Effects Of Conditional Location Preference And Expressions Of DA And D1R In VTA、PFC And NAC Brain Regions Of Mice Addicted To METH Were Injected With MLT At St36

Objective: MEth is highly addictive,can cause damage to the human body and is difficult to quit.MLT has a variety of functions,and has a protective effect on dopaminergic neurons.It is widely used in the study of nervous system diseases.Therefore,we used the method of acupoint injection with MLT at ST36 to treat addicted mice that had established METH-induced conditioned place preference,observed the effect of the therapy on the regression of conditioned place preference in METH-addicted mice,and explored whether the therapy had an effect by regulating the expression of DA and D1 R in the brain regions of VTA,PFC and NAC to achieve the effect of rehabilitation,it provides a theoretical basis for the clinical promotion of acupuncture detoxification.Method:Part 1 The efficacy of MLT acupoint injection preconditioning intervention in METH-dependent miceExperimental method:Adaptation phase: Used CPP box to select 100 mice without obvious CPP preference,record the residence time of mice in each box within 15 minutes,and take the residence time of mice on the natural non preference side of the box as the CPP value for statistics.One hundred mice were randomly divided into the control group,the model group,the normal saline acupoint injection group,and the MLT subcutaneous injection group,and the MLT acupoint injection group.Modeling stage:Mice in all groups except the control group were restricted to the non preferred side box for 30 minutes after intraperitoneal injection of METH(1 mg/kg)on the 1st,3rd,5th and 7th day;After intraperitoneal injection of 1 mg/kg normal saline on the 2nd,4th,6th and 8th days,it was limited to the preferred side box for 30 minutes;The CPP value was detected and counted on the 9th day.Treatment phase:(1)the control group and the model group: no treatment.(2)the normal saline acupoint injection group: mice were injected with 10 ul normal saline was injected into ST36 on both sides of the mice respectively.(3)the MLT subcutaneous injection group: 20 ul of 1 mg/kg MLT was subcutaneously injected into the back of the mice.(4)the MLT acupoint injection group: mice were given 10 ul of MLT(1 mg/kg)acupoint injection into ST36 bilaterally.The CPP values were detected and counted on the 13 th and 17 th days.10 mice in each group were randomly selected to take brain tissue for cryopreservation.Observation content:the degree of regression of mice ’preference for METH was evaluated by the change of CPP value at each stage of each group of mice.Part 2 Based on MLDS research at the molecular level1.Experimental materials: brain tissue collected on the 13 th and 17 th days of Part 1.2.Observation content: HE staining was used to observe the morphological changes of neurons and Nissl staining were used to count the neurons in the VTA,PFC and NAC brain regions of mice in each group on the13 th and 17 th day;The expression of DA in VTA,PFC and NAC brain regions of mice in each group were detected by ELISA on the 13 th and 17 th day;WB was used to detect the relative expression of D1 R in PFC and NAC brain regions of mice in each group on the 13 th and 17 th day.Result:Part 1 The efficacy of MLT acupoint injection preconditioning intervention in METH-dependent miceCPP test resultsModeling stage: Except for the control group,the CPP values of the mice in each group were significantly higher than those in the adaptation phase(P<0.01),which means that Modeling is successful.Treatment stage: On the 13 th and 17 th days,the CPP test results were similar.The results showed that the CPP values of mice in each group all subsided,and the preference of the MLT acupoint injection group subsided more significantly,and the CPP value was significantly lower than that of the model group(P<0.01);On the 17 th day,the CPP value of mice in each group was less than 450 s,and the preference changed significantly.The preference regression speed in each group:The MLT acupoint injection group>The MLT subcutaneous injection group>The normal saline acupoint injection group>The model group.Part 2 Based on MLDS research at the molecular levelHE staining resultsMorphological analysis: The results of HE staining on the 13 th and 17 th days showed that the gap between neurons in VTA,PFC and NAC brain areas of the model group was widened,the cytoplasm and nucleus were not clearly stained,the nuclei were pyknotic,and some cells showed vacuolar changes.In addition,neurons in various brain areas of the model group,the normal saline acupoint injection group and the MLT subcutaneous injection group were damaged to varying degrees,but neurons in various brain areas of the MLT acupoint injection group were more densely distributed,and the structure and morphology of nerve cells were better than those of the model group,the normal saline acupoint injection group and the MLT subcutaneous injection group were clearer and the morphological effect was better,indicating that acupoint injection MLT at ST36 could improve the morphological structure of neurons in VTA,PFC and NAC brain regions and reduce the degree of neuronal damage in each brain region.Nissl staining resultsAnalysis of neuron counts: On the 13 th and 17 th days,the results of neuron count in VTA,PFC and NAC brain areas of mice in each group were similar.Compared with the control group,the number of neurons in the model group decreased significantly(P<0.01);The number of neurons in the MLT acupoint injection group and the MLT subcutaneous injection group was significantly higher than that in the model group(P<0.01,P<0.01),and there was no significant difference between the MLT acupoint injection group and the control group(P>0.05).It shows that acupoint injection MLT at ST36 can reduce the loss of neurons in various brain regions.The results of ELISA: On the 13 th and 17 th days,the DA expression trend in VTA,PFC and NAC brain regions of mice in each group was similar,and on the 13 th day: The expression of DA in each brain area of mice in the model group,the normal saline acupoint injection group and the MLT subcutaneous injection group was significantly lower than that in the normal control group(P<0.01,P<0.01,P<0.01);However,the expression of DA in the MLT acupoint injection group was no significant difference with the control group(P>0.05).The test results on the 17 th day showed that the expression of DA in the VTA brain region of the mice in each group except the model group and the normal saline acupoint injection group was close to that of the normal control group(P>0.05,P>0.05).There was no significant difference(P>0.05),except for the model group,the expression of DA in the NAC brain region of the mice in each group was close to that of the normal control group(P>0.05).It shows that acupoint injection MLT at ST36 has a better effect on the up regulation of DA expression in VTA,PFC and NAC brain regions.The results of WB: Day 13: the expression levels of D1 R in PFC and NAC brain region of the mice in the model group,the normal saline acupoint injection group,the MLT subcutaneous injection group and the MLT acupoint injection group were significantly higher than those in the normal control group(P<0.01,P<0.01,P<0.01,P<0.01);Compared with the model group,the expression of D1 R in each brain region of the mice in the normal saline acupoint injection group,the MLT subcutaneous injection group and the MLT acupoint injection group decreased to varying degrees(P<0.01,P<0.01,P<0.01);the D1 R expression in the MLT subcutaneous injection group was significantly lower than that in the normal saline acupoint injection group(PFC: P<0.05,NAC: P<0.01),but still higher than the MLT acupoint injection group(P<0.01).The general trend of the detection results on the 17 th day was similar to that on the 13 th day.At this time,the expression of D1 R in the MLT acupoint injection group had recovered,and there was no significant difference with the control group(P>0.05).It shows that acupoint injection MLT at ST36 has a better effect on the down-regulation of the relative expression of D1 R in PFC and NAC brain regions than the normal saline acupoint injection group and the MLT subcutaneous injection group.Conclusion:1.MLT acupoint injection into ST36 can accelerates the regression of METH-induced conditioned place preference in mice and relieve the drug dependence of mice on METH,and the effect of MLT acupoint injection is better than that of MLT subcutaneous injection.2.MLT acupoint injection into ST36 can reduce the nerve damage caused by METH.3.MLT acupoint injection into ST36 may reduce the drug dependence of METH in mice by affecting the expression of DA and D1 R in MLDS.