Clinical Observations And Metabolomics Study Of Medicinal Moxibustion On CV4 In Cold Coagulation Blood Stasis Dysmenorrhea

Objective:1.Using Wenjing Tang as the basic formula for medicinal moxibustion,this essay aims to explore the metabolomics study and the clinical efficacy that medicinal moxibustion on CV4 cure cold coagulation blood stasis dysmenorrhea.2.This essay conduct animal experiments and use LC-MS techniques to reveal the molecular biology mechanism of medicinal moxibustion on CV4 for cold coagulation blood stasis dysmenorrhea,providing scientific basis for clinical practice.Methods:1 Clinical trial research90 PD patients with cold coagulation blood stasis were divided into a 20min group,a40min group and a positive drug group according to the random number table method,with 30 patients in each group.The 20min group was cured for medicinal moxibustion on CV4 and mild technique for 20min,the 40min group changed the treatment time to 40 min.The positive drug group was given ibuprofen sustained release capsule,0.3g/time,BID.All patients were treated from day 1 to day 3 of their menstrual period,which will last for three months.Using the VAS scale,the efficacy of pain relief was evaluated,the efficacy of cold relief was evaluated using the symptom scale,and the efficacy of blood activation was evaluated using color ultrasound.2 Basic experimental researchForty SPF-grade Wistar female rats were acclimatized and fed for 7 days and then randomly divided into groups C,M,20 min,40 min,and Y;8/group.Saline was used for subcutaneous injection and gastric gavage in the control group,and estradiol benzoate and oxytocin as well as local freezing were used in the remaining 4 groups to establish a cold-congealed,blood-stasis PD model.After 2 h of modeling each day,the 20min group was cured for medicinal moxibustion on CV4 and mild technique for 20min,the 40min group changed the treatment time to 40 min.The positive drug group was given ibuprofen sustained release capsule solution,0.06g/kg for 10 consecutive days.On the 11th day,each group of rats was injected intraperitoneally with uterotonin,and after observing the torsional response of the rats for 30 min.Observe the morphology of the uterus and ovaries using HE technology,screen for differential metabolites in the control group,model group,and 40 minutes using plasma metabolomics,and measure the signaling pathways related to metabolites using ELISA and Western blot.Results:1 Clinical research（1）Total effective rate:The efficacy rate was 93.33%in the 20minute group,with a non-efficacy rate of 6.67%;40minute group 96.67%and 3.33%;positive drug group83.33%and 16.67%.（2）VAS rating scale and symptomatic rating scale:In the VAS score results,Both the20min group,the 40min group and the positive drug group could reduce the VAS total score and showed significant differences（P<0.01）.There was no difference in the VAS results between the positive drug group and the 40min group（P>0.05）.In the syndrome score results,20min group,40min group and positive drug group can reduce the total score and showed significant difference（P<0.01）.The total score of 40min group was lower than the positive drug and significantly different（P<0.01）.（3）Gynecological ultrasound:For the uterine artery peak systolic flow velocity PSV,resistance index RI,blood beat index PI,peak systolic/diastolic peak velocity S/D results,compared with that before treatment,the three groups decreased after treatment,there were significant differences（P<0.01）;compared with the values of PSV,RI,PI,and S/D,the 40min group was decreased,and there were significant differences（P<0.01）,all of the20-min groups were elevated but not statistically significant（P>0.05）.From the diastolic blood velocity EDV results,the values increased after treatment in the three groups,and the results were statistical significant（P<0.01）;compared with the EDV value in the positive drug group,the 40min group increased and the 20min group decreased,but not statistically significant（P>0.05）.2 Animal experimental research（1）Experimental results of basic inspection:Anal temperature:Post-treatment as compared to group M,there was difference in the 20min group（P<0.05）,40min group and C group showed significant differences（P<0.01）.Twist body reaction related indicators:in the twist number results,compared with the M group,there was difference in the 20min group（P<0.05）,40min group and Y group showed significant differences（P<0.01）.In the Twister score results,compared with the M group,there was difference in the 20min group（P<0.05）,40min group and Y group showed significant differences（P<0.01）.In the torsion latency results,the torsion latency of the other groups was significantly different compared with the M group（P<0.01）.Uterine index:Compared with the M group,the uterine index was significantly different in the other group（P<0.01）.（2）Pathological tissue section test results:Except for group C,the uterus and ovary had different degrees of damage,among which,40min group had the least damage,group Y was lighter,group 20min was heavier,and M group had the most damage.（3）Non-targeted metabolomics results:5-HETE,Gamma-Linolenic acid,Prostaglandin F2a,Prostaglandin E2,and Alpha-dimorphecolic acid showed significant differences in the occurrence of PD and during the treatment course.By analyzing the above differential metabolite enrichment pathway,we found that arachidonic acid metabolism pathway,PPAR metabolism pathway and linoleic acid metabolism pathway had significant differences in the onset and treatment of cold coagulation and blood stasis PD.（4）ELISA results:Compared with group M,the serum levels of COX-2,PGF_2α,IL-6,and IL-1βwere reduced in the remaining groups of rats with significant differences（P<0.01）.In the PGE₂results,compared with group M,the serum levels of PGE₂were elevated in the remaining groups of rats with significant differences（P<0.01）.（5）Westem blot results:In the COX-2 and PGE₂results,the contents in the remaining rats were significantly different compared with the M group（P<0.01）.Conclusion:1 Clinical efficacyMedicinal moxibustion on CV4 is effective in the treatment of PD with cold coagulation and blood stasis,which can reduce pain,relieve symptoms and improve uterine arterial blood flow of patients.And optimal treatment time is 40 min.2 Mechanism of actionThe mechanism of medicinal moxibustion on CV4 is effective in the treatment of PD with cold coagulation and blood stasis may be realized through the COX-2/PGE₂signaling pathway in arachidonic acid metabolism.