| ObjectivePerimenopausal panic disorder(PPD)is characterized by recurrent unexpected panic attacks in perimenopausal women,and its pathogenesis may be related to serotonin metabolism dysfunction,leading to an imbalance between brain excitation and inhibition.Based on the clinical findings of PPD patients and the clinical intervention effect of acupoint embedding,this study constructed an animal model of PPD and explored the biological markers of serotonin metabolism pathway changes and excitation/inhibition imbalance,as well as the molecular mechanism of ERK-CREB-BDNF changes,focusing on the prefrontal cortex,hippocampus,and amygdala of the "fear center".The study also investigated the therapeutic mechanism and brain nerve effects of acupoint embedding for PPD,providing experimental evidence for the prevention and treatment of PPD.MethodsIn this study,a combined animal model of denervation and panic was used to simulate the pathological characteristics of patients with perimenopausal panic disorder.The animal model of perimenopausal panic disorder was modeled by bilateral ovarian removal in 3month-old female SD rats,and the model of panic was modeled by natural enemy predation stress combined with bystander electric shock.Five groups were grouped in this study,in which the OVX group underwent simple bilateral ovarian removal,the sham group underwent skin incision without ovarian removal,and the model group underwent panic modeling after denuded surgery.The animals of the denuded panic model were randomly divided into model group,medicine group,and acupoint catgut embedding(Acup)group.Acupuncture point burial treatment was performed after successful modeling,and the points were Guanyuan,ShenShu and Sanyinjiao,and the intervention was performed once a week for 4 consecutive times.In the medicine group,5 mg/ml sertraline solution was administered orally according to body weight,once daily for 4 weeks.This study can be divided into 4 parts:(1)Part I:The rats were divided into three groups,namely,the OVX group,the sham group and the(desensitized panic)model group.The differences between the model group and the rats in the OVX and sham groups were compared by vaginal smear.ELISA was used to determine the levels of sex hormones estradiol(E2),follicle-stimulating hormone(FSH)and luteinizing hormone(LH)in the serum.Sugar-water preference test,open-field test and elevated cross-maze test were used to determine the modeling status of the desensitized panic rats.(2)Part II:the experimental animals were divided into OVX group,sham group,model group,medicine group and Acup group.The improvement of anxiety-like behaviors of desensitized rats by acupoint burial and medicine intervention was observed by open-field and elevated cross-maze experiments;the modulation of serum sex hormones E2,FSH and LH was detected by Elisa;the effect of medicine and acupoint burial on the fear of the prefrontal cortex,hippocampus and amygdala was assessed by HE staining and Nissl staining.The neuronal morphology and cell degeneration and necrosis in the prefrontal cortex,hippocampus,and amygdala of the fear loop were evaluated by eosinhematoxylin staining and Nissl staining.(3)Part III:The experimental animals were divided into the OVX group,sham group,model group,medicine group,and Acup group.The levels of tryptophan metabolites in the prefrontal cortex,hippocampus,amygdala and serum of rats in each group were detected by high performance liquid chromatographymass spectrometry(LC-MS/MS)targeting tryptophan metabolism to determine the biomarkers of central and peripheral tryptophan metabolism in desensitized and frightened rats,and to assess the regulatory effects of medicine and acupoint catgut embedding on tryptophan metabolism;Western Blot technique was used to detect The relative protein expression of tryptophan pathway catalase IDO 1/2,TDO,AADAT,KMO,and TPH1/2 was measured by protein immunoblotting(Western Blot)to assess the activity of tryptophan pathway catalase in the depressed state of panic,and the effect of drugs and acupuncture point burial on their catalase activity.(4)Part IV:Western Blot technique was used to detect the expression and phosphorylation of ERK-CREB-BDNF pathway related proteins in the prefrontal,hippocampus and amygdala of each group of rats,to further elucidate the neurological mechanisms in the brain of desensitized and frightened rats,and to explore the central neurological regulation of acupoint burial and drugs at the molecular level.ResultsResults of experiment 11.Vaginal smear results showed irregular changes in the motility cycle in de-ovulated female rats,or the appearance of inter-motility periods or motility periods lasting several days,etc.It can be considered that the motility cycle is disturbed and the animals are in perimenopause.2.Sex hormone results.① Compared with the sham group,the serum estrogen content of the rats in the OVX and model groups was reduced(P<0.05).②Serum follicle-stimulating hormone levels were increased in the OVX and model groups compared with the sham group(P<0.05).③Serum luteinizing hormone levels were increased in the OVX and model groups compared with the sham group(P<0.05).3、Behavioral results(1)Before modeling:①Open field test results:There was no statistically significant difference(P>0.05)in the total distance traveled,central distance,central time,and activity counts among the groups.② Elevated plus maze test results:There was no statistically significant difference(P>0.05)in the percentage of time spent in the open arms,percentage of distance traveled in the open arms,and percentage of entries into the open arms among the groups.(2)After modeling:①Open field test results:Compared with the castrated group,the model group showed a significant decrease in total distance traveled,central distance,central time,and activity counts in the open field test(P<0.05).② Elevated plus maze test results:Compared with the castrated group,the model group showed a significant decrease in the percentage of time spent in the open arms,percentage of distance traveled in the open arms,and percentage of entries into the open arms(P<0.05).(3)Before and after modeling:①Open field test results:Before and after modeling,there was no statistically significant difference(P>0.05)in the open field test results between the castrated group and the sham surgery group.However,the model group showed a significant decrease in total distance traveled,central distance,central time,and activity counts after modeling(P<0.05).② Elevated plus maze test results:Before and after modeling,there was no statistically significant difference(P>0.05)in the elevated plus maze test results between the castrated group and the sham surgery group.However,the model group showed a significant decrease in the percentage of time spent in the open arms,percentage of distance traveled in the open arms,and percentage of entries into the open arms after modeling(P<0.05).(4)Sugar-water preference experimentAfter modeling,there was no statistically significant difference in the sucrose preference value among the castrated group,sham surgery group,and model group of rats(P>0.05).Results of Experiment 21.Behavioral results(1)Before the intervention:① Open field test results:Compared with the castrated group,the model group,acupoint embedding group,and drug group showed a significant decrease in total distance traveled,central distance,central time,and activity counts in the open field test(P<0.05).There was no statistically significant difference(P>0.05)between the acupoint embedding group and drug group compared with the model group.② Elevated plus maze test results:Compared with the castrated group,the model group,acupoint embedding group,and drug group showed a significant decrease in the percentage of time spent in the open arms,percentage of distance traveled in the open arms,and percentage of entries into the open arms(P<0.05).There was no statistically significant difference(P>0.05)between the acupoint embedding group and drug group compared with the model group.(2)After the intervention:① Open field test results:Compared with the model group,the acupoint embedding group and drug group showed a significant increase in total distance traveled,central distance,central time,and activity counts in the open field test(P<0.05).② Elevated plus maze test results:Compared with the model group,the acupoint embedding group and drug group showed a significant increase in the percentage of time spent in the open arms,percentage of distance traveled in the open arms,and percentage of entries into the open arms(P<0.05).(3)Before and after modeling:① Open field test results:After intervention,the acupoint embedding group and drug group showed a significant increase in total distance traveled,central distance,central time,and activity counts in the open field test(P<0.05).②Elevated plus maze test results:After intervention,the acupoint embedding group and drug group showed a significant increase in the percentage of time spent in the open arms,percentage of distance traveled in the open arms,and percentage of entries into the open arms(P<0.05).2.Serum sex hormone results:① Compared with the model group,the acupoint embedding group and drug group showed a significant increase in serum estrogen levels(P<0.05).Compared with the acupoint embedding group,the drug group showed a significant decrease in serum estrogen levels(P<0.05).② Compared with the model group,the acupoint embedding group and drug group showed a significant decrease in serum follicle-stimulating hormone levels(P<0.05),and there was no statistically significant difference(P>0.05)between the acupoint embedding group and drug group.③ Compared with the model group,the acupoint embedding group and drug group showed a significant decrease in serum progesterone levels(P<0.05),and there was no statistically significant difference(P>0.05)between the acupoint embedding group and drug group.3、Pathological results(1)HE staining:2-4 layers of pyramidal cells in CA1,CA3 and DG areas of the hippocampus in the deconditioned and sham groups were seen in full morphology,with regular and uniform distribution of prefrontal cortical pyramidal cells and granule cells and clear nuclei.In the model group,the number of layers of cortical and hippocampal pyramidal cells was reduced,the nuclei were shrunken,the cells were stacked and scattered,the intercellular space was sparse,and even some pyramidal cells were lost;the number of neuronal cells in the amygdala was increased,but the nucleoli were not clear and the shrunken cells were increased.(2)Nissl staining:the number of layers of cortical and hippocampal pyramidal cells in the model group was reduced,the distribution was sparse,the cell staining was lighter,the nuclei were unclear,the number of intracellular Nissl vesicles was reduced,and a small number of cytostomes were vacuolated;the number of amygdala pyramidal cells was increased,the distribution was more in the BLA area,but the nuclei were missing more,and the overall coloring was darker.Experiment 3 results1.Principal component analysis:in the principal component analysis of different tissue samples,PCI=25.98%and PC2=12.31%for cortical samples;PC1=18.6%and PC2=14.07%for hippocampal samples;PC1=18.05%and PC2=15.34%for amygdala samples;PC1=19.1%and PC2=1 1.68%for serum samples.2.Differential metabolites:① Prefrontal cortex:Compared with the castrated group,the model group showed a decrease in IP,IPA,L-KYN,and KYNA.The acupoint embedding group and drug group showed an increase in IP and IPA compared with the model group,and the acupoint embedding group showed a decrease in SER,while the drug group showed an increase in L-KYN and KYNA.②Hippocampus:Compared with the castrated group,the model group showed a decrease in IP and IPA,and an increase in SER.The acupoint embedding group and drug group showed an increase in IP and IPA compared with the model group.③Amygdala:Compared with the castrated group,the model group showed a decrease in IP,IPA,and PA.The acupoint embedding group and drug group showed an increase in IP and IPA compared with the model group.④ Serum:Compared with the castrated group,the model group showed a decrease in IP and IPA.The acupoint embedding group showed a decrease in TRM compared with the model group,while the drug group showed an increase in IP and IPA compared with the model group.3.Kynurenine metabolites.(1)Prefrontal cortex:①KYN/TRP results:Compared with the castrated group,the sham surgery group showed a decrease in KYN/TRP ratio(P=0.018).②KYNA/KYN results:no significant difference among groups(P>0.05).③QA/KYN results:no statistical difference in comparison among groups(P>0.05).④KYNA/QA results:Compared with the castrated group,the model group showed a decrease in KYNA/QA ratio(P=0.045).⑤5-HTP/TRP results,no statistical difference among groups(P>0.05).⑥ SER/5-HTP results:Compared with the castrated group,the model group showed an increase in SER/5-HTP ratio(P=0.039).Compared with the model group,the drug group and acupoint embedding group showed a decrease in SER/5-HTP ratio(P=0.007,0.011).(2)Hippocampus:① KYN/TRP results:Compared with the castrated group,the model group showed an increase in KYN/TRP ratio(P=0.025).②KYNA/KYN results:Compared with the model group,the drug group and acupoint embedding group showed a decrease in KYNA/KYN ratio,and the difference was statistically significant(P=0.045,0.042).③QA/KYN results:Compared with the castrated group,the model group showed an increase in QA/KYN ratio(P=0.027).Compared with the model group,the drug group and acupoint embedding group showed a decrease in QA/KYN ratio,and the difference was statistically significant(P=0.041,0.036).④KYNA/QA results There was no statistically significant difference(P>0.05)in the comparison between groups.⑤5-HTP/TRP results:no statistical difference among groups(P>0.05).⑥ SER/5-HTP results;Compared with the castrated group,the model group showed an increase in SER/5-HTP ratio(P=0.002).Compared with the model group,the drug group and acupoint embedding group showed a decrease in SER/5-HTP ratio,and the difference was statistically significant(P=0.008,0.008).(3)Amygdala:①KYN/TRP results:no statistical difference among groups(P>0.05).②KYNA/KYN results:There was no statistically significant difference(P>0.05)in the comparison between groups.③QA/KYN results:Compared with the model group,the drug group showed a decrease in QA/KYN ratio(P=0.031).④KYNA/QA results:There was no statistically significant difference(P>0.05)in the comparison between groups.⑤5HTP/TRP results:Compared with the castrated group,the sham surgery group showed an increase in 5-HTP/TRP ratio,and the difference was statistically significant(P=0.134).⑥SER/5-HTP results:Compared with the castrated group,the model group showed an increase in SER/5-HTP ratio(P=0.032).Compared with the model group,the drug group and acupoint embedding group showed a decrease in SER/5-HTP ratio,and the difference was statistically significant(P=0.031,0.021).(4)Serum:①KYN/TRP results:Compared with the castrated group,the model group showed an increase in KYN/TRP ratio(P=0.042).②KYNA/KYN results:there was no significant difference among the groups(P>0.05).③QA/KYN results;Compared with the model group.the drug group showed a decrease in QA/KYN ratio(P=0.049).④KYNA/QA results:there was no significant difference among the groups(P>0.05).⑤ 5-HTP/TRP results:no statistical difference among groups(P>0.05).⑥SER/5-HTP results:Compared with the drug group,the acupoint embedding group showed an increase in SER/5-HTP ratio(P=0.022).4.Tryptophan metabolizing enzyme results(1)Prefrontal cortex:①IDO1 protein:no statistical difference was seen among the groups(P>0.05).②IDO2 protein:Compared with the castrated group,the sham surgery group showed a decrease in IDO2 protein expression(P=0.002);compared with the model group,the drug group showed a decrease in IDO2 protein expression(P=0.026);compared with the drug group,the acupoint embedding group showed an increase in IDO2 protein expression,and the difference was statistically significant(P<0.05).③TDO protein:no statistical difference was seen among TDO groups(P>0.05).④ AADAT protein:Compared with the castrated group,the sham surgery group showed an increase in AADAT protein expression(P<0.001);compared with the model group,the drug group showed an increase in AADAT protein expression,and the difference was statistically significant(P=0.042).⑤ KMO protein:Compared with the castrated group,the model group showed an increase in KMO protein expression(P=0.0001);compared with the model group,the drug group and acupoint embedding group showed a decrease in KMO protein expression(P=0.005,0.004).⑥ TPH protein:no statistical difference was seen among the groups(P>0.05).⑦TPH2 protein:Compared with the castrated group,the model group showed a decrease in TPH2 protein expression(P=0.038).(2)Hippocampus:① IDO1 protein:Compared with the castrated group,the model group showed an increase in IDO1 protein expression(P=0.002).②IDO2 protein:no statistical difference was seen among the groups(P>0.05).③TDO protein:no statistical difference was seen among the groups(P>0.05).④ AADAT protein:Compared with the castrated group,the model group showed an increase in AADAT protein expression(P=0.001);compared with the model group,the drug group and acupoint embedding group showed a decrease in AADAT protein expression(P=0.029,0.044).⑤KMO protein:Compared with the castrated group,the model group showed an increase in KMO protein expression(P=0.007);compared with the model group,the acupoint embedding group showed a decrease in KMO protein expression(P=0.031).⑥TPH protein:There was no statistically significant difference(P>0.05)in the comparison between groups.⑦ TPH2 protein:no statistical difference was seen among the groups(P>0.05).(3)Amygdala:① IDO1 protein:Compared with the castrated group,the model group showed an increase in IDO1 protein expression(P=0.012).②IDO2 protein:Compared with the castrated group,the model group showed a decrease in IDO2 protein expression(P=0.017);compared with the drug group,the acupoint embedding group showed a decrease in IDO2 protein expression(P<0.05).③TDO protein:Compared with the castrated group,the sham surgery group showed a decrease in TDO protein expression(P=0.017);compared with the model group,the drug group showed an increase in TDO protein expression(P=0.031);compared with the drug group,the acupoint embedding group showed a decrease in TDO protein expression(P=0.0498).④AADAT protein:no statistical difference was seen among the groups(P>0.05).⑤KMO protein:Compared with the castrated group,the model group showed an increase in KMO protein expression(P=0.043).⑥ TPH protein:no statistical difference was seen among the groups(P>0.05).⑦ TPH2 protein:no statistical difference was seen in the comparison among groups(P>0.05).5.The results of the multidimensional scaling analysis(1)Tryptophan metabolites:a)Parallel profile test:KYN/TRP,QA/KYN,KYNA/KYN,KYNA/QA,5-HTP/TRP,and SER/5-HTP profiles were parallel to each other(P>0.05).b)Overlapping profile test:KYN/TRP,5-HTP/TRP,and SER/5-HTP profiles did not overlap(P<0.05),while QA/KYN,KYNA/KYN,and KYNA/QA profiles overlapped(P>0.05).c)Horizontal profile test:QA/KYN and KYNA/QA profiles were not horizontal(P<0.05),while KYNA/KYN profile was horizontal(P>0.05).(2)Tryptophan metabolizing enzymes:a)Parallel profile test:IDNO,TDO,KMO,TPH1,and TPH2 protein profiles were parallel to each other(P>0.05),while ID02 and AADAT protein profiles were not parallel(P<0.05).b)Overlapping profile test:TDO protein profile did not overlap(P<0.05),while IDNO,KMO,TPH1,and TPH2 protein profiles overlapped(P>0.05).c)Horizontal profile test:KMO and TPH2 protein profiles were not horizontal(P<0.05),while IDNO and TPH1 protein profiles were horizontal(P>0.05).Experiment 4 results1.ERK-CREB-BDNF protein results(1)Prefrontal cortex:①CREB protein expression was lower in the model group compared to the castration group(P=0.045);CREB protein expression was higher in the suture group compared to the model group(P<0.01);and CREB protein expression was higher in the suture group compared to the drug group(P<0.01).②P-CREB protein expression was lower in the drug group compared to the model group(P=0.041).③ There was no statistically significant difference in ERK protein expression between groups(P>0.05).④ There was no statistically significant difference in P-ERK protein expression between groups(P>0.05).⑤BDNF protein expression was lower in the model group compared to the castration group(P=0.0047);BDNF protein expression was higher in the suture group compared to the model group(P=0.028).⑥The P-ERK/ERK ratio was higher in the model group compared to the castration group(P=0.041).⑦The P-CREB/CREB ratio was lower in the drug group and suture group compared to the model group(P=0.0095,0.0003).(2)Hippocampus:① CREB protein expression was higher in the drug group and suture group compared to the model group(P=0.018,0.031).② There was no statistically significant difference in P-CREB protein expression between groups(P>0.05).③ERK protein expression was higher in the drug group and suture group compared to the model group(P=0.0002,0.043);ERK protein expression was lower in the suture group compared to the drug group(P=0.018).④P-ERK protein expression was higher in the drug group compared to the model group(P=0.042);P-ERK protein expression was lower in the suture group compared to the drug group(P=0.047).⑤BDNF protein expression was lower in the model group compared to the castration group(P=0.011);BDNF protein expression was higher in the drug group and suture group compared to the model group(P=0.0003.0.0407).⑥The PERK/ERK ratio was higher in the model group compared to the castration group(P=0.032);the P-ERK/ERK ratio was lower in the drug group and suture group compared to the model group(P=0.010,0.034).⑦The P-CREB/CREB ratio was higher in the model group compared to the castration group(P=0.016).(3)Amygdala:①CREB protein expression was lower in the model group compared to the castration group(P=0.003);CREB protein expression was higher in the drug group compared to the model group(P=0.02).②There was no statistically significant difference in P-CREB protein expression between groups(P>0.05).③There was no statistically significant difference in ERK protein expression between groups(P>0.05).④P-ERK protein expression was lower in the suture group compared to the model group(P=0.006);P-ERK protein expression was lower in the suture group compared to the drug group(P=0.010).⑤BDNF protein expression was higher in the drug group compared to the model group(P=0.045).⑥The P-ERK/ERK ratio was higher in the model group compared to the castration group(P=0.010);the P-ERK/ERK ratio was lower in the drug group and suture group compared to the model group(P=0.044,<0.0001);the P-ERK/ERK ratio was lower in the suture group compared to the drug group(P=0.002).⑦ The P-CREB/CREB ratio was higher in the model group compared to the castration group(P=0.0005);the PCREB/CREB ratio was lower in the drug group compared to the model group(P=0.014).2.The results of the multidimensional scaling analysis① Parallel profile analysis:BDNF,CREB,ERK,and P-ERK protein profiles were not parallel(P<0.05),while P-CREB,P-CREB ratio,and P-ERK ratio profiles were parallel to each other(P>0.05).②Coincident profile analysis:P-CREB,P-CREB ratio,and P-ERK ratio profiles were coincident(P>0.05).③Horizontal profile analysis:P-CREB profile was horizontal(P>0.05),while P-CREB ratio and P-ERK ratio profiles were not horizontal(P<0.05).Conclusion:(1)OVX panic model rats exhibit hormonal imbalances and anxiety-like behavior.Acupoint embedding can improve the disrupted hormonal levels and alleviate anxiety-like behavior in castration-induced fear model rats,as well as improve the pathological structure of the fear circuit brain regions(cortex,hippocampus,and amygdala).(2)The central tryptophan metabolism pathway is disrupted in castration-induced fear model rats,mainly characterized by abnormal elevation of QA,decreased KYNA/QA ratio,and increased activity of the metabolic enzyme KMO.Acupoint embedding can inhibit the activity of the tryptophan metabolism catalytic enzyme KMO,regulate tryptophan metabolism products,maintain the dynamic balance of KYNA/QA,thereby inhibiting neuronal overexcitation and correcting the metabolic abnormalities in the fear center.(3)The central P-ERK and P-CREB phosphorylation are abnormally activated in the cortex,hippocampus,and amygdala of castration-induced fear model rats.Acupoint embedding can inhibit the overactivation of ERK-CREB phosphorylation in the cortex,hippocampus,and amygdala,and promote BDNF expression to protect neuronal regeneration and improve synaptic plasticity,thereby regulating brain neural activity. |
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