Effects Of Navel Therapy On Transient Receptor Potential Vanilloid Channels And Toll-like Receptors Of Rats With Primary Dysmenorrhea Due To Cold Coagulation And Blood Stasis

OBJECTIVE:To explore the signaling pathways of analgesic and anti-inflammatory effects of navel therapy,through observing impact of navel therapy on relative expression of TRPV1,TRPV2,TRPV4,TLR2 and TLR4 in hypothalamus and spinal cord of dysmenorrhea rats with cold coagulation and blood stasis syndrome.METHODS:Fifty adult female Wistar rats were randomly divided into control group,model group,herb-partition moxibustion on navel group,drug coving navel group and moxibustion on navel group.The control group was not provided treatment.The other groups were treated by subcutaneous injection of estradiol benzoate into the femur combined with systemic freezing,to establish the model of primary dysmenorrhea of cold coagulation and blood stasis type.The rats were injected once a day for 10 days,and were cryopreserved for 4 hours every day from the first day to the fifth day.The three treatment groups were treated with corresponding navel therapy from the sixth day to the tenth day,also once a day for 5 days.After the 10th day of modeling and treatment,all rats were injected oxytocin into tail vein.The writhing reaction of all rats was observed and the samples were sacrificed.The expression of TRPV1,TRPV2,TRPV4,TLR2,TLR4 protein and mRNA in hypothalamus and spinal cord of rats were detected by immunofluorescence,protein immunoblotting and real-time fluorescence quantitative PCR,and effects of navel therapy on the expression of TRPV1,TRPV2,TRPV4,TLR2 and TLR4 were discussed.RESULTS:1.Effect of navel therapy on the expression of TRPV1,TRPV2,TRPV4 protein and mRNA in hypothalamus and spinal cord of rats with dysmenorrhea due to cold coagulation and blood stasisThe expression of TRPV1,TRPV2,TRPV4 protein and mRNA in hypothalamus"of model group was significantly higher than that of blank group(P<0.01),and was significantly lower after navel therapy(P<0.01 or P<0.05).The expression of TRPV1 and TRPV2 protein in moxibustion on navel group was lower than that in herb-partition moxibustion on navel group and drug coving navel group;the expression of TRPV4 protein in moxibustion on navel group and herb-partition moxibustion on navel group was lower than that in drug coving navel group.The expression of TRPV1 mRNA in moxibustion on navel group was lower than that in herb-partition moxibustion on navel group(P<0.01)and drug coving navel group(P<0.05);the expression of TRPV2 mRNA in herb-partition moxibustion on navel group and drug covering navel group was lower than that in moxibustion on navel group(P<0.01);and the expression of TRPV4 mRNA in moxibustion on navel group and herb-partition moxibustion on navel group was lower than that in drug coving navel group(P<0.01).The expression of TRPV1,TRPV2,TRPV4 protein and mRNA in spinal cord of model group was significantly higher than that of control group(P<0.01),and was lover after navel therapy(P<0.01).The expression of TRPV1 protein in moxibustion on navel group and drug coving navel group was lower than that in herb-partition moxibustion on navel group(P<0.01);the expression of TRPV2 protein in moxibustion on navel group and herb-partition moxibustion on navel group was lower than that in drug coving navel group.The expression of TRPV4 protein level in moxibustion on navel group was significantly lower than that of herb-partition moxibustion on navel group(P<0.01)and drug coving navel group(P<0.01).The expression of TRPV1,TRPV2 and TRPV4 mRNA in moxibustion on navel group was significantly lower than that in herb-partition moxibustion on navel group(P<0.01)and drug coving navel group(P<0.01).2.Effect of navel therapy on the expression of TLR2 and TLR4 protein and mRNA in spinal cord of rats with dysmenorrhea due to cold coagulation and blood stasisThe expression of TLR2,TLR4 protein and mRNA in hypothalamus of model group was significantly higher than that of control group(P<0.01),and decreased after navel therapy(P<0.01 or P<0.05).The expression of TLR2 and TLR4 protein in moxibustion on navel group was lower than that in herb-partition moxibustion on navel group(P<0.01 or P<0.05),and that in herb-partition moxibustion on navel group was lower than that in drug coving navel group(P<0.01).The expression of TLR2 mRNA in moxibustion on navel group was lower than that in herb-partition moxibustion on navel group and drug coving navel group(P<0.01);the expression of TLR4 mRNA in drug coving navel group was lower than that in herb-partition moxibustion on navel group(P<0.01)and moxibustion on navel group(P<0.01).The expression of TLR2,TLR4 protein and mRNA in spinal cord of model group was significantly higher than that of control group(P<0.01),while significantly lower after navel therapy(P<0.01).The expression of TLR2 protein in herb-partition moxibustion on navel group and the drug coving navel group was significantly lower than that in moxibustion on navel group(P<0.01).The expression of TLR4 protein in moxibustion on navel group and drug coving navel group was significantly lower than that in herb-partition moxibustion on navel group(P<0.05 and P<0.01).The expression of TLR2 mRNA in moxibustion on navel group was lower than that in herb-partition moxibustion on navel group and drug coving navel group(P<0.01);the expression of TLR4 mRNA in drug coving navel group was lower than that in herb-partition moxibustion on navel group(P<0.01)and moxibustion on navel group(P<0.01).CONCLUSION:1.Navel therapy can reduce the expression of TRPV1,TRPV2 and TRPV4 in hypothalamus and spinal cord,inhibit their participation in generating and conducting pathological pain,and achieve analgesic effect on dysmenorrhea.2.Navel therapy can inhibit inflammation caused by dysmenorrhea through restraining expression levels of TLR2 and TLR4 of hypothalamus and spinal cord,which can mediate MCs immune response,to achieve analgesic effect.3.Herb-partition moxibustion on navel therapy is superior to drug covering navel therapy and moxibustion on navel therapy in alleviating dysmenorrhea symptoms in rats,but its regulating effects on the expression of TRPV1,TRPV2,TRPV4,TLR2 and TLR4 in hypothalamus and spinal cord of rats are less obvious than that of moxibustion on navel therapy,which means that besides TRPVs and TLRs pathways,herb-partition moxibustion on navel therapy may produced analgesic effect through other signal pathways.