.Testing result of Lee’s index, liver-to-body ratio, fat-to-body ratio of M, F, A and BC group: The Lee’s index and fat-to-body ratio of A group were lower than that of M group,which had significant differences (P<0.05, P<0.05).The liver-to-body ratio of A group were also lower than that of M group.2.Testing result of blood of M, F,A and BC group:2.1Testing result of blood lipid of M, F,A and BC group: The level of TC, TG, LDL of M, F and A group were higher than that of BC group,which had significant differences (P<0.05).The blood lipid level among M, F and A group were close, there was no significant difference.2.2Testing result of serum apoB, ox-LDL level of M, F and M, F,A and BC group: The apoB,ox-LDL content of A group were lower than that in M group,which had significant difference (P<0.05). The ox-LDL content of BC group were significantly lower than other groups.3.Testing result of aortasinus sections of M, F and A group:3.1Aortasinus sections of HE staining of M, F and A group: The size of aortic plaque and plaque-to-aorta ratio of A group were lower than that in M group,which had significant difference (P<0.05). Cells count in an unit view of aortasinus’ intima of A group was more than that in M group,which had significant difference (P<0.05). Cells count in an unit view of aortasinus’ tunica media of A group was more than that in M group.The maximum thickness of collagen fiber layer of A group was more than that in M group(P<0.05).Based on the status,acupuncture can reduce the lipid plaque area, inhibit apoptosis and necrosis of cells under the intima, the migration of smooth muscle cells and degradation of collagen fibers, significantly inhibited the development of plaque.3.2Aortasinus sections of PPARy immunohistochemical staining of M, F and A group: Cells expressing PPARy protein in an unit view of aortasinus’ intima of A group were more than that in M group. Cells expressing PPARy protein in an unit view of aortasinus’ tunica media of A group were more than that in M group,which had significant difference (P<0.05). Based on these,acupuncture can improve expression of PPARy protein in plaque of aortasinus.3.3Aortasinus sections of LDLR immunohistochemical staining of M, F and A group: Cells expressing LDLR protein in an unit view of aortasinus’ intima of A group were similar to that in M group,which had no significant difference.Based on that,acupuncture had no obvious effect on expression of LDLR protein in plaque of aortasinus.4.Testing result of liver sections of M, F,A and BC group:4.1Liver sections of HE staining of M, F,A and BC group: Hepatic cell count in an unit view of hepatic acinus zone3in A group was more than that in M group,which had significant difference (P<0.05). Hepatic cells of hepatic acinus zone3in A group did not appear large particle size fatty droplets,which were appeared hepatic cells of M group. Based on these, acupuncture can inhibit apoptosis and necrosis of liver cells,reduce dimension of lipid droplets and improve the inflammatory state.4.2Liver sections of LDLR immunohistochemical staining of M, F,A and BC group: Hepatic cells expressing LDLR protein in an unit view of hepatic acinus zone3in A group was more than that in M group,which had significant difference (P<0.05). Based on that,acupuncture can improve the expression of LDLR protein in liver cells.5.Testing result of liver’s LDLR protein with Western blotting hybridization of M, F,A and BC group: Acupuncture can improve the expression of LDLR protein in liver.ConclusionAcupuncture can prevent development of plaque by regulating lipid and energy metabolism of liver and blood vessels,to prevent oxidation,fight inflammation, inhibit smooth muscle cell migration, proliferation and prevent plaque rupture, Inhibit apoptosis and necrosis of cells,etc. Acupuncture can play therapeutic effect by reinforcing spleen,replenishing qi,reducing phlegm and removing blood stasis from TCM perspective. |
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