The Interventional Effect And Possible Mechanisms Of Baicalin On Atherosclerosis In ApoE-deficient Mice Fed A High Cholesterol Diet

Part I Effect of baicalin on atherosclerotic plaques in ApoE-/- mice fed a high cholesterol dietObjective:To observe the effect of baicalin on inhibiting the progress of atherosclerotic plaques.Methods:Ten wild type C57BL/6J mice (six weeks old, male) and twenty ApoE-/-mice on C57BL/6J background (six weeks old, male) were purchased and fed two weeks to adapt the environment. At the age of eight weeks old, C57BL/6J mice fed a normal chow daily received gavage with normal saline as a normal control group (NC group, n=10). The ApoE-/- mice fed a high cholesterol (HC) diet were randomly assigned to two groups:the model group in which mice daily received gavage with normal saline (HC group, n=10) and the treatment group in which mice daily received gavage with baicalin (100mg/kg) (BAI group, n=10). Mice were treated for twelve weeks with free access to drinking water and feedstuff. At the end of treatment, mice were euthanized, and hearts were rapidly removed. The heart with aortic root was prepared for frozen sections in optimal cutting temperature (OCT) compound. Oil red O staining was performed to detect plaques.Results:After twelve-week treatments, we found no lipids depositions on arterial wall of C57BL/6J mice in NC group. ApoE-/- mice in HC group exhibited extensive atheromatous plaques adhering to the inner surface of arterial wall. However, baicalin treatment significantly decreased the area of plaques in ApoE-/- mice.Conclusion:Baicalin significantly attenuates the progress of atheromatous plaques in ApoE-/- mice fed a HC diet.Part ? Effect of baicalin on serum lipids and key factors involved in cholesterol metabolism in ApoE-/- mice fed a high cholesterol dietObjective:To investigate the effect of baicalin on decreasing lipids and regulating key factors involved in the process of cholesterol metabolism.Methods:Ten wild type C57BL/6J mice (six weeks old, male) and twenty ApoE-/-mice on C57BL/6J background (six weeks old, male) were purchased and fed two weeks to adapt the environment. At the age of eight weeks old, C57BL/6J mice fed a normal chow daily received gavage with normal saline as a normal control group (NC group, n=10). The ApoE-/- mice fed a high cholesterol (HC) diet were randomly assigned to two groups:the model group in which mice daily received gavage with normal saline (HC group, n=10) and the treatment group in which mice daily received gavage with baicalin (100mg/kg) (BAI group, n=10). Mice were treated for twelve weeks with free access to drinking water and feedstuff. At the end of treatment, mice were anesthetized, and blood was collected to test serum levels of total cholesterol (TC), triglycerides (TG), and high density lipoprotein cholesterol (HDL-C) using an automatic biochemistry analyzer. Low density lipoprotein cholesterol (LDL-C) concentrations were calculated by Friedewald formula. After mice were euthanized, livers were rapidly removed. Real time RT-PCR was used to detecting 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reducase, LDL receptor (LDLR), proprotein convertase subtilisin kexin (PCSK) 9, liver X receptor (LXR), ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1).Results:After twelve-week treatments, ApoE-/- mice in HC group exhibited much higher levels of TC and LDL-C, and much lower levels of HDL-C compared with C57BL/6J mice. Baicalin treatment seemingly decreased TC and LDL-C but had no statistical significance. Serum levels of TG among three groups had no differences. The mRNA detection on key factors involved in lipid metabolism showed that baicalin could dramatically upregulate mRNA expression of LXR, ABCA1 and ABCG1 with no affecting on HMG-CoA reducase, LDLR and PCSK9.Conclusion:Twelve-week treatment of Baicalin does not decrease serum levels of lipids. However it significantly upregulates the mRNA expression of key factors (LXR, ABCA1 and ABCG1) which are important for cholesterol efflux. This probably promotes the inhibitory effect of baicalin on plaque formation.Part ? Effect of baicalin on regulating the number and function of regulatory T cells in ApoE-/- mice fed a high cholesterol dietObjective:To investigate whether baicalin can increase the number and function of regulatory T cells in ApoE-/- mice fed a high cholesterol diet.Methods:Ten wild type C57BL/6J mice (six weeks old, male) and twenty ApoE-/-mice on C57BL/6J background (six weeks old, male) were purchased and fed two weeks to adapt the environment. At the age of eight weeks old, C57BL/6J mice fed a normal chow daily received gavage with normal saline as a normal control group (NC group, n=10). The ApoE-/- mice fed a high cholesterol (HC) diet were randomly assigned to two groups:the model group in which mice daily received gavage with normal saline (HC group, n=10) and the treatment group in which mice daily received gavage with baicalin (100mg/kg) (BAI group, n=10). Mice were treated for twelve weeks with free access to drinking water and feedstuff. At the end of treatment, mice were anesthetized, and blood was collected to test serum levels of transforming growth factor beta (TGF-0) and interleukin 10 (IL-10) using enzyme-linked immunosorbent assay (ELISA). After mice were euthanized, spleens, hearts and thoracoabdominal aortas were rapidly removed. Mononuclear cells isolated from fresh spleens were adjusted to a suspending liquid with a density of 106 cells/mL. Primary antibodies including FITC-conjugated anti-CD4, APC-conjugated anti-CD25 and PE/Cy5-conjugated anti-Foxp3 antibodies were used to mark regulatory T cells (Tregs). The proportion of Tregs was analyzed by flow cytometry and FlowJo software. Expression of forkhead box p3 (Foxp3), the key regulator of Tregs, was detected using real time RT-PCR and western blot in aortas, and immunohistochemistry (IHC) in cryosections of aortic sinus.Results:Serum levels of TGF-? and IL-1 had no statistical difference between NC group and HC group. Baicalin treatment significantly increased serum levels of TGF-? and IL-10. Proportion of Tregs in splenic T cells from HC group was significant increased compared with that from NC group. However, baicalin treatment further increased Tregs proportion in splenic T cells. Moreover, mRNA and protein expression of Foxp3 in aortas from HC group was higher than that from NC group, but much lower than that from baicalin treatment group. IHC staining showed a few Tregs in plaques of mice from HC group and a significantly increased number of Tregs in plaques of mice from BAI group.Conclusion:Baicalin upregulates expression of Foxp3 both at the transcriptional and translational level, and significantly increases the number and function of Tregs in ApoE-/-mice fed a HC diet.Part ? Effect of baicalin on regulating Foxp3 at post-translational levelObjective:To investigate whether baicalin promotes Foxp3 acetylation at post-translational level in ApoE-/- mice fed a high cholesterol diet.Methods:Twenty-five wild type C57BL/6J mice (six weeks old, male) and forty ApoE-/-mice on C57BL/6J background (six weeks old, male) were purchased and fed two weeks to adapt the environment. At the age of eight weeks old, C57BL/6J mice fed a normal chow daily received gavage with normal saline as a normal control group (NC group, n=25). The ApoE-/- mice fed a high cholesterol (HC) diet were randomly assigned to two groups:the model group in which mice daily received gavage with normal saline (HC group, n=25) and the treatment group in which mice daily received gavage with baicalin (100mg/kg) (BAI group, n=15). Mice were treated for twelve weeks with free access to drinking water and feedstuff. At the age of 8,12,16 and 20 weeks, real time RT-PCR was used to compare the mRNA changes of histone deacetylase (HDAC) 9 and HDAC6 in aorta between NC group and HC group. We also detected mRNA and protein expression of HDAC9 and HDAC6 in aorta from BAI group at the end of treatment. After mice were euthanized, spleens, hearts and thoracoabdominal aortas were rapidly removed. Mononuclear cells isolated from fresh spleens were used for immunoprecipitation (IP) and western blot to test the acetylation of Foxp3. Immunofluorescence (IF) double staining was used to test the acetylation of Foxp3 in aortic sinus.Results:Compared with NC group, HDAC9 and 6 were increased continuously in aorta from HC group, and much higher than that from NC group in the middle and later period. Baicalin could significantly downregulate the mRNA and protein expression of HDAC9 and 6. Both IP and IF assay showed that Foxp3 acetylation was decreased in HC group and increased in BAI group compared with that in NC group.Conclusion:Baicalin significantly promotes Foxp3 acetylation at post-translational level partly via downregulating expression of HDAC9 and 6.