The Effect Of Function Of Mast Cells On Navel Therapy Of Treating Dysmenorrhea Rats With Cold Coagulation And Blood Stasis Syndrome

OBJECTIVE:1.To observe navel therapy's effect on writhing response of dysmenorrhea rats with cold coagulation and blood stasis syndrome,to provide laboratory basis for analgesic effect of navel therapy.2.To investigate the relationship between function of mast cells(MCs)in Shenque(CV8)region,uterus and analgesic effect of navel therapy.3.To explore the signaling pathway of analgesic effect of navel therapy,through observing impact of navel therapy on relative expression of TLR2 and TLR4 in uterus of dysmenorrhea rats with cold coagulation and blood stasis syndrome.METHODS:90 healthy female wistar rats were randomly divided into 9 groups:the control group,the model group,the drug covering navel group,the herb-partition moxibustion on the navel group,the moxibustion on navel group,the disodium cromoglycate(DSC)+model group,the DSC+drug covering navel group,the DSC+herb-partition moxibustion on the navel group,the DSC+moxibustion on navel group.Every groups prepared for dysmenorrhea with congealing cold and blood stasis syndrome by systemic freezing combining injection of estradiol benzoate except control group.To give navel therapy groups the corresponding treatment from the sixth day after injected estradiol.Injection of DSC groups were given subcutaneous injection of 20ul DSC in SV8 region before navel therapy,all the treatment once time a day,for 5 days in total.After treatment on the tenth day,injected oxytocin through tail vein for every groups,then observed writhing response of every groups.After extraction of SV8 region and uterus,using modified toluidine blue staining to test the MCs number and degranulation,to observe impact of navel therapy on function of MCs.The expression of TLR2,TLR4 proteins in uterus of rats were detected by using Western blot method.The expression of TLR2mRNA,TLR4mRNA in uterus of rats were detected by using Quantitative Real-time PCR(qRT-PCR)method,in order to observe navel therapy effect on relationship between TLR2,TLR4 and MCs.RESULTS:1.The impact of navel therapy on cold-coagulation blood-stasis and writhing response of every groupsCompared with model group,the drug covering navel group,the herb-partition moxibustion on the navel group and the moxibustion on navel group(navel therapy groups)could relieve cold-coagulation blood-stasis.At the same time,the navel therapy groups could prolong incubation period,reduce frequency of writhing response,lower scores of writhing response,and those of herb-partition moxibustion on the navel group were more significant than drug covering navel group and moxibustion on navel therapy group(P<0.05).After function of MCs was inhibited,the writhing response of DSC+model group was weaker than model group(P<0.05),the analgesia effect were significantly weakened in DSC+navel therapy groups,especially in herb-partition moxibustion on the navel group(P<0.01).2.The influence of navel therapy on function of MCs in SV8 region and uterus of every groupsCompared with control group and model group,the MCs counts and degranulation ratio in SV8 region of navel therapy groups increased(P<0.05).There were no differences between navel therapy groups in terms of MCs counts in SV8 region,but in aspect of degranulation ratio in SV8 region of navel therapy groups,the herb-partition moxibustion on the navel group was significantly more than drug covering navel group and moxibustion on navel group(P<0.01).After function of MCs was inhibited,The MCs counts and degranulation ratio in SV8 region of DSC+navel therapy groups were lower than navel therapy groups(P<0.05).Compared with model group,the MCs counts and degranulation ratio in uterus of navel therapy groups decreased significantly(P<0.01).Comparisons of navel therapy groups,MCs degranulation ratio of herb-partition moxibustion on the navel group were lower than drug covering navel group and moxibustion on navel group(P<0.01).After injection of DSC,The MCs counts and degranulation of DSC+model group were lower than model group(P<0.05),while those of DSC+navel therapy groups increased,especially DSC+herb-partition moxibustion on the navel group and DSC+moxibustion on navel group(P<0.05),but DSC+model group were above both of groups.3.Western blot was applied to observe the impact of navel therapy on TLR2,TLR4 protein expression levels in uterus of every groupsCompared with control group,TLR2,TLR4 protein expression levels in uterus of model group significantly increased(P<0.01),those of navel groups also increased respectively,but were lower than model group(P<0.01).Comparison of navel groups,TLR2,TLR4 protein expression levels gradually decreased in the drug covering navel group,the moxibustion on navel group,the herb-partition moxibustion on the navel group(P<0.05).After injection of DSC,TLR2,TLR4 protein expression levels of DSC+model group were lower than model group(P<0.01).TLR2 protein expression levels of navel therapy groups were higher than navel therapy groups(P<0.05),but still lower than DSC+model group.There were no differences between DSC+navel groups in terms of TLR2 protein expression levels.TLR4 protein expression levels of DSC+herb-partition moxibustion on the navel group and DSC+moxibustion on navel group increased,but were lower than DSC+model group.Comparison of DSC+navel therapy groups,TLR4 protein expression levels of DSC+herb-partition moxibustion on the navel group were minimum(P<0.01).4.qRT-PCR was applied to observe the impact of navel therapy on TLR2mRNA,TLR4mRNA expression levels in uterus of every groupsTLR2mRNA,TLR4mRNA expression levels in uterus of model group and navel therapy groups were significantly more than control group(P<0.05),those of navel therapy groups were lower than model group(P<0.01).Comparisons of navel therapy groups,TLR2mRNA expression levels of herb-partition moxibustion on the navel group and moxibustion on navel group were lower than drug covering navel group(P<0.01).TLR4mRNA expression levels of herb-partition moxibustion on the navel group were the minimum.After injection of DSC,TLR2mRNA,TLR4mRNA expression levels of DSC+model group were lower than model group(P<0.01).TLR2mRNA,TLR4mRNA expression levels of DSC+navel therapy groups were increased in various degrees,but still lower than DSC+model group.Comparisons of navel therapy groups,TLR2mRNA,TLR4mRNA expression levels of DSC+herb-partition moxibustion on the navel group and DSC+moxibustion on navel group were lower than DSC+drug covering navel group(P<0.05).There were no significant differences between DSC+herb-partition moxibustion on the navel group and DSC+moxibustion on navel group.CONCLUSION:1.The navel therapy can significantly relieve pain and manifestation of rats caused by cold coagulation and blood stasis syndrome.2.MCs of SV8 region play an important role for analgesia effect of navel therapy.The degranulation of MCs in SV8 region take part in initiating process of analgesia,While DSC can inhibit this process.3.Navel therapy can relieve pain caused by dysmenorrhea through restraining expression levels of TLR2 and TLR4 of uterus,which can mediate MCs immune response.While DSC can weaken analgesia effect of navel therapy by inhibiting function of MCs in SV8 region.4.Compared with drug covering navel group and moxibustion on navel group,Herb-partition moxibustion on the navel group has an advantage of activation of MCs in SV8 region and relieve pain caused by dysmenorrhea.