Effect Of Early Intervention With Moxibustion On Shenshu Point On AD-Like Rats Induced By Ovariectomized And D-galactose

Objective:(1)To observe the Alzheimer’s disease-like behavioral and pathological changes of rats being ovariectomized and injected with D-gal;(2)To observe the protective effect of moxibustion at Shenshu acupoint onthe loss of neurons in AD-like rats and its effect on ERK1/2 signaling pathway;(3)To study the mechanism of moxibustion at Shenshu acupoint to improve the ability of learning and memory and neuron loss in AD-like rats induced by activating hippocampal ERK / CREB signaling pathway in AD-like rats.Methods:Female SD rats aged 12 weeks were randomly divided into normal group(Con),sham operation group(Sham)and model group(VOX+D-gal),with 10 rats in each group.The bilateral ovaries of VOX+D-gal rats were removed,and the anti-infective treatment of penicillin was performed for 3 days.On the 4th day,D-galactose was intraperitoneally injected at a dose of 150 mg/kg/d,and only the ovarian fat was removed from the rats in Sham group.On the same time,0.9% normal saline was intraperitoneally injected at the same dose as VOX+D-gal group.After 12 weeks of modeling,the three groups of rats were subjected to behavioral tests such as Morris water maze,open field test,Sucrose preference test and ELISA to detect serum estrogen levels.The level of Aβ1-42 in hippocampus was detected by immunohistochemistry and the phosphorylation level of tau protein in hippocampus at PHF-1 was detected by western blot;In the second experiment,60 female SD rats aged 12 weeks were randomly selected and divided into normal group,sham operation group,model group,“Shens hu” group,“Weishu ”group,and non-acupoint group,with 10 rats in each group.Three days after the operation,Alzheimer was established by injecting D-gal(D-galactose)at a dose of 150 mg/kg/d for 90 days,The sham operation group was only removed off peripheral ovarian fat,and injected 0.9% physiological saline intraperitoneally at the same dose for 90 days.The Shenshu group,Weishu group,and non-point group were given suspended moxibustion from the 3rd day after the ovariectomy.The experimenter used a custom moxa with a diameter of about 6mm to suspend moxibustion 2~3cm above “Shenshu”,“Weishu”and“non-acupoint”on both sides of the rat to keep the local temperature at 41±0.5℃.Each acupoint starts at 9:00 am every day for 10 minutes one day,one course of treatment lasted for 5 days and the interval between courses was 2 days,12 consecutive courses were performed.No intervention was performed in normal groupnor the sham operation group,but restraint treatment was given in the same way as others.After modeling and intervention,immunohistochemical method was used to detect the expression of Aβ1-42 and estrogen receptor α in hippocampus of each group of rats;nissl staining was used to analyze the number and morphological changes of hippocampal neurons in each group.The protein expressions of p-CREB,p-ERK,ERK,and CREB in hippocampus of each group were detected by immunoblotting.Serum estradiol levels were detected by ELISA in rats at 24 hours after operation and after intervention;In experiment three,40 female SD rats of 12 weeks old were randomly divided into normal group,model group,Shenshu group and inhibitor group,10 rats in each group.Except for the normal group,the other three groups all had bilateral ovaries removed,and 3 days after surgery,an AD-like rat model was established by intraperitoneal injection of D-gal90 d at a dose of 150 mg / kg / d daily.From the 3rd day onwards,suspend moxibustion at the "Shenshu" points on both sides 1 to 2 cm above the skin.The moxibustion method and time were the same as in Experiment 2.The intervention method of the inhibitor group was based on the Shenshu group.The selective ERα receptor antagonist methylpiperidine(MPP)was injected subcutaneously every week at a dose of 0.25 mg / kg body weight,subcutaneously,once a week / w,a total of 12 w.The intervention methods of the other groups are the same as Experiment 2,and the detection indexes are the same as Experiment 2.Results: Experiment 1 shows the escape latency of each group decreased over time.From the second day of study period,compared with the normal group,the escape latency of the model group was significantly prolonged for 5 consecutive days.At the 14 th day of memory testing,compared with the normal group,the number of consecutive crossings of the model group decreased significantly(P <0.001).In the open field test,compared with the Sham group and the Con group,the number of upper limb lifts、 the movement time、 the total distance and the number of crossing grids within 5 minutes decreased significantly in the VOX+D-gal group(P<0.01).In the Sucrose preference test,the saccharide preference rate of VOX+Dgal group was significantly lower than that of Sham group and Con group(P<0.01).Serum estradiol test results showed that compared with Sham group and Con group,serum estradiol levels in VOX+D-gal group were significantly decreased in 24 h and 12 w after surgery(P<0.01).The results of immunohistochemistry showed that the level of Aβ1-42 in hippocampus of model group was significantly higher than that of sham o perationgroup and normal group(P<0.01).The results of western blot sh owed that the phosphorylation level of tau protein in model group was significantly higher than that in sham operation group and normal group(P <0.01);Experiment 2 shows that compared with the normal group,the re was no significant differences inthe indicators of the sham operation group.After modeling,Aβ1-42 expression was significantly increased in hippocampus(P <0.001).Neurons in the hippocampal region of the model group were disorderly arranged,lightly colored,and the cell bodies were vacuolated.The number of Nissl bodies in the hippocampal CA1 and CA3 regions decreased significantly(p<0.001).P-ERK/ERK,P-CREB/CREB levels decreased significantly(P<0.001);After 12 weeks of moxibustiont reatment,The expression of Aβ1-42 in hippocampus of rats in Shenshu group was significantly reduced(P <0.01),and the expression of Aβ1-42 in Shenshu group was significantly lower than that in Weishu group and non-acupoint group(P <0.01,P <0.01).There was no significant difference in total ERK nor CREB in each group;The number of neurons in hippocampal CA1 and CA3 areas of Shenshu group and Weishu group was significantly increased(p <0.05,p <0.01),and the levels of P-ERK / ERK and P-CREB / CREB in Shenshu group were significantly increased(P <0.001,P <0.001),the levels of P-ERK / ERK and P-CREB / CREB in the Weishu group increased significantly(P <0.01,P <0.05);the escape latency of the Shenshu group was significantly shorter than that of the Weishu group and the non-Meridian non-acupoint group(P <0.05),the number of neurons in hippocampal CA1,CA3 area was significantly higher than that of Weishu group and non-menstrual non-a cupoint group(p <0.01),the levels of P-ERK / ERK,P-CREB / CREB were significantly higher than that of Weishu group Non-meridian and non-acupoint group(P <0.05,P <0.01;Experiment 3 shows that After three months of intervention,there was no significant difference in the total amount of ERK and CREB in the hippocampus of each group,and the escape latency of each group decreased with time gone.Start from the learning stage Day2,the escape latency of the model group was significantly prolonged for five consecutive days compared with the normal group(P <0.001),the number of crossing platforms decreased significa ntly(P <0.01),and the expression of Aβ1-42 in the hippocampus area increased significantly(P < 0.001),neurons in the hippocampal region are disorderly arranged,lightly colored,most of the cell bodies are vacuolated,and the number of Nissl bodies is scarce.The number of main neurons in the CA1 and CA3 areas has decreased significantly(p <0.001).P-ERK and The level of P-CREB decreased significantly(P <0.001),and the expression of ERα in the hippocampus decreased significantly(P <0.01).From the 3rd day after treatment,the number of crossing platforms in the Shenshu group increased significantly compared with the model group(P <0.01).),The escape latency was significantly shortened for 5 consecutive days(P <0.001),the expression of Aβ1-42 in the hippocampus was significantly reduced(P <0.001),the number of neurons in the CA1 and CA3 regions was significantly increased(p <0.01),P-ERK/T-ERK,P-CREB/T-CREB levels were significantly increased(P <0.001),and ERα expression in the hippocampus area was significantly increased(P <0.05).Compared with the Shenshu group,the escape latency of the inhibitor group was prolonged for 5 consecutive days.The number of crossing the platform decreased significantly(P <0.01),and the expression of Aβ1-42 increased significantly.High(P <0.001),the number of neurons in CA1 and CA3 area was significantly less than that in Shenshu group and normal group(p <0.05,p <0.01),P-ERK/T-ERK,P-CREB/T-CREB levels Significantly increased(P <0.01,P <0.05)and the expression of ERa was significantly lower than that in the Shenshu group(P <0.01).Conclusion: VOX and D-gal injection rats have decreased learning and memory ability,accompanied by depressive symptoms,and the con tent of Aβ amyloid and phosphorylated tau protein increased significantly,which is an ideal model of senile dementia;Moxibustion Shenshu acu point may reverse the neuron loss in AD-like rats induced by ovarian injection and D-galactose intraperitoneal injection,and its neuroprotective effect may be related to activation of ERK1/2 signaling pathway in hippocampus;Moxibustion Shenshu points may improve learning and memory ability and neuron loss in AD-like rats by activating the ERα-dependent ERK / CREB signaling pathway in the hippocampus of AD rats.