| AIM:Based on the mechanism of TRPV1-mediated warming moxibustion effect,the effect of repeated thermal stimulation on the EC state and its function under inflammatory state is clearly defined.METHODS:Human umbilical vein endothelial cells(HUVEC)cultured in vitro.1?g/ml lipopolysaccharide(LPS)stimulates cells to produce an inflammatory environment for 1hour.Intervention method:the cells were stimulated continuously at 43? for 4 minutes,with intervals of 1 minute,and were repeated 5,9 and 13 stimulations,respectively.Experimental intervention method:5?g/ml capsicum(TRPV1 antagonist,Capz)was added to the cell culture solution,and then repeated at 43?thermal stimulation.Grouping:Experiment 1:blank group,model group,5 times group,9 times group,13 times group.Experiment 2:blank group,control group+Capz,5 times group+Capz,9 times group+Capz,13 times group+Capz.Evaluation indicators:MTT assay was used to detect cell status,immunofluorescence assay and Western blot was used to detect the expression of HUVEC proliferation-related signal pathway protein(NF-?B)after intervention in each group to detect the cell's own functional state;enzyme-linked adsorption test(ELISA)The kit detects the mRNA expression of HUVEC cell adhesion molecules(ICAM-1,VCAM-1)and nitric oxide synthase(eNOS)after intervention in each group to detect endothelial cell function.RESULTS:1.The effect of repeated thermal stimulation on HUVEC's own function under the action of LPS.From the MTT experiment,after adding 1?g/ml LPS,the cell vitality of each group decreased compared with the blank group.Compared with the model group,the cell vitality decreased significantly in the 5 times group and the 13 times group(P<0.05),but not significantly in the 9 times group(P>0.05).Compared with repeated 43? thermal stimulation,the cell vitality of the 13 times group was the highest(P<0.05).Cell vitality decreased after 1?g/ml LPS,indicating successful modeling.Continuous stimulation at 43? for 4 minutes,interval of 1 minute,repeated 9 times is the optimal thermal stimulation parameter,which also shows that it has a protective effect on EC.Compared with the blank group,the expression of NF-?B in the model group and the three groups of thermal stimulation increased(P<0.05).Compared with the model group,the expression of NF-?B in the three groups of thermal stimulation decreased,among which the 9 times group is the most obvious(P<0.05).Combined with the WB experiment results,since WB has only one set of data,we look at the trend,after adding LPS,NF-?B protein showed an increasing state.After repeated 43? thermal stimulation,the expression of NF-?B protein decreased.And its trend was consistent with the results of immunofluorescence experiments.2.The influence of repeated thermal stimulation on the function of HUVEC under the action of LPS:From the expression of adhesion molecules,the addition of 1 ?g/ml LPS increased the expression of ICAM-1mRNA and VCAM-1 mRNA.After different repeated thermal stimulation,the mRNA expressions of ICAM-1 and VCAM-1 decreased in three experimental groups(P<0.01).The expression of ICAM-1 mRNA in the 9 times group was the lowest(P<0.001).The expression of VCAM-1 mRNA did not change significantly(P>0.05)suggesting that different repeated thermal stimuli have a protective effect on inflammatory HUVEC.In addition,43? continuous stimulation for 4 minutes,1 minute interval,repeated 9 anti-inflammatory effects is the best.In terms of eNOS mRNA expression,after different repeated thermal stimulation,the expression of eNOS mRNA in the three groups of experiments decreased(P<0.01).The expression of eNOS mRNA in the 9th group was the lowest(P<0.001).The expression of VCAM-1 mRNA did not change significantly(P>0.05),suggesting different repetitions thermal stimuli have a protective effect on inflammatory HUVEC.In addition,43? continuous stimulation for 4 minutes,1 minutes interval,repeated 9 anti-inflammatory effects is the best.3.The influence of Capz's on HUVEC's own function under the action of LPS:From the MTT experiment results,after adding 5?g/ml Capz from external sources ?Compared with the group,the cell vitality of the 9 times group decreased after adding 5?g/ml Capz(P<0.05);? Comparison between groups:Compared with the control group,the cell vitality of the 5 times and 13 times groups was lower after adding 5?g/ml Capz(P<0.05,P<0.01).Compared with the three groups,the difference in cell viality after adding 5?g/ml was statistically significant(P<0.05;P<0.01).Judging from the expression of NF-?B:after adding 5?g/ml Capz,the expression of NF-?B in the four groups all decreased;from the perspective of the reduction,the 9 times group was the largest.The 13 times group was not significantly decreased,suggesting continuous stimulation at 43?,the anti-inflammatory effect may be the most stable after repeating 9 times at 4 minutes and 1 minute intervals.4.The influence of Capz's effect on the active function of HUVEC under the action of LPS:From the expression of adhesion molecules,after adding 5?g/ml of Capz,?Compared with the group,the expression of ICAM-1mRNA and VCAM-1mRNA both decreased 0.01;P<0.05);? Comparison between groups:Compared with the control group,the expression levels of ICAM-1mRNA and VCAM-1mRNA in the three groups of repeated thermal stimulation groups all decreased(P<0.01;P<0.05).Compared with different repeated thermal stimulation,the expression level of ICAM-1mRNA decreased(P<0.01).There is no statistical difference in the expression of VCAM-1mRNA.From the perspective of eNOS mRNA expression,after adding 5?g/ml Capz,? Compared with the group,the expression of eNOS mRNA decreased(P<0.01;P<0.05);?Compared between the groups:Compared with the control group,the expression of eNOS mRNA decreased(P<0.01;P<0.05).Compared with different repeated thermal stimuli,the expression of eNOS mRNA decreased(P<0.01).The results suggest that repeated thermal stimuli affect the anti-inflammatory effect of HUVEC through TRPV1.CONCLUSION:1.Repeated thermal stimulation can achieve the protection of HUVEC in the inflammatory state.2.Repeated thermal stimulation can promote the anti-inflammatory effect of HUVEC in the inflammatory state. |
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