| Background:Recently,many researches reported that the incidence of metabolic disease such as hyperlipidemia、adiposity and atherosclerosis-related vascular disorders like stroke increased remarkably,even attacked younger individuals more often.Since the life styles、eating habits or the age structure of our country have changed,risks brought by atherosclerosis-related diseases were becoming more and more critical,leading a heavy load to society and large numbers of families.Therefore,the demand for subject study on atherosclerosis-related diseases turned more urgent.Accumulating epidemiological investigations showed compelling evidence that atherosclerosis-related diseases is closely allied to cold seasons like winter or colder ambient temperature.Some scholars found out a direct link between the mortality rate of atherosclerosis-related diseases and Winter,after finished statistical analysing about the death information on the deceased with atherosclerosis-related vascular disorders,but the mechanism still unknown.Some researchers provided a explanation that cold exposure could promote the progress of atherosclerosis-related diseases through vasoconstriction and blood pressure increasing,which couldn’t demonstrate the cardiovascular events without higher blood pressure.Several fundamental researchs considered that cold exposure could elevate serum lipid level,induce vessel inflammatory response,aggravate atherosclerosis,and finally,increase the atherosclerosis-related cardiovascular events.On the other hand,there’s a totally opposite view pointed out that we should devided the cold exposure into intermittent cold exposure(last for several hours only each day)and continuous cold exposure(last for more than 24 hours)by different stimulating time,both of which controlled the secretion of inflammatory factor in different ways,producing different effects on the atherosclerosis progress:the former aggrevated the formation of atherosclerosis plaque,while the latter lessened the severity of atherosclerosis.The viewpoint was supported by a study published on "Cell Metabolism" last year.All in all,no final conclusion has been reached on how cold exposure influenced atherosclerosis progress yet.As is known,adipose tissue’s an significant human endocrine organ,the state of which plays a decisive role of the lipid metabolism.Professor Spiegelman from Harvard University proposed that the white adipose tissue could transform into the BAT-like tissue(which also called "BRITE")under certain conditions,accompanying phenotypic and function conversion,promoting the expression of uncoupling protein 1 and lipolysis,alleviating the lipid-related metabolic diseases.Majority of studies affirmed that WAT-BRITE could activated by sport、menthol stimulation and cold exposure,the first two were good for decreasing blood lipids level,therefore,reduced weight.For further studying,we provided an novel idea that maybe cold exposure could also reduce blood fat and influence the formation of atherosclerosis plaque by converting WAT to BRITE and augmenting BAT activation.Statistical results pointed out that cold ambient temperature could change the TC、LDL-C、TG levels,meanwhile,increase the incidence of hyperlipidemia,indicating that there may be some substantial associations between cold exposure and lipid levels.However,only a handful of clinical trials conducted made the association unsure yet.Still,there’s no unified verdict on whether cold exposure could increase the incidence of hyperlipidemia and atherosclerosis-related diseases or not.Io order to figure out how cold exposure effects on individuals’ blood lipids,we designed clinical trail collecting data on volunteers’ serum lipid levels before and after cold exposure,besides,we testeds the blood pressure and Ang Ⅱ level exploring whether cold exposure increased atherosclerosis incident through cold-caused elevation of blood pressure or not.After that,we conducted an animal experiment on mice supposed to address how cold exposure effectted on the phenotypic characters and function of WAT or BAT、the lipid levels、inflammatory reaction、the foundation of atherosclerosis plaque and discussed mechanisms of all above.What’s more,the conclusion on menthol’s function about lessening blood lipids and losing weight were quite certain,but the pity is that no one further discussed the possibility on if menthol could influence the AS progress through it’s function on lipid metabolic.As is known,menthol is extracted from a kind of Chinese traditional medicine named mint,maybe we can develop into modern research investigating the potential pharmacology value of mint on influencing the AS progress.Methods:Experiment Ⅰ(Clinical Trail):We recruited volunteers,randomly divided them into two groups like 18 C cold exposure group and 25℃ normal temperature control group according to the proportion of 5:1,then tested volunteers’ serum lipid,blood pressure,Ang Ⅱ level before and after cold exposure.Finally,collected data and did statistical analysis.Experiment Ⅱ(Animal Experiment):According to different experiments’ aims and requirements,we chose the classic AS mouse model,ApoE-/-mice,to study and use C57BL/6J mice as a control,which were randomly divided into control group(30℃)and cold exposure group(4℃),recorded body weight and food-intake of these mice.After completed stimulation conditions,we collected experimental samples:saved the serum in 1.5ml EP tubes at-80℃,and stored the sample in liquid nitrogen immediately or 4%paraformaldehyde as experimental need until use.Use biochemical process testing the blood lipids,HE staining observing adipocyte morphology,immunohistochemical testing the uncoupling protein 1 and inflammatory factor positive staining,Sirius red stain checking collagenous fiber s expression and western-blot technique detecting uncoupling protein 1’s content.Ultimately,statistical analysis of the data.Results:Experiment Ⅰ(Clinical Trail):Cold exposure increased volunteers’ TC and LDL-C levels,but the results were not statistically significant.Cold exposure had no significant influence on the TG content.Cold exposure had an upward trend in blood pressure and Ang Ⅱ levels,but the results about Ang Ⅱlevels and systolic pressure were not statistically significant,while the diastolic pressure results were statistically different.Experiment Ⅱ(Animal Experiment):1.Influence by 4 weeks/8 weeks cold exposure on mice’s WAT-BTITE conversion and BAT activation.Both ApoE-/-mice and C57BL/6J mice,in comparison with 30 ℃ control groups’inguen white adipose tissue(sWAT)and scapular brown adipose tissue(iBAT),4 weeks/8 weeks cold exposure(4 C)diminished the average size of inguen white adipose cells(iWAC)and scapular brown adipose cells(sBAC),increased the number of adipocytes,turned the HE staining darker,at the same time,increased the positive staining area of UCP1 by immunohistochemical,improved the expression of UCP1 which was showed in western-blot result.Whereas,in comparison with 30 ℃ control group’s epididumis white adipose tissue(eWAT),cold exposure(4 C)seemed did not work on it.2.Influence by 4 weeks/8 weeks cold exposure on mice’s weight and blood lipids level.In comparison with 300C control groups,4 weeks/8 weeks cold exposure(4℃)reduce the weight of C57BL/6J mice but not ApoE-/-mice,besides,4 weeks/8 weeks cold exposure(4 ℃)approximately lowered the blood lipids in both ApoE-/-mice and C57BL/6J mice.3.8 weeks cold exposure alleviated the inflammatory response and reduced the area of AS plaque in aorta of ApoE-/-mice.In comparison with 30 C control groups,8 weeks cold exposure lowered the expression of inflammatory factor such as CD31,MCP1,MMP2 in aorta of ApoE-/-mice,meanwhile,reduced the area of AS plaque.4.Influence by 4 weeks/8 weeks menthol stimulation on ApoE-/-mice’s adipose tissue.In comparison with 30 C control groups,ApoE-/-mice’s sWAT and iBAT,4 weeks/8 weeks menthol stimulation diminished the average size of adipocytes、increased the number of adipocytes and turned the HE staining darker of them,which did not happen in eWAT.8 weeks menthol stimulation turned the positive staining of UCP1 more obvious by immunohistochemical in iBAT,but not in sWAT and eWAT.By contrast,4 weeks menthol stimulation increased the positive staining area of UCP1 by immunohistochemical in sWAT.5.Influence by menthol stimulation on ApoE-/-mice’s blood lipids level and AS plaque.In comparison with 300C control groups,4 weeks/8 weeks menthol stimulation approximately lowered the blood lipids in ApoE-/-mice.Meanwhile,8 weeks menthol stimulation reduced the area of AS plaque obviously.6.Influence by 4 weeks/8 weeks cold exposure and menthol stimulation on the expression of TRPM8 in ApoE-/-mice.4 weeks cold exposure and menthol stimulation turned the positive staining of TRPM8 in sWAT and iBAT darker by immunohistochemical,while the change became slightly by 8 weeks cold exposure and menthol stimulation.furthermore,changes mentioned above did not occur in eWAT.Conclusions:Experiment Ⅰ(Clinical Trail):1.Cold exposure had no obvious effects on the blood lipids.2.Cold exposure had no obvious effect on systolic pressure,but could increase the diastolic pressure in volunteers.3.Cold exposure didn’t increase the Ang II levels in volunteers.Experiment Ⅱ(Animal Experiment):1.4 weeks/8 weeks cold exposure caused the BRITE of sWAT and activation of iBAT in both ApoE-/-mice and C57BL/6J mice,but no significant changes in eWAT.2.4 weeks/8 weeks cold exposure decreased the blood lipids in both ApoE-/-mice and C57BL/6J mice.3.8 weeks cold exposure lessened the inflammatory response as well as the area of AS plaque in aorta of ApoE-/-mice.4.4 weeks/8 weeks cold exposure decreased the weight of C57BL/6J mice,but not ApoE-/-mice.8 weeks cold exposure increased the food intake of C57BL/6J mice.5.8 weeks menthol stimulation caused the BRITE of sWAT and activation of iBAT but not eWAT in ApoE-/-mice.6.8 weeks menthol stimulation decreased the blood lipids as well as area of AS plaque in ApoE-/-mice.7.4 weeks cold exposure and menthol stimulation increased the TRPM8 expression in sWAT and iBAT,which only expressed slightly in 8 weeks cold exposure and menthol stimulation groups,besides,no TRPM8 expression were found in eWAT. |
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