Effects Of Different Low Temperatures On Rat Dermal Microvascular Endothelial Cells And Mechanism Of Trpm8Involved In The The Process

BackgroundAs a harmful environmental agent for human health, cold can cause a series ofdiseases in the body. The non-freezing injury, such as chilblains, trench foot andfrostbite is the most common disease. Previous studies indicated subcutaneousmicrocirculation dysfunction may be the capital factor for development of thenon-freezing injury, and cold-induced endothelial cells functional disorder may be theoriginal agent.Therefor, many scholar suggested endothelial cells were the firstexcited and vulnerable site when human exposed to Low temperature environment. Inaddition, chronic exposure to different low temperature could keep human skin indifferent state. Normally, the body feel comfortable when skin temperature is32℃;when the temperature below29℃,human would feel uncomfortable; if thetemperature below28℃,human may feel cool; cold and pain feeling usually occurswhen the skin temperature below17℃,chilblains might happen potentially; when thetemperature drop at12℃,numbness could occur and immersion foot or trench footmight happen at this time; below8℃,pain sensation completely lost; as soon as theskin temperature below0℃,the morphological and subcellular structures of vascularendothelial cells would be damaged; tissues can be frozen when skin temperaturebelow-2℃.All these phenomenons suggest that during the process of cold injury ofdistal extremities, subcutaneous microvascular endothelial cells may be affect bydifferent temperatures, then generate different effect. As the unique vascularendothelial cells that contact with ambient temperature closest, dermal microvascularendothelial cells(DMVECs) may receive different temperatures stimulus in everydaylife. However, the molecular mechanisms about DMVECs receive low temperaturestimulus and low temperature effects on DMVECs are still unclear. Fortunately, with the discovery of transient receptor potential family(TRPs),we may find a way tocomprehend the mechanisms. TRP channels are non-selective cation channels, whichcan be activated by various agents and trigger diverse change of cellular physiologicalfunction. Moreover, many studies found that members of TRPs such asTRPV1,V2,V3,V4and TRPA1,TRPM8can help people sense environmentaltemperature change.TRPM8can be activated by28℃to8℃.This temperature rangeresemble the hypothermia range of human skin when the body exposed into cold zone.Based on these theroies, the present study proposed to establish a rat DMVECs coldexposure model in vitro, then investigated the distinct effects of different temperatureson rat DMVEC, namely, to study whether rat DMVECs can receive differenttemperatures stimulus and generate different effects. In addition, we also investigatedif rat DMVECs can express active TRPM8channel, then studied the molecularmechanism of low temperature effect on rat DMVECs. The elucidation of thesemechanisms would provide us a new clue to comprehend the mechanisms of coldinjury, find new way to diagnosis and treat cold injury and develop new drug that canpromote cold acclimatization of people who go into cold area.Objective：Through establish a cell cold exposure model with rat dermal microvascularendothelial cell(DMVECs) in vitro, to investigate the distinct effects of differenttemperatures on DMVECs and the mechanisms of transient receptor potential cationchannel subfamily M member8involved in these process.Methods:1. Purification and identification of rat DMVECs: Rat dermal microvascularendothelial cells were isolated from cell suspension by discontinuous densitygradients Percoll solutions, then the cells were identified with morphologicalobservation, Factor Ⅷ immunofluorescence staining,CD31immunofluorescencestaining and lectin binding assays.2. Establishment of rat DMVECs cold exposure model: Use a stable cell coldexposure platform, rat DMVECs were exposed to37℃、28℃、12℃and0℃respectively for24h,then evaluating the model through morphological observation determination of cell growth rate and lactate dehydrogenase activity in the supernatantafter treatment.3. Effects of different low temperatures on mRNA expression of relatedendothelium cytokines of rat DMVECs: Total RNA of different groups were extractedwith RNA extraction kit after treatment，then the mRNA expression of relatedendothelium cytokines including VEGF,AQP-1,ET-1,ICAM-1,IL-1β,IL-6and TNF-αwere semi-quantitative analyzed by RT-PCR method.4. Identification of TRPM8expression in rat DMVECs: Primers of rat TRPM8were designed with primer-designing software, then the mRNA expression of TRPM8in rat DMVECs were identified by RT-PCR method and the sequence of RT-PCRproduction was analyzed simultaneous; In addition, the protein expression of TRPM8in rat DMVECs was identified with double immunofluorescence staining method.5. Clarification of mechanism of TRPM8involved in the process of lowtemperature effect on rat DMVECs: Rat DMVECs were exposed into low temperaturefor12h after the TRPM8channel in the cells was blocked with differentconcentrations of TRPM8specific blocker AMTB (25μmol/Land75μmol/L), then themRNA expression of VEGF,ET-1,IL-6and TRPM8in rat DMVECs weresemi-quantitative analyzed through RT-PCR method.Results:1. Purification and identification of rat DMVECs: Cells between21%-35%density were harvested and cultured. The cultured cells were confluent on the fifthday and exhibited a typical “cobblestone” structure. The results ofimmunofluorescence staining of rat factor VIII-related antigen and CD31protein werepositive. Lectin binding assay also showed positive result. These results indicated thecells that purified by discontinuous density gradient Percoll solution were ratDMVECs.2. Establishment of rat DMVECs cold exposure model: After24h exposure atdifferent temperatures，the change of cellular morphous of28℃group was slight.The morphous of12℃group exhibited “fibroblast-like structure”, but the cellular structure was intact.0℃group exhibited apparent shrinkage. The growth rate of28℃group,12℃group and0℃group have decreased by4%,10%and20%respectively(P<0.05),and the disparity between different low temperature groups werestatistically significant. the LDH activity(U/L) in the supernatant of28℃group(54.17±3.02),12℃group(64.66±3.03) and0℃group(82.13±10.91) wasstatistically significant compared with37℃(12.23±3.03) group(P＜0.05).Theseresults indicated the cold exposure model was successfully established. In addition,rat DMVECs can exhibited different effects after different low temperatures exposure.3. Effects of different low temperatures on mRNA expression of relatedendothelium cytokines of rat DMVECs: After exposed at37℃,28℃,12℃and0℃for24h, the results of mRNA expression of different cytokines in rat DMVECs asfollows:①The relative expression levels of VEGF mRNA in37℃group,28℃group,12℃group and0℃group were0.64±0.01,0.86±0.04,1.04±0.04and0.64±0.01,compared with37℃group, the mRNA expression of28℃and12℃group were unregulated (P<0.05),but there was no significant change in0℃group.This result indicated that28℃and12℃can increase the expression of VEGF mRNAin rat DMVECs;②The relative expression levels of AQP-1mRNA in37℃group,28℃group,12℃group and0℃group were1.45±0.04,1.72±0.04,1.77±0.03and1.37±0.06,compared with37℃group, the mRNA expression of28℃and12℃groupwere unregulated (P<0.05), but there was no significant change in0℃group. Thisresult indicated that28℃and12℃can increase the expression of AQP-1mRNA inrat DMVECs;③The relative expression levels of ET-1mRNA in37℃group,28℃group,12℃group and0℃group were0.87±0.02,0.85±0.04,1.04±0.04and0.45±0.02, compared with37℃group, the mRNA expression of12℃group wassignificant unregulated(P＜0.01), the mRNA expression of0℃group was significantdownregulated(P＜0.01),but there was no significant change in28℃group. Thisresult indicated that12℃can increase but0℃would decrease the expression of ET-1mRNA in rat DMVECs；④The relative expression levels of ICAM-1mRNA in37℃group,28℃group,12℃group and0℃group were1.04±0.02,1.24±0.01,1.50± 0.04and1.01±0.01, compared with37℃group, the mRNA expression of28℃and12℃group was unregulated(P＜0.05),but there was no significant change in0℃group. This result indicated that28℃and12℃can increase the expression of ICAM-1mRNA in rat DMVECs；⑤The relative expression levels of IL-1β mRNA in37℃group,28℃group,12℃group and0℃group were0.67±0.02,0.37±0.01,0.38±0.03and0.46±0.02,, compared with37℃group, the mRNA expression of alllow temperature group was downregulated(P＜0.05).This result indicated that28℃,12℃and0℃can decrease the expression of IL-1β mRNA in rat DMVECs；⑥The relative expression levels of IL-6mRNA in37℃group,28℃group,12℃group and0℃group were0.89±0.02,0.89±0.02,1.02±0.03and0.67±0.03,compared with37℃group, the mRNA expression of12℃group was significantunregulated(P＜0.01), the mRNA expression of0℃group was significantdownregulated(P＜0.01),but there was no significant change in28℃group. Thisresult indicated that12℃can increase but℃decrease the expression of IL-6mRNAin rat DMVECs;⑦The relative expression levels of TNF-α mRNA in37℃group,28℃group,12℃group and0℃group were0.14±0.01,0.26±0.02,0.15±0.02and0.21±0.02,, compared with37℃group, the mRNA expression of28℃and0℃group was significant unregulated(P＜0.01), but there was no significant change in12℃group. This result indicated that28℃and0℃can increase the expression ofTNF-α mRNA in rat DMVECs;4. Identification of TRPM8expression in rat DMVECs: RT-PCR result showedthat there was an obvious band at the purpose site(216bp) and the sequence resultshowed the product can match with the sequence of rat TRPM8in Genebank. Inaddition, the result of double immunofluorescence staining was positive.These resultsindicated rat DMVECs can express TRPM8mRNA and protein.5. Clarification of mechanism of TRPM8involved in the process of lowtemperature effect on rat DMVECs: After treatment with AMTB for12h at12℃，theresults of mRNA expression change of different cytokine in rat DMVECs as follows:①the relative expression levels of VEGF mRNA in the0h group,12h group, DMSOgroup25μmol/L AMTB group and75μmol/L AMTB group were 0.94±0.01,1.02±0.03,1.02±0.02,0.94±0.01and0.93±0.02, compared with DMSOgroup, the expression of VEGF mRNA of25μmol/L AMTB group and75μmol/LAMTB group was downregulated(P<0.05).This result indicated that TRPM8may beinvolved in the regulation of VEGF mRNA in rat DMVECs after exposed to12℃.②the relative expression levels of ET-1mRNA in the0h group,12h group, DMSOgroup25μmol/L AMTB group and75μmol/L AMTB group were0.73±0.03,0.94±0.01,0.96±0.02,0.73±0.01and0.53±0.02, compared with DMSO group, theexpression of ET-1mRNA of25μmol/L AMTB group and75μmol/L AMTB groupwas significant downregulated(P<0.01).This result indicated that TRPM8may beinvolved in the regulation of ET-1mRNA in rat DMVECs after exposed to12℃;③the relative expression levels of ET-1mRNA in the0h group,12h group, DMSOgroup25μmol/L AMTB group and75μmol/L AMTB group were0.26±0.03,0.57±0.04,0.62±0.02,0.67±0.01and0.77±0.01, compared with DMSO group, theexpression of IL-6mRNA of75μmol/L AMTB group was significantdownregulated(P<0.01).This result indicated that TRPM8may be involved in theregulation of IL-6mRNA in rat DMVECs after exposed to12℃;④the relativeexpression levels of TRPM8mRNA in the0h group,12h group, DMSO group25μmol/L AMTB group and75μmol/L AMTB group were0.54±0.01,0.74±0.01,0.76±0.03,0.67±0.01and0.54±0.01, compared with DMSO group, the expression ofTRPM8mRNA of25μmol/L AMTB group and75μmol/L AMTB group wassignificant downregulated(P<0.01).This result indicated that TRPM8may be involvedin the regulation of TRPM8mRNA in rat DMVECs after exposed to12℃.Conclusion：1. Rat dermal microvascular endothelial cells can be successfully preparedthrough discontinuous density gradient segregating method.2. Different temperatures can result in different effects on rat DMVECsfunction, including cell morphous, cell growth rate, membrane integrity and theregulation of different cytokines expression. These results indicated low temperaturecould influence rat DMVECs directly. The rat DMVECs may have the ability toreceive different temperatures stimulus. 3. Positive and active expression of TRPM8channels can be identified in ratDMVECs.TRPM8may be involved in the process of12℃effects on the expressionof different cytokine。4. Low temperature-induced expression changes of relative cytokine in ratDMVECs may depend on TRPM8channel.