Protective Effects Of Flavonoids From Polygonum Capitate On Blood Lipid Disorder,Liver Injury And Atherosclerosis In Rats

Background:Polygomun capitutam(PC)is a traditional medicinal plant of the Miao nationality.It has protective and preventive effects on many diseases and biological regulation.Flavonoids are the main active constituents of Polygonum capitate,which can extract many kinds of Flavonoids from Polygonum capitum.Flavonoids have polyphenolic structure in chemical structure.This structure gives them the function of anti-oxidation,inhibition of inflammation and regulation of blood lipid level.Flavonoids in many plants can protect cancer,cardiovascular disease,gastrointestinal diseases,nervous system related syndromes such as depression,epilepsy,Alzheimer's disease and neurodegenerative diseases.In addition,flavonoids in plants can also significantly regulate blood lipid levels in humans and animals.Hyperlipidemia is a risk factor for liver disease and atherosclerosis(AS).The protective effects of Flavonoids from Polygonum capitum on blood lipid disorder,liver injury and atherosclerosis in rats were studied by building a rat model of high fat and a AS model.Part one:Protection of Flavonoids from Polygonum capitate on dyslipidemia and liver injury in hyperlipidemic rats.Objective:To study the protective effects of Flavonoids from Polygonum capitate on dyslipidemia and liver injury in rats with hyperlipidemia.Methods:Ultrasonic extraction of Flavonoids from Polygonum capitate was carried out.The experimental animals were 7 month old SPF grade male Wistar rats,which were divided into blank control group,model group,low dose Polygonum capitum,middle dose group of Polygonum capitum,high dose of Polygonum capitum and positive control group,10 rats in each group.The rats in the blank control group were fed normal diet,while the rest groups were fed high-fat diet for 30 days.After the high fat model,the low dose group of Polygonum capitum,the middle dose group of Polygonum capitum and the high dose group of Polygonum capitum were treated with 1.4mg/kg-bw,2.8mg/kg-bw and 5.6mg/kg-bw respectively.The positive control group was given the Atorvastatin of 125mg/kg.d,the control group and the model group were intragastric 401m/kg daily saline.After 28 days of intervention,the rats were killed after a fasting and no water 12h.ELISA kit was used to detect the activity of alanine aminotransferase(alanine aminotransferase,ALT)and aspartate aminotransferase(aspartate aminotransferase,AST)in the plasma of each group.The content of serum total cholesterol(total cholesterol,TC),total glycerol three fat(triglyceride,TG),high density lipoprotein(high-density lipoprotein,HDL),low density lipoprotein(low density lipoprotein,LDL)were detected on the automatic biochemical analyzer.The activity of superoxide dismutase(superoxidedismutase,SOD),the activity of glutathione peroxidase(glutathione peroxidase,GSH-Px),the content of malondialdehyde(malondialdehyde,MDA)and the content of catalase(catalase,CAT)in liver homogenate were detected by hydroxylamine method,colorimetric method,TBA method and visible light method.The levels of interleukin-1 beta(interleukin 1 beta,IL-1 beta),interleukin-18(interleukin 6,IL-18),tumor necrosis factor(Tumor necrosis factor-alpha,TNF-alpha)and interleukin-6(6))were detected by ELISA kit.Liver biopsy was made by HE staining,and liver injury was observed by electron microscope.After taking the intact liver,the liver index was calculated by liver weight(g)/body weight(100g).Results:1.Blood lipid level:The levels of TC,TG and LDL in the model group were significantly higher than those in the normal control group,and HDL was significantly lower than that in the normal control group.The level of TC and LDL in the middle,high dose group and positive control group of Polygonum capitum was significantly lower than that of the model group.The level of TG in the low,middle and high dose groups of Polygonum capitum and the positive control group was significantly lower than that of the model group.The HDL level of the high dose group of Polygonum capitum and the positive control group was significantly higher than that of the model group.2.Liver function enzyme:The level of ALT and AST in the model group was significantly higher than that in the normal control group.The level of serum ALT and AST in the low,middle and high dose groups of Polygonum capitum was significantly lower than that of the model group.3.Liver tissue section:The fat vacuoles and slight liver damage were seen in the liver tissue section of the model group.The high dose group of Polygonum capitum and the positive control group had no fat vacuoles,liver injury and liver fibrosis.4.Liver index:The liver index of the model group was significantly higher than that in the normal control group.The liver index of the low,middle,high dose and positive control groups of Polygonum capitum was significantly lower than that of the model group.5.Oxidative stress index:The activity of SOD,CAT and GSH-Px in model group was significantly lower than that in normal control group,and MDA activity was significantly higher than that in normal control group.The activity of SOD in middle,high dose group and positive control group of Polygonum capitum was significantly higher than that of model group.The activity of CAT and GSH-Px in high dose and positive control group of Polygonum capitum was significantly higher than that in model group,and the activity of MDA was lower than that of model group.6.Inflammatory indices:The level of IL-1 beta,IL-6,IL-18 and TNF-alpha in the model group was significantly higher than that in the normal control group.The IL-1 beta level of the low,middle and high dose groups of Polygonum capitum and the positive control group was significantly lower than that of the model group.The level of IL-6 and TNF-a in the high dose group of Polygonum capitum and the positive control group was significantly lower than that in the model group.The middle and high dose groups of Polygonum capitum were in the high dose group.The level of IL-18 in positive control group was significantly lower than that in model group.Conclusion:High fat model dry prognosis,the increase of blood lipid level,liver function enzyme,liver pathological damage,liver inflammatory factors and oxidative stress levels increase,suggesting that hyperlipidemia can lead to liver injury and liver inflammation and oxidative stress.Flavonoids extracted from Polygonum capitate can improve the blood lipid level,inflammatory factors and oxidative stress in rats,and protect the liver of rats.The second part:The effect and mechanism of Flavonoids from Polygonum capitum on atherosclerosis in rats.Objective:To study the protective effect of Flavonoids from Polygonum capitum on atherosclerosis,and the relationship between flavonoids and NF-kappa B/PI3K/AKT signaling pathway in Polygonum capitum,and explore the possible regulatory mechanism in the atherosclerosis model rats.Methods:The experimental animals were divided into 6 groups:normal control group,AS model group,low dose Polygonum capitum,middle dose group of Polygonum capitum,high dose of Polygonum capitum and 6 groups of positive control group,10 male rats in each group.The normal group was fed normal diet,and the other 5 groups were modeled.Vitamin D 3 million 600 thousand IU/kg was injected intraperitoneally once before feeding,and then fed high-fat diet for 8 weeks.ELISA kit was used to detect the content of Prostacyclin2(PGI2),thromboxane A2(Thromboxane2,TXA2)and endothelin-1(Endothelin1,ET-1)in the plasma of each group.The blood viscosity(high,medium and low shear rate),plasma viscosity and hematocrit(HCT)were measured by blood rheology analyzer.The Medlab bioinformatics amplification acquisition system was used to detect the diastolic function of thoracic aorta.HE staining of rat thoracic aorta was performed under 400 electron microscope.Image-Pro Plus 6 software was used to analyze the HE staining results and detect the intima media thickness of the aorta.The expression level of PI3K protein(p85 and P110),AKT protein and NF-kappa B(p65)protein in the thoracic aorta of rat was detected by Western blot method.The relative expression levels of p85,AKT and p65 mRNA were detected by RT-PCR.Results:1.Blood lipid level:The levels of TC,TG and LDL in model group were significantly higher than those in normal control group.The level of TC and TG in the low,middle,high dose and positive control groups of Polygonum capitum was significantly lower than that of the AS model group.The LDL level in the middle,high dose group and the positive control group of Polygonum capitum was significantly lower than that of the model group.2.Blood inflammatory index:The level of IL-6 and TNF-alpha in the AS model group was significantly higher than that in the normal control group.The levels of IL-6 and TNF-a in the middle,high dose and positive control groups of Polygonum capitum were significantly lower than those in the AS model group.3.Comparison of hemorheology indexes:Blood viscosity,plasma viscosity and HCT of AS model group were significantly higher than those of normal control group.In the middle dose group,the whole blood viscosity and HCT of low cut edge rate were significantly lower than those of AS model group.The whole blood viscosity,plasma viscosity and HCT of high dose group and positive control group were significantly lower than those of AS model group.4.Vascular endothelial active factor:The level of TXA2 and ET-1 in the AS model group was significantly higher than that in the normal control group.The level of TXA2 and ET-1 in the high dose group of Polygonum capitum and the positive control group was significantly lower than that in the AS model group.5.The function of thoracic aorta diastolic function:With the increase of Ach concentration,the difference of endothelia relaxation of each group gradually increased.When the concentration of Ach was 10-5mol/L and 10-4mol/L,the difference was statistically significant.The vasodilation of vascular endothelial cells in the AS model group was significantly lower than that in the normal control group.The vascular endothelial vasodilation in the middle,high dose group and the positive control group was significantly higher than that of the AS model group.6.Aorta HE staining results:AS model group aortic stenosis,intima thickening,significant atherosclerotic lesions.The degree of atherosclerosis in aorta was reduced in low and medium dose groups.There was no significant injury of aorta in high dose group and positive control group.7.The thickness of the medial membrane of the aorta:The thickness of the middle membrane in the AS model group was significantly higher than that in the normal control group.The thickness of the middle middle membrane in the high dose group of Polygonum capitum and the positive control group was significantly lower than that in the model group.8.Signal pathway related protein:The relative expression level of P110 protein,p65 protein and Akt protein in AS model group was significantly higher than that in normal control group.The relative expression level of P110 protein in high dose group of Polygonum capitum was significantly lower than that of AS model group.The relative expression level of p65 protein in high dose group of Polygonum Polygonum and positive control group was significantly lower than that of AS model group.The relative expression level of Akt protein in Polygonum capitate,high dose group and positive control group was significantly lower than that in AS model group.9.Signal pathway-related mRNA:The relative expression levels of p85 mRNA,p65 mRNA and Akt mRNA in AS model group were significantly higher than those in normal control group.The relative expression levels of p85 mRNA in high-dose group and positive control group were significantly lower than those in AS model group.Significantly lower than the AS model group.Conclusion:The flavonoids in Polygonum capitum can reduce the level of blood lipid,the level of inflammatory factors,blood viscosity and hematocrit,can reduce the level of vasoactive factors and improve the diastolic function of the aorta,and can reduce the thickness of the middle membrane of the aorta and improve the degree of atherosclerosis in rats.It can reduce the levels of PI3K-p85,PI3K-p110,Akt and NF-kappa B-p65 in the aorta,suggesting that it has a regulatory effect on PI3K/Akt/NF-kappa B signaling pathway.