| ObjectiveHyperlipidemia(HLP)can be defined as elevation of serum total cholesterol(TC)and/or triglyceride(TG)due to abnormal lipid transport or metabolism.Cardiovascular diseases(CVD)are disorders of the heart and blood vessels which consist of coronary heart disease,cerebrovascular disease,rheumatic heart disease and other conditions.HLP is one of the important causes of CVD.CVD mortality rate still ranked first among all diseases in the world.Effective treatment of hyperlipidemia can reduce the morbidity and mortality of CVD.Statins are commonly used to treat hyperlipidemia in clinic at present.however,long-term use of statins can cause side effects such as rhabdomyolysis,hepatotoxicity and so on.Therefore,it is of great significance to find safe and effective hypolipidemic drugs.Garlic is the bulb of Allium in liliaceae.It and its extracts have many biological effects,such as antimicrobial,antiviral,antihypertensive,hypolipidemic,prevention and treatment of atherosclerosis based on some studies.Garlic oil contains many organic sulfur compounds,such as diallyl sulfur(DAS),diallyl disulfide(DADS)and diallyl trisulfide(DATS).It is not clear which organic sulfur component is the effective component to reduce blood lipid in garlic oil.In the first part of the experiment,hyperlipidemia model was established by feeding Wistar rats with high-fat diet according to the latest"Assisted Lipid Reduction Function Test Method[2012]107" issued by the State Food and Drug Administration of China.The hypolipidemic action of three components were studied and compared in this part.It was found that DADS had a good hypolipidemic effect based on the experiment data,but its function and mechanism were not yet available.In the second part,we used the same hyperlipidemia model to explore the hypolipidemic effect and its mechanism of DADS.The intervention effects of low,medium and high doses of DADS were evaluated by related blood lipid and liver indices,and the mechanisms of DADS was further explored from the perspective of metabolism and molecular biology to provide scientific experiments for the prevention and treatment of hyperlipidemia.It also provides a theoretical basis for the further development and utilization of DADS.Chapter 1:Evaluation and comparison of the hypolipidemic effects of DAS,DADS,DATS and garlic oil in in hyperlipidemic ratsMethods1.High-fat feed20.0%sucrose,15.0%lard,1.2%cholesterol and 0.2%sodium cholate were added to the maintenance feed,and 1.2%calcium hydrogen phosphate,0.8%stone powder and 5.0%casein were added to meet the national standard of maintenance feed.2.Grouping and processing of experimental animals70 healthy male Wistar rats,weighing 200± 20g,after acclimation for 7 days,were randomly divided into solvent control group(10 rats)and experimental group(60 rats)according to their body weight.The solvent control group was fed with normal diet and the experimental group was fed with high-fat diet.One week later,the experimental group was randomly divided into high-fat model group,DAS intervention group(160mg/kg.bw),DADS intervention group(60mg/kg.bw),DATS intervention group(40mg/kg.bw),garlic oil intervention group(100mg/kg.bw)and simvastatin control group(4mg/kg.bw).DAS,DADS and DATS were diluted with peanut oil respectively.Solvent control group and high fat model group were given equal volume peanut oil.The volume of peanut oil was 1ml/kg.bw by gavage,once a day for 4 weeks.The same parts of the left lobe of the liver were cut out as specimens for homogenate and oil red O staining.The rest of the liver were frozen in the refrigerator at-80 C.3.Measurement of body weight and food utilizationThe dosage was adjusted according to the body weight which was weighed once a week during the experiment.Weekly food intake was recorded and food utilization rate was calculated according to the following formula:food utilization rate per week=average food intake per animal per week(g)/weight gain per week(g)*100%.4.Measurement of liver coefficientThe liver coefficient was calculated according to the following formula:liver coefficient = liver mass(g)/last body weight(g)*100%.5.Determination of serum biochemical indicatorsAfter the rats were executed by decapitation,blood samples were collected and centrifuged at 3500 r/min rpm in room temperature for 15 minutes to separate serum.The contents of TC,TG,HDL-C and LDL-C were determined by automatic biochemical analyzer.6.Determination of TG and TC in Liver50 mg and 100 mg in the same part of the left lobe of the liver were weighed by weight reduction method.The concentrations of TG and TC were measured according to the requirements of the kit,and the total protein concentration in the homogenate of liver was corrected by BCA method.7.Oil red O staining of liverThe same part of the left lobe of the liver was harvested and frozen at 10 um was prepared.The distribution and area of lipid droplets in liver were observed.Image-Pro-Plus 6.0 software was used to analyze the area ratio of lipid droplets stained with oil red O[area ratio = lipid droplet area/slice area*100%].Results1.Changes of body weight and liver coefficient in ratsThere was no significant difference in body weight and liver coefficient between the hyperlipidemia group and solvent control group.Compared with the hyperlipidemia model group,there was no significant difference in body weight and liver coefficient among the intervention groups of DAS,DADS,DATS and garlic oil.2.Changes in food utilization rate of ratsCompared with the hyperlipidemia model group,the total food utilization rate of the garlic oil intervention group decreased by 14.81%(P<0.05),and there was no significant difference among the other groups.3.Changes of serum lipid-related indicators in ratsCompared with the solvent control group,the serum TC,TG and LDL-C levels in the hyperlipidemia model group increased by 76.57%,115.16%and 43.40%,respectively(P<0.01).Compared with the hyperlipidemia model group,serum TG of DAS group decreased by 25.88%(P<0.05),HDL-C of DAS group increased by 52.57%(P<0.01);serum TC,TG and LDL-C of DADS group decreased by 23.70%,48.88%and 41.04%(P<0.01),and HDL-C of DADS group increased by 33.67%(P<0.01);serum TC,TG and LDL-C of DATS group decreased by 17.79%,43.71%and 27.11%(P<0.01 or 0.05),and HDL-C of DATS group increased by 29.04%(P<0.01).96%(P<0.01);TC and TG in serum of garlic oil group decreased by 16.17%and 44.65%(P<0.01 or 0.05),HDL-C of garlic oil group increased by 32.52%(P<0.01).4.Changes of liver TG and TC in ratsCompared with the solvent control group,the liver TG and TC concentrations in the hyperlipidemia model group increased by 133.84%and 164.32%(P<0.01).Compared with the hyperlipidemia model group,TC and TG in the liver of the DAS group decreased 52.68%and 36.74%(P<0.01),TC and TG in the liver of the DADS group decreased 63.64%and 51.75%(P<0.01),TC and TG in the liver of the DATS group decreased 61.43%and 53.91%(P<0.01),and TC and TG in the liver the garlic oil decreased 59.23%and 5.91%(P<0.01).5.Changes of liver oil red O stainingCompared with the solvent control group,red lipid droplets with large amount and area could be seen in the hyperlipidemia model group under the microscope.Compared with the hyperlipidemia model group,there were no obvious lipid droplets in the field of vision in DADS group,and a few lipid droplets in DATS group.The number and area of lipid droplets in DAS,garlic oil and simvastatin intervention group were larger than those in other intervention groups.Image Pro-Plus 6.0 software was used to analyze the area ratio of oil red O staining lipid droplets.Compared with the hyperlipidemia model group,the DAS,DADS,DATS and garlic oil intervention group decreased by 56.10%,96.29%,90.04%and 81.18%(P<0.01 or 0.05).Conclusion1.The serum TC,TG and LDL-C in the hyperlipidemia model group increased significantly after 4 weeks of high-fat diet,which indicated that the establishment of mixed hyperlipidemia model was successful.2.DADS,DATS and garlic oil can reduce the levels of TC,TG and LDL-C in serum of hyperlipidemic animals,and have good hypolipidemic effects.3.Comparing the hypolipidemic effects of DADS,DATS and garlic oil,DADS showed the best effect of hypolipidemic.Chapter 2:Study on the hypolipidemic effects and mechanism of DADS in hyperlipidemia RatsMethods1.Grouping and processing of experimental animalsafter acclimation for 7 days,84 healthy male Wistar rats weighing 200±20g,were randomly divided into solvent control group(12 rats),DADS control group(12 rats)and experimental group(60 rats).The solvent control group was fed with normal diet and the experimental groups were fed with high-fat diet.After 7 days,the animals in the experimental group were randomly divided into hyperlipidemia model group,DADS low,medium and high dose intervention groups(15,30,60 mg/kg.bw)and simvastatin control group(8 mg/kg.bw)after the determination of serum TC,TG,HDL-C and LDL-C.DADS was diluted with peanut oil and simvastatin was made into suspension with distilled water.Solvent control group,DADS control group and hyperlipidemia model group were given peanut oil of equal volume.The volume of peanut oil was 1ml/kg.bw by gavage,once a day for 5 weeks.After weighing,the same parts of the left lobe of the liver were cut off as samples for preparation of homogenate,HE staining(10%formalin solution immersion)and oil red O staining.The rest of the liver was frozen in the refrigerator at-80℃.Fast separation of epididymal fat,brown fat,subcutaneous fat,mesenteric fat and perirenal fat,weighing and frozen in-80℃ refrigerator.The measurements of body weight,food utilization rate,liver coefficient,serum biochemical parameters,liver TG and TC content and liver oil red O staining are the same as the Chapter 1.2.HE staining of liverThe paraffin sections were dewaxed,stained with Mayer’s hematoxylin for 1 minute,washed with tap water for 5-10 minutes,differentiated with 75%ethanol-hydrochloric acid solution for 1-2 minutes,washed with tap water for 0.5-1 minutes,re-dyed with eosin dye solution for 1 minute and washed with tap water for 0.5-1 minute and dehydrated and transparent with ethanol gradient.After sealing,the BX53 Olympus microscope was used to observe and collect the images.3.The detection of MDA concentration in serum and liver of ratsAfter the rats were decapitated,the blood was collected and centrifuged at 3500 r/min under room temperature for 15 minutes.The supernatant was removed into a new centrifugal tube and diluted by 2 times of saline.The liver was homogenized with ice PBS at the ratio of 1:9(mg:μL).The supernatant was centrifuged for 10 minutes at 10,000 g-12,000 g to be used for subsequent determination.After diluted by 2 times PBS,the samples were detected with a kit.4.Determination of SOD activity in serum and liver of ratsThe blood were collected by anticoagulant tube and mixed drastically.The supernatant was removed after centrifugation for 10 minutes at 4℃,600 g,diluted twice with proper saline solution,and then tested.Preparation of liver samples:Take appropriate tissue samples and homogenize them at 4℃ according to the proportion of 100 μL of SOD sample solution added to every 10 mg of tissue.The homogenate was centrifuged for 3-5 minutes at 4℃ for at 12,000 g.The supernatant concentrations were determined by kit.5.Determination of GSH and GSSG concentrations in serum and liver of ratsConcentrations of GSH and GSSG in serum and liver were detected by commercial kit.6.Detection of liver related-protein expression by Western blotWestern blot was used to detect the expression levels of oxidative stress-related proteins and lipid metabolism-related proteins in the liver of rats.Image Pro-Plus 6.0 was used to analyze the integral optical density of the strips on the film and calibrated by β-actin.Results1.General condition and weight change of ratsDuring the experiment,there was no significant difference in the mean body weight of rats.2.Changes in food utilization rate of laboratory animalsThere was no significant difference in the mean of total food utilization rate between the first,second and third weeks.3.Organ Coefficient of ratsCompared with the solvent control group and the hyperlipidemia model group,the liver coefficient of the DADS high-dose intervention group increased(P<0.01).Compared with solvent control group and hyperlipidemia model group,the spleen coefficient of DADS control group and simvastatin control group increased(P<0.01).4.Fat coefficients of different parts of ratsCompared with the solvent control group,the fat coefficients of peri-renal,epididymis,intestinal and subcutaneous in the hyperlipidemia model group increased by 21.06%,13.14%,41.79%and 24.46%(P>0.05 or<0.01).Compared with the hyperlipidemia model group,the high-dose DADS intervention group decreased by 25.61%(P<0.01);the low,medium and high-dose DADS intervention group decreased by 14.78%,10.24%and 26.90%(P<0.01 or 0.05 ox P>0.05);the low,medium and high-dose DADS intervention group decreased by 5.68%,10.65%and 22.38%(P<0.01 or 0.05 or P>0.05).The subcutaneous fat coefficient of DADS low,medium and high dose intervention group decreased by 30.81%,15.78%and 50.63%(P<0.01 or 0.05 or P>0.05).5.Serum lipid-related indexes and atherosclerosis index in experimental animalsCompared with the solvent control group,the concentrations of TG,TC and LDL-C in the hyperlipidemia model group increased by 76.26%,32.42%and 83.72%(P<0.01),and AI increased by 51.39%(P<0.01).Compared with the hyperlipidemia group,the TG concentration in the low,medium and high dose intervention group of DADS decreased by 28.69%,30.32%and 46.59%(P<0.01 or 0.05),the TC concentration in the low,medium and high dose intervention group of DADS decreased by 18.87%,21.51%and 24.90%(P<0.01),the HDL-C concentration in the medium and high dose intervention group of DADS increased by 17.22%(P<0.01)and 28.97%(P<0.05),and the LDL-C concentration in the low,medium and high dose intervention group of DADS decreased by 29.28%,34.60%and 47.81%(P<0.01);AI in low,medium and high DADS intervention groups decreased by 38.70%,49.16%and 59.54%respectively(P<0.01).6.Liver TG and TC concentrationCompared with the solvent control group,the liver TG and TC concentrations in the hyperlipidemia model group increased by 152.47%and 156.76%(P<0.01).Compared with the hyperlipidemia model group,the TG concentration in the low,medium and high dose intervention group of DADS decreased by 42.99%,51.25%and 53.29%(P<0.01),while the TC concentration in the low,medium and high dose intervention group of DADS decreased by 54.03%,62.78%and 63.43%(P<0.01).7.HE staining of liverAfter HE staining,the morphology of liver cells and the structure of hepatic lobules in solvent control group were normal.Diffuse steatosis of hepatocytes was observed in rats with hyperlipidemia model group.The degree of hepatic steatosis was mild in each intervention group of DADS.A few hepatocytes had vesicular lipid droplets,some hepatocytes showed mild turbidity and hepatic sinus narrowed slightly.The high dose group of DADS showed significant improvement with clear hepatic sinus,neat arrangement of hepatic cord and no inflammatory cell infiltration.8.Oil red O staining of liverThe liver cells of solvent control group and DADS control group were uniformly arranged without visible lipid droplets.Compared with the solvent control group,there were more red lipid droplets in the hyperlipidemia model group under the microscope.Compared with the hyperlipidemia model group,the red lipid droplets of DADS intervention groups and simvastatin control group decreased in varying degrees,and there was no obvious lipid droplets of DADS high-dose group.Image Pro-Plus 6.0 software analyzed the area ratio of oil red O stained lipid droplets.Compared with hyperlipidemia model group,DADS low,medium and high dose intervention group and simvastatin control group decreased 74.36%,93.20%,98.93%and 72.73%(P<0.01),9.Oxidative stress indicators of liver in ratsCompared with solvent control group,MDA concentration increased by 2241.88%(P<0.01),SOD activity decreased by 19.92%(P<0.05),GSH concentration decreased by 45.96%(P<0.05)and GSSG concentration decreased by 74.07%(P<0.05)in the hyperlipidemia model group.Compared with the hyperlipidemia model group,the MDA concentration of DADS medium,high dose intervention group and simvastatin control group decreased by 21.27%,44.62%and 26.38%(P<0.01 or 0.05),the SOD activity of DADS high dose intervention group increased by 23.39%(P<0.05),and the GSH concentration of DADS low,medium and high dose intervention group increased by 16.76%,41.92%and 49.50%(P<0.01 or 0.05).10.Oxidative stress indicators of serum in ratsCompared with solvent control group,MDA concentration increased by 140.55%(P<0.01)and SOD activity decreased by 56.08%(P<0.01)in hyperlipidemia model group.Compared with the hyperlipidemia model group,MDA concentration in the low,medium and high dose intervention group of DADS decreased by 45.25%,53.19%and 57.96%(P<0.01 or 0.05),SOD activity in the low,medium and high dose intervention group of DADS increased by 101.83%,102.37%and 117.95%(P<0.01),GSH concentration in the low,medium and high dose intervention group of DADS increased by 89.41%,119.62%and 146.48%(P<0.01)9 GSSG concentration in the low,medium and high dose intervention group of DADS increased by 50.58%,97.54%and 111.82%(P<0.01).11.Expression of oxidative stress-related proteins in hepatocytesCompared with the solvent control group,the relative expression of Nrf2 protein decreased by 9.86%(P>0.01);the expression of NQO1 decreased by 39.95%(P>0.05);the expression of y-GCSc decreased by 36.12%(P<0.05);the expression of y-GCSm increased by 169.58%(P<0.05);and the relative expression of SOD-1 protein decreased by 29.95%(P>0.05)in the liver of the hyperlipidemia model group.Compared with the hyperlipidemia model group,the relative expression of Nrf2 protein in the liver of the low,medium and high dose intervention groups of DADS increased by 158.93%,56.35%and 71.28%(P<0.01 or 0.05),the expression of NQO1 increased by 505.01%,600.67%and 1024.94%(P<0.01 or 0.05 or P>0.05,the expression of y-GCSc increased by 71.77%,67.78%and 91.34%(P<0.01 or 0.05),and the expression of SOD-1 increased by 102.20%,136.86%and 113.55%(P<0.01 or 0.05).12.Expression of fatty acid metabolism-related proteins in hepatocytesCompared with the solvent control group,the expression of p-SREBP-lc protein increased by 45.56%(P<0.01),the expression of n-SREBP-lc protein increased by 93.48%(P<0.01),and the expression of PPAR-alpha protein decreased by 17.44%(P<0.05)in the liver of the hyperlipidemia model group.Compared with the hyperlipidemia model group,the expression of p-SREBP-1c protein decreased by 34.68%,75.52%and 63.48%(P<0.01),the expression of n-SREBP-1c protein decreased by 46.52%,69.83%and 71.28%(P<0.01)in the liver of the low,medium and high dose of DADS intervention group.The expression of PPAR-a increased by 42.78%and 119.81%in the low and high dose of DADS intervention groups(P<0.05 or P>0.05).Conclusion1.The serum TC,TG and LDL-C in the hyperlipidemia model group increased significantly after 5 weeks of high-fat dietary,whichdemonstrated the success of the establishment of mixed hyperlipidemia model.2.The concentrations of TC,TG and LDL-C in serum of rats fed with high-fat dietary were decreased in all dosage groups of DADS,which proved that DADS had well hypolipidemic effect.3.DADS improved the level of oxidative stress in animal liver,thereby reduced lipid peroxidation,which is related to activating of Keapl/Nrf2 pathway and up-regulating the expression levels of downstream protein-related proteins NQO1,gamma-GCSc and SOD-1.4.DADS may inhibit the synthesis of liver TG and related lipids by down-regulating the expression of SREBP-lc and accelerate the decomposition of TG by up-regulating the expression of PPAR-a in liver.Thereby,hyperlipidemia in experimental animals was alleviated. |
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