| Lipid metabolism is divided into anabolism and catabolism.The liver acts as a very important organ in the process of lipid metabolism.It acts as a transfer station in the process of fat metabolism,thus maintaining the decomposition and synthesis of fat in an orderly manner.When the balance between lipid synthesis and decomposition is disturbed,lipid metabolism disorders will occur.When excessive lipids are deposited in the liver,the occurrence of fatty liver will be induced.When fatty acids and their binding transporters are not transported to other tissues in time,the retention of blood can lead to atherosclerosis,which is the result of lipid metabolism disorders and oxidative stress.Atherosclerosis is the pathological basis of many cardiovascular and cerebrovascular diseases,but because the early clinical symptoms of the disease are not obvious,the condition is relatively hidden,and once the patient is found to be mostly in the middle and late stages of the disease,it is difficult to reverse.Atherosclerosis plaque stage can lead to the formation of local vascular thrombosis,plaque shedding often causes embolism phenomenon,which causes the occurrence of cardiovascular and cerebrovascular accidents.At present,there is no specific drug for the treatment of this disease in clinic,and many statins are used as adjuvant therapy,so prevention is the key to delay the occurrence of AS.The occurrence of non-alcoholic fatty liver is closely related to abnormal lipid metabolism.At the same time,there are clinical reports that the occurrence of non-alcoholic fatty liver will accelerate the formation of AS.It is believed that the deposition of abdominal and liver adipose tissue will aggravate the production of AS.It is suggested that obese and high-fat people should undergo routine abdominal color doppler ultrasonography,and that patients with confirmed non-alcoholic fatty liver should undergo neck color doppler ultrasonography to the early detection and intervention of AS.Hyperlipidemia is considered to be an important risk factor for the development of AS and nonalcoholic fatty liver disease.Previous research by the research team found that anhua dark tea can play a good role in reducing blood lipids in rats with high-fat diet,and found that anhua dark tea can inhibit expression of aortic inflammatory factors.Tea polyphenols,theaflavins,and caffeine rich in dark tea have a regulating effect on lipid metabolism,and the catechin-like components of dark tea produced during the fermentation of the scorpion are found to have statin-like components.Flavonoids in anhua dark tea have good anti-coagulation,promoting the activity of fibrinolytic system and inhibiting platelet formation.Dark tea has always been loved by the Northwest minority because of its good greasy effect,and there are investigations and studies that found that the incidence of cardiovascular and cerebrovascular diseases of ethnic minorities who love to drink dark tea is much lower than that of the mainland.Is it related to drinking dark tea?Can anhua dark tea also prevent and treat AS and non-alcoholic fatty liver?We don’t know,so we need to further verify and provide scientific evidence through relevant animal experiments and molecular biology experiments,in order to better reveal the medicinal value of dark tea.The experimental study is divided into five parts.The first part is to explore the methods of gene identification and model application of APOE knockout mice;the second part is mainly to explore the preventive effect of anhua dark tea on atherosclerosis;the third part is mainly to explore the therapeutic effect of anhua dark tea on atherosclerosis.The fourth part mainly discusses the preventive effect of anhua dark tea on non-alcoholic fatty liver;the fifth part mainly discusses the therapeutic effect of anhua dark tea on non-alcoholic fatty liver.Objective:To explore the mechanism of anhua dark tea in preventing atherosclerosis and non-alcoholic fatty liver formation.At the same time,the correlation between non-alcoholic fatty liver and atherosclerosis were studied.Methods:（1）APOE-/-male mice introduced from the Biomodel Center of Nanjing University were raised in the SPF Central Laboratory of Hunan University of Traditional Chinese Medicine.The DNA information was obtained by boiling lysis method,and the genotype was identified by PCR.（2）Fifty male APOE-/-mice of 8 weeks old were randomly divided into model group,atorvastatin group and high,medium and low dose groups of anhua dark tea.The high,middle and low doses of anhua dark tea were administered intragastrically at a dose of2.16 g.kg-1.d-1,1.44 g.kg-1.d-1,0.72 g.kg-1.d-1 respectively.The atorvastatin group was given 10 mg kg-1.d-1 drug for intragastric intervention.The model group was given the same dose of normal saline for gastric perfusion.The rats were fed with high-fat diet and continued to intervene for 17 weeks.Ten C57 male wild mice of the same background at 8 weeks of age were used as a blank control group for continuous feeding for 17 weeks,and an equal dose of normal saline was administered at the same time.At the end of the experiment,the mice in each group were used to take serum for the detection of blood lipids and related inflammatory factors.The aorta of mice were taken for HE staining and plaque area was calculated.The expression of TLR4、MyD88、NF-kB P50、NF-kB P65、MMP-9、TIMP-1、IL-1βin plaques were detected by immunohistochemistry.The gene expressions of TLR4,MyD88,NF-kB,IL-beta and ABCA1 in arterial tissues were detected by fluorescence quantitative RT-qPCR.（3）Fifty-five eight-week-old male APOE-/-mice were fed with western diet for 13weeks continuously.Another ten eight-week-old male C57 mice as normal group were fed with nomal diet for 13 weeks continuously.After 13 weeks,five mice were randomly selected for HE staining to determine the model.Then the mouse were randomly divided into model group,atorvastatin group and anhua dark tea of high,medium and low dose groups.they were given saline,atorvastatin with 10 mg.kg-1.d-1,2.16 g.kg-d-1,1.44 g.kg-1.d-1,0.72 g.kg-1.d-1 dose of anhua dark tea by gavage respectively.the blank group were given equal doses of saline by gavage,at the same time,the APOE-/-mice were continued to be fed with western diet for 8 weeks.at the end of experiment,take the eyeball for blood and separate the serum for the detect the blood lipids and related inflammatory factors.The aorta of mice were taken for HE staining and plaque area were calculated.The expression of TLR4、MyD88、NF-kB P50、NF-kB P65、MMP-9、TIMP-1、IL-1βin plaques were detected by immunohistochemistry.The gene expressions of TLR4,MyD88,NF-kB,IL-beta and ABCA1 in arterial tissues were detected by fluorescence quantitative PCR.（4）Fifty male APOE-/-mice of 8 weeks old were randomly divided into model group,atorvastatin group and high,medium and low dose groups of anhua dark tea.The high,middle and low doses of anhua dark tea were administered intragastrically at a dose of 2.16 g.kg-1.d-1,1.44 g.kg-1.d-1,0.72 g.kg-1.d-1 respectively.The atorvastatin group was given 10 mg kg-1.d-1 drug for intragastric intervention.The model group was given the same dose of normal saline for gastric perfusion.The rats were fed with high-fat diet and continued to intervene for 17 weeks.Ten C57 male wild mice of the same background at 8 weeks of age were used as a blank control group for continuous feeding for 17 weeks,and an equal dose of normal saline was administered at the same time.At the end of the experiment,the mice in each group were used to take serum for the detection of blood lipids.The body weight,liver weight,spleen weight and abdominal fat weight were weighed and the liver index was calculated.the tissue of liver were homogenized for detection of transaminase and oxidative stress indicators in homogenate.the liver of mice were taken for HE staining.In this experiment,HMGCR,PPAR-gamma and SCD-1 were selected to observe the effect of anhua black tea on lipid synthesis;LDLR was used to observe the effect of anhua dark tea on lipid uptake;ABCA1 was used to observe the effect of anhua dark tea on lipid output;and IL-beta was used to observe the effect of anhua dark tea intervention on liver inflammation.（5）Fifty-five eight-week-old male APOE-/-mice were fed with western diet for 13 weeks continuously.Another ten eight-week-old male C57 mice as normal group were fed with nomal diet for 13 weeks continuously.After 13 weeks,five mice of APOE-mice were randomly selected for HE staining to determine the model.Then the mouse were randomly divided into model group,atorvastatin group and anhua dark tea of high,medium and low dose groups.they were given saline,atorvastatin with 10 mg.kg-1.d-1,2.16 g.kg-d-1,1.44 g.kg-1.d-1,0.72 g.kg-1.d-1 dose of anhua dark tea by gavage respectively.the blank group were given equal doses of saline by gavage,at the same time,the APOE-/-mice were continued to be fed with western diet for 8 weeks.at the end of experiment,take the eyeball for blood and separate the serum for the detect the blood lipids and related inflammatory factors.The body weight,liver weight,spleen weight and abdominal fat weight were weighed and the liver index was calculated.the tissue of liver were homogenized for detection of transaminase and oxidative stress indicators in homogenate.the liver of mice were taken for HE staining.In this experiment,HMGCR,PPAR-gamma and SCD-1 were selected to observe the effect of anhua dark tea on lipid synthesis;LDLR was used to observe the effect of anhua dark tea on lipid uptake;ABCA1 was used to observe the effect of anhua dark tea on lipid output;and IL-beta was used to observe the effect of anhua dark tea intervention on liver inflammation.Results:（1）The astrocyte gel electrophoresis experiment showed that the target band of the knockout mice was consistent with the expected size of the target band.（2）the blood lipid level of model group mice was significantly higher than that of the blank control group.The white plaque of porcelain was visible near the aortic valve in model group,and a large amount of cholesterol crystals and foam cells appeared in the microscope.It is suggested that the model of atherosclerosis is successfully established.Compared with the blank group,the contents of TC,TG,LDL and HDL in serum of model group mice increased（P<0.05）;compared with model group,the contents of TG and LDL-C in different dosage groups of atorvastatin and dark tea decreased,while the contents of TC in atorvastatin group decreased（P<0.05）;compared with atorvastatin,the contents of TG and HDL in high and middle dosage groups of dark tea decreased（P<0.05）.Compared with the blank group,the serum levels of OX-LDL,IL-1beta,TNF-a,MCP-1 and hs-CRP in the model group increased（P<0.05）;compared with the model group,the serum levels of OX-LDL,IL-1beta,TNF-a,MCP-1 and hs-CRP in the different dosage groups of atorvastatin and black tea decreased（P<0.05）.Immunohistochemical experiments showed that TLR4,MyD88,MMP-9,NF-kB P50and NF-kB P65 were the strongest expressions in the aorta of model mice,while TIMP-1 was low.The expression of TLR4,MyD88,MMP-9,NF-kB P50 and NF-kB P65 in the aorta of model group mice was mainly concentrated in the artery endothelial cell plaque.The expression of TLR4,MyD88,MMP-9,NF-kB P50 and NF-kB P65 in the high and medium dose dark tea prevention group and atorvastatin prevention group was significantly lower than that in model group,However,the expression of TIMP-1 was higher（P<0.05）.RT-q PCR results showed that compared with model group,the gene expression of TLR4,MyD88,NF-kB,IL-beta in artery of mice in different doses of dark tea prevention group decreased,while the gene expression of ABCA1 increased.Compared with different doses of dark tea prevention group,the gene expression of TLR4,MyD88 and NF-kB in middle doses prevention group was the lowest.（3）The blood lipid level of model group was significantly higher than that of blank control group.The white plaque of porcelain was visible near the aortic valve in model group.A large number of cholesterol crystals and foam cells appeared in the microscope,it is suggested that the model of atherosclerosis is successfully established.Compared with the blank group,the serum levels of TC,TG,LDL and HDL in the model group increased（P<0.05）;compared with the model group,the levels of TG in the different dosage groups of dark tea decreased（P<0.05）;compared with atorvastatin,the levels of TG and HDL in the high and medium dosage groups of dark tea decreased（P<0.05）.Compared with the blank group,the serum concentrations of OX-LDL,MCP-1,TNF-alpha,hs-CRP and IL-1beta in the model group increased（P<0.05）;compared with the model group,the serum concentrations of OX-LDL,MCP-1,TNF-alpha,hs-CRP and IL-1beta in the different dosage groups of atorvastatin and black tea decreased（P<0.05）.The expression of TLR4,MyD88,MMP-9,NF-kB P50 and NF-kB P65 in the aorta of model group mice was the strongest,while TIMP-1 was low.The expression of TLR4,MyD88,MMP-9,NF-kB P50 and NF-kB P65 in the aorta of model group mice was mainly concentrated in the artery endothelial cell plaque.The expression of TLR4,MyD88,MMP-9,NF-kB P50 and NF-kB P65 in the high and medium dose black tea prevention group and atorvastatin prevention group was significantly lower than that in model group,However,the expression of TIMP-1 was higher（P<0.05）.RT-q PCR results showed that compared with model group,the gene expression of TLR4,MyD88,NF-kB and IL-beta in arteries of mice treated with different doses of anhua dark Tea decreased（P<0.05）.The expression of TLR4 gene was the lowest in the high and middle dose groups,MYD88 was the lowest in the high dose group,and the expression of NF-KB and IL-beta was the lowest in the middle dose group（P<0.05）.（4）The liver of the model group showed yellow greasy sensation with naked eyes,and vacuoles of different sizes were observed under the microscope,suggesting that the non-alcoholic fatty liver model was successfully prepared.Compared with the blank group,the levels of TC,TG,LDL and HDL in serum of model group mice increased（P<0.05）;compared with model group,the levels of TG and LDL-C in different dosage groups of atorvastatin and black tea decreased,while the levels of TC in atorvastatin group decreased（P<0.05）;compared with atorvastatin,the levels of TG and HDL in high and medium dosage groups of black tea decreased（P<0.05）.Compared with the model group,the body weight,liver weight,spleen weight and abdominal fat weight of mice in the blank control group,atorvastatin group and dark tea of high,medium and low dose group decreased（P<0.05）;compared with atorvastatin,the body weight,liver weight,spleen weight,abdominal fat weight and liver index of black tea high and medium dose group decreased（P<0.05）.Compared with the blank group,the vitality of ALT and AST in the liver of the model group increased（P<0.05）;compared with the model group,the vitality of ALT and AST in the liver of the atorvastatin group,high,medium and low dose prevention groups of anhua dark tea were lower（P<0.05）,and the vitality of ALT in the medium-dose group of dark tea was the lowest（P<0.05）.Compared with the blank group,the SOD and GSH-PX vitality in the liver of the model group decreased and the MDA contents increased（P<0.05）;compared with the model group,the SOD and GSH-PX vitality in the liver of the high,medium and low dose dark tea group increased,while the MDA contents decreased（P<0.05）;compared with the atorvastatin group,the SOD contents of the high dose black tea group increased（P<0.05）.Compared with the model group,the gene expression of HMGCR,SCD-1,PPAR-gamma and IL-βin the liver of mice in different doses of anhua dark Tea prevention group decreased,while the gene expression of ABCA1 increased,and LDLR had no difference with the model group.Compared with different dose groups,the expression of HMGCR was the lowest in medium dose prevention group（P<0.05）,while the expression of SCD-1 and PPAR-gamma in liver was the lowest in high and medium dose prevention group（P<0.05）.（5）The liver of the model group showed yellow greasy sensation with naked eyes,and vacuoles of different sizes were observed under the microscope,suggesting that the non-alcoholic fatty liver model was successfully prepared.Compared with the blank group,the serum levels of TC,TG,LDL and HDL in the model group increased（P<0.05）;compared with the model group,the levels of TG in the different dosage groups of black tea decreased（P<0.05）;compared with atorvastatin,the levels of TG and HDL in the high and medium dosage groups of black tea decreased（P<0.05）.Compared with the model group,the body weight,liver weight,abdominal fat weight,and liver index of the blank control group,dark tea of high and medium dose treatment group decreased（P<0.05）,The liver weight and liver index of the atorvastatin group decreased（P<0.05）.Compared with atorvastatin,the body weight and abdominal adipose tissue quality of the high and middle dose dark tea group decreased（P<0.05）,and the liver index of the middle dose group decreased（P<0.05）.and the body weight of the low dose treatment group was decreased（P<0.05）.Compared with the blank group,the vitality of ALT and AST in the liver of the model group was lower（P<0.05）;compared with the model group,the vitality of ALT and AST in the liver of the drug group was lower,and the vitality of ALT in the medium dose group of dark tea was the lowest,while that in the high dose group was the lowest（P<0.05）;compared with the atorvastatin group,the vitality of ALT and AST in the liver of the high and medium dose group of dark tea was lower（P<0.05）.Compared with the blank group,the vitality of SOD and GSH-PX in the liver of the model group decreased,while the content of MDA increased（P<0.05）;compared with the model group,the vitality of SOD and GSH-PX in the liver of the drug group increased and the content of MDA decreased,with the highest SOD in the high-dose group of dark tea,the lowest MDA in the medium-dose group of dark tea and the highest GSH-PX vitality in the atorvastatin group（P<0.05）.Compared with the model group,the gene expression of HMGCR,SCD-1,PPAR-gamma and IL-beta in the liver of mice in different doses of Anhua dark tea treatment group decreased（P<0.05）,while the gene expression of ABCA1increased（P<0.05）.The gene expression of LDLR in Anhua dark tea treatment group was not different from that in model group（P>0.05）.Conclusion:（1）DNA was obtained by boiling and lysing the mouse tail method,and DNA identification by agarose gel electrophoresis is an ideal method for rapidly and accurately identifying mouse gene types.（2）The mechanism of anhua dark tea in preventing and controlling AS formation may be related to lipid-lowering,anti-inflammatory,anti-oxidation and inhibition of TLR4/MyD88/NF-kB pathway.anhua dark tea can enhance the expression of TIMP-1 by inhibiting the expression of MMP-9 to play a stable role.（3）The mechanism of prevention and treatment of non-alcoholic fatty liver formation by anhua dark Tea may be through inhibiting the expression of HMGCR,SCD-1 and PPAR-gamma to reduce lipid synthesis,increasing the expression of ABCA 1 to promote lipid metabolism,and reducing oxidative damage through anti-inflammatory and anti-oxidation.Nonalcoholic fatty liver disease is positively associated with the onset of atherosclerosis. |
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