Effects Of Direct Needling And Transcutaneous Needling Of “Sanyinjiao” Acupoint On OT, AVP And Its Receptors In Rats With Dysmenorrhea Caused By Cold Stagnation Syndrom

Needling technique is one of the important factors that may affect the effect of acupuncture points.The angle,direction and depth of needling are the three elements of filiform needling.Some studies have found that different needling angles can have different effects on the acupuncture therapeutic results by stimulating different tissues.In acupuncture and moxibustion clinic,some patients did not like strong needling sensation Transverse needling has advantage of less pain and gentle needling sensation,and easy to be accepted in large number of patients who are afraid of needling sensation.At present,there is no research systematically comparing the analgesic effect and mechanism of perpendicular needling and transverse needling at point "Sanyinjiao"(SP 6).Previous research by our research group found that the analgesic effect at SP 6 on primary dysmenorrhea is positive and has certain specificity.Different stimulation methods at SP 6 have different effects on its analgesic effect.The research results showed that strong stimulation intensity(deep insertion with thick needle with manipulation,similar to Deqi)at SP 6 had better analgesia effect for cold congealing dysmenorrhea rats compared with gentle stimulation intensity(shallow insertion of thin needle without manipulation,no Deqi).The underlying mechanism may be related to the fact that the strong stimulation intensity needling method activiated the analgesic substances in both peripheral and center systems.Based on our previous reseacrhes,this study observed the immediate analgesic effect of perpendicular needling and transverse needling at SP 6 in cold congealing dysmenorrhea rats,and explored its possible analgesic mechanism from the changes of uterine contraction related substances and their receptors in the peripheral and central,so as to provide experimental basis for optimizing the acupuncture treatment scheme for primary dysmenorrhea.Objective:To compare the effects of perpendicular needling and transverse needling at SP 6 on uterine contraction,writhing response,concentration and expression of oxytocin(OT),arginine vasopressin(AVP)and their receptors in serum,uterine tissue,spinal cord and hypothalamus in cold congealing dysmenorrhea rats,and to explore the possible analgesic mechanism.Methods:Experiment 1:Thirty two 3-month-old sexually mature unmated estrus SD female rats were randomly divided into blank control group,cold congealing dysmenorrhea model group,perpendicular needling group and transverse needling group with 8 rats in each group.The whole body freezing method was combined with abdominal subcutaneous injection of estradiol benzoate to prepare the model.On the 1st and 10th day,estradiol benzoate was injected with the dosage of 0.5 mg in each animal,and on the 2nd to 9th day with the dosage of 0.2 mg in each animal.After the last injection of estradiol for 1 hour,all rats in the model group and two acupuncture groups were injected intraperitoneally with oxytocin(2 U/each animal).After the injection,the rats in three modeling groups were placed in a freezer at-25℃for 4 hours with a 5 seconds interval between every 2 hours for ventilation.In the blank control group,only 0.9%NaCl solution was injected subcutaneously and intraperitoneally.After injection of oxytocin on the 10th day of model preparation SP 6 was needled 4-5 mm perpendicularly or transversely(with the needle tip pointing to the abdomen along the spleen channel)bilaterally in 2 acupuncture groups.The needles were retained for 20 minutes without manipulations in both groups.The rats in blank control group and the model group were fastened by the same method and without any treatment.The BL-420S biological function experimental system was used to observe the number and amplitude of uterine smooth muscle contraction waves,and the changes of uterine activity.Experiment 2:Forty female SD rats were divided into blank control group,cold coagulation dysmenorrhea model group,perpendicular needling group and transverse needling group with 10 rats in each group according to the random number table method.The methods of modeling preparation and acupuncture intervention were the same as experiment 1.The latency period and writhing score were observed within 20 minutes after injection of oxytocin.The concentrations of oxytocin(OT)and arginine vasopressin(AVP)in serum,uterine tissues,spinal cord and hypothalamus of rats in each group were detected by ELISA.The protein expressions of OTR and AVPR1A in uterine tissues and spinal cord,OTR and AVPR1B in hypothalamus were detected by Western blot.The expression of OTR,AVPR1A mRNAs in uterine tissues and spinal cord,OTR,AVPR1B mRNAs in hypothalamus was detected by Real Time PCR.Results:1.The effect of perpendicular needling and transverse needling at "Sanyinjiao" on uterine contraction in cold congealing dysmenorrhea ratsCompared with the blank control group,the number of uterine contraction waves in the model group was increased significantly(P<0.01),and the uterine activity intensity was increased significantly(P<0.01),the peak value of uterine contractions showed an increasing trend(P>0.05).Compared with the model group,the number of uterine contractions was decreased both in the perpendicular needling group and the transverse needling group(P<0.05),the uterine activity was decreased significantly in the perpendicular and transver needling groups(P<0.01,P<0.05),the peak value of uterine contractions showed a downward trend in 2 acupuncture groups(P>0.05).There were no significant differences on the degree of uterine contraction between 2 acupuncture groups(P>0.05).2.The effect of perpendicular needling and transverse needling at "Sanyinjiao" on writhing response in cold congealing dysmenorrhea ratsCompared with the blank control group,the writhing latency in the model group was shortened(P<0.05),and the writhing score was significantly increased(P<0.01).Compared with the model group,the writhing latency was significantly prolonged(P<0.01)and the writhing score was significantly decreased(P<0.01)in 2 acupuncture groups.There were no significant differences of writhing latency and writhing score between 2 acupuncture groups(P>0.05).3.The effect of perpendicular needling and transverse needling at "Sanyinjiao" on oxytocin and its receptor in cold congealing dysmenorrhea ratsCompared with the blank control group,the OT concentrations were increased significantly in serum and uterine tissues(P<0.01)and decreased significantly in spinal cord and hypothalamus(P<0.01)in the model group,the expressions of OTR in protein and mRNA levels were increased significantly in uterine tissues(P<0.01)and decreased in spinal cord and hypothalamus(P<0.01,P<0.05)in the model group.Compared with the model group,the serum OT concentration was decreased significantly(P<0.01)and the protein expression of OTR was increased in hypothalamus(P<0.05)in the perpendicular group;while the OT concentrations were decreased significantly in uterine tissues(P<0.01)and increased in spinal cord and hypothalamus both 2 needling groups(P<0.01,P<0.05),as well as the protein and mRNA expressions of OTR were decreased in uterine tissues(P<0.01,P<0.05).There were no significant differences in OT concentration,OTR protein and mRNA expressions between 2 acupuncture groups(P>0.05).4.The effect of perpendicular needling and transverse needling at "Sanyinjiao" on arginine vasopressin and its receptor in cold congealing dysmenorrhea ratsCompared with the blank control group,the AVP concentrations were increased in serum and uterine tissues(P<0.05,P<0.01)and decreased in spinal cord and hypothalamus(P<0.01,P<0.05)in the model group;the expressions of AVPR1A in protein and its mRNA levels were increased in uterine tissues(P<0.01,P<0.05),decreased in spinal cord(P<0.05,P<0.01)and decreased in hypothalamus(P<0.01)in the model group.Compared with the model group,the serum AVP concentration was decreased(P<0.05)and the protein expression of AVPR1B was increased in hypothalamus(P<0.05)in the perpendicular group;while the AVP concentrations were decreased significantly in uterine tissues(P<0.01)and increased in hypothalamus both 2 needling groups(P<0.05),as well as the protein and mRNA expressions of AVPR1A were decreased in uterine tissues(P<0.01,P<0.05).There were no significant differences in AVP concentration,AVP1A,AVP1B protein and mRNA expressions between 2 acupuncture groups(P>0.05).Conclusions:1.Both perpendicular needling and transverse needling at "Sanyinjiao" can reduce the activity of uterine smooth muscle,prolong the writhing latency,and reduce the writhing score,showing immediate analgesic effect in cold congealing dysmenorrhea rats.2.The underlying mechanism may be related to the effects of acupuncture in regulating OT,AVP and their receptors differently in peripheral and central regions.Both needling methods can reduce the concentrations and expressions of OT,AVP and their receptors in uterine tissues,moreover,its central mechanism may be more related to the up-regulation of the levels and expressions of OT and AVP in hypothalamus.The perpendicular needling method preliminarily shows a relatively wider effect in regulating peripheral and central OT,AVP and their receptors.