| Objective:In this experiment,10ug/4u16-OHDA solution was injected into the unilateral striatum,and the DA content in the striatum was reduced by 30%～40%at 2 weeks after modeling,suggesting that the modeling was successful.To explore the pathogenesis of depression in the prodromal stage of PD,the TCM syndrome characteristics of rats with 30%～40%reduction in DA content in the prodromal stage of PD were determined by prescription test.The oxidative stress damaged the function of DA neurons,resulting in a decrease in DA levels in the substantia nigra and striatum and a decrease in hippocampal neurotransmitter levels.To compare the effects of acupuncture,traditional Chinese medicine,and acupuncture combined with medicine under the guidance of ’Tiaoshen Changzhi’ theory on the level of the striatum and total antioxidant level T-AOC in substantia nigra,the levels of MLT,SIP,MDA,SOD,GSH,and GSH-PX in substantia nigra and the depression-like behavior of rats.It was preliminarily verified that the acupuncture intervention under the guidance of the theory of ’Tiaoshen Changzhi’ could improve the depression-like behavior of PD precursor by anti-oxidative stress.The possible associated mechanisms were further explored by detecting the expression of related proteins and genes in the Keapl/Nrf2/HO-1 signaling pathway.Methods:1.The rat model of 30%～40%reduction of DA content in PD precursor was established by unilateral striatum injection of different doses of 6-OHDA solution.128 SD rats were divided into sham operation group,6ug/4ul group,8ug/4ul group,10ug/4ul group,32 rats in each group.The behavioral changes of rats were observed at 1w,2w,3w and 8w,including sleep evaluation,sucrose solution preference rate and buried food experiment.The content of DA in striatum of rats at each time point was detected by Elisa method.2.Through the improvement of rat appearance characterization,striatum DA content,Morphological changes of substantia nigra tissue,plasma viscosity,and hippocampal neurotransmitters by different prescriptions,the TCM syndrome characteristics of rat models with 30%～40%reduction of DA content in PD prodromal stage were judged.48 SD rats were randomly divided into the normal group,sham operation group,model group,Chaihu Jia Longgu Muli Decoction group,and Xiaoyao San group.Tongqiao Huoxue Decoction and Ditan Decoction group,8 rats in each group.After 14 days of modeling,the sham operation and model groups were given normal saline for 28 days.The other three groups were given traditional Chinese medicine for 28 days.After treatment,the characterization of rats was observed,The content of DA and metabolites in the striatum was detected by the Elisa method.HE staining was used to observe the pathology of substantia nigra in rats.The levels of TXB2,6-keto-PGF1a in plasma,5-HT,and NE in the hippocampus were detected by the Elisa method.3.To observe the effects of an acupuncture-medicine intervention under the guidance of ’Tiaoshen Changzhi’ theory on oxidative stress markers and DA levels in substantia nigra and striatum of rats with 30%～40%reduction of DA content in PD precursor period.48 SD rats were divided into a normal group,sham operation group,model group,Chaihu Jia Longgu Muli Decoction group,acupuncture group,acupuncture-medication group,and 8 rats in each group.After 14 days of modeling,the sham operation group and the model group were given normal saline for 28 days.The Chaihu Jia Longgu Muli Decoction group was given intragastric administration of traditional Chinese medicine for 28 days.The acupuncture group was treated with Tiaoshen Changzhi’ three-six-nine acupuncture for 28 days,and the acupuncture-medicine group was treated with Tiaoshen Changzhi’ three-six-nine acupuncture and Chaihu Jia Longgu Muli Decoction for 28 days.After the treatment,the behavioral changes of the rats were observed.The levels of MDA、MLT、SIP、SOD、GSH、GSH-Px in the substantia nigra of the rats were detected by Elisa method.The level of T-AOC and DA in the striatum of rats was detected by the Elisa method,and the expression of TH-positive neurons in the substantia nigra of rats was detected by immunohistochemistry.4.To observe the effect of acupuncture and medicine intervention under the guidance of ’Tiaoshen Changzhi’ theory on oxidative stress in rats with a 30%～40%reduction of DA content in PD prodromal period.48 SD rats were randomly divided into the normal group,sham operation group,model group,Chaihu Jia Longgu Muli Decoction group,acupuncture group,and acupuncture-medication group,with 8 rats in each group.After 14 days of modeling,the sham operation group and the model group were given normal saline for 28 days.Chaihu Jia Longgu Muli Decoction group was given traditional Chinese medicine by gavage for 28 days.The acupuncture group was treated with "Tiaoshen Changzhi" three-six-nine acupuncture for 28 days.The acupuncture-medication group was treated with "Tiaoshen Changzhi"three-six-nine acupuncture and Chaihu Jia Longgu Muli Decoction for 28 days.After treatment,Western blot and RT-PCR were used to detect the expression of Keapl,NQO1,Nrf2,HO-1,SOD,GSH,GSH-PX,proteins,and genes in the Keapl/Nrf2/HO-1 signaling pathway in the substantia nigra.Results:1.Behavioral results:One week after modeling,compared with the sham operation group,there was no significant difference in sleep latency,sleep duration,sucrose solution preference rate,and buried food experiment between 6ug/4ul group,8ug/4ul group and 10ug/4ul group(P>0.05).At 2 weeks,3 weeks,and 8 weeks after modeling,compared with the sham operation group,6ug/4ul group,and 8ug/4ul group,the sleep latency of rats in 10ug/4ul group was prolonged,the sleep duration was shortened,the preference rate of sucrose solution was decreased,and the time to find food was significantly prolonged,all of which were statistically significant(P<0.01,P<0.05).In the 10ug/4ul group,compared with 1w,2w,3w,and 8w,the sleep latency of rats was prolonged,the sleep duration was shortened,the preference rate of sucrose solution was decreased,and the time of looking for food was prolonged,all of which were statistically significant(P<0.05).There was no significant difference in sleep latency,sleep duration,sucrose solution preference rate,and buried food between the experimental groups at 2-week,3-week,and 8-week(P>0.05).2.In the sham operation group,there was no significant difference in the total DA content between the two groups at 1-week,2-week,3-week,and 8-week(P>0.05).In the 6ug/4ul group,8ug/4ul group,and 10ug/4ul group,the total content of DA in the striatum of rats was significantly reduced at 2 weeks after modeling(P<0.05).After modeling 2-week,3-week,and 8-week,compared with the sham operation group,6ug/4ul group,and 8ug/4ul group,the sleep latency of rats in 10ug/4ul group was significantly prolonged,the sleep duration was shortened,the preference rate of sucrose solution was decreased,and the time to find food was significantly prolonged,with statistically significant differences(P<0.01,P<0.05).In the 10ug/4ul group,compared with 1 w after modeling,After modeling at 2-week,3-week,and 8-week,the sleep latency of rats was prolonged,the sleep duration was shortened,the sucrose solution preference rate was decreased(P<0.05),and the time to find food was prolonged,the differences were statistically significant(P<0.05).There was no significant difference in sleep latency,sleep duration,sucrose solution preference rate,and buried food between the experimental groups at 2 weeks,3-week,and 8 weeks after modeling(P>0.05).In the sham operation group,there was no significant difference in the decrease of DA content in rats at 1w,2w,3w,and 8w after modeling(P>0.05).In the 6ug/4ul group,8ug/4ul group,10ug/4ul three groups,modeling 1w and 2w,3w,8w comparison,modeling 2w,3w,8w,rat striatum DA content decreased significantly(P<0.05).There was no significant difference in DA content between the two groups at 2w,3w,and 8w(P>0.05).3.Characterization of rats:Compared with the normal group,the activity status,emotional sleep,diet stool,skin,and hair scores of the model group were significantly increased(P<0.01).Compared with the model group,the activity status,emotional sleep,diet,and stool scores of the Chaihu jia Longgu Muli decoction group were decreased(P<0.05).The skin hair score of rats in the Chaihu jia Longgu Muli decoction group was significantly lower(P<0.01);compared with the model group,there was no significant difference in the scores of the rats in the Xiaoyao San group and the Tongqiao Huoxue Decoction combined with Ditan Decoction group(P>0.05).4.Striatum Elisa:Compared with the normal group,there was no significant difference in the sham operation group(P>0.05).DA,HVA,and DOPAC in the striatum of the model group were significantly reduced(P<0.01).Compared with the model group,the DA,HVA,and DOPAC in the striatum of the Chaihu jia Longmu group,Xiaoyaosan group,and Tongqiao plus Ditan group were significantly increased(P<0.01).Compared with the Tongqiaohe Ditan group,DA,HVA,and DOPAC in the striatum of the Chaihu Jialongmu group were significantly increased(P<0.01).Compared with the Xiaoyaosan group,DA,HVA,and DOPAC in the striatum of the Chaihu jia Longmu group were significantly increased(P<0.01).The Xiaoyaosan group was higher than the Tongqiaohe Ditan group(P<0.05).5.HE staining in Substantia nigra:Compared with the normal group,the morphology of neurons in the substantia nigra of the model group was irregular,the cytoplasm was swollen and deeply stained,the nucleus was pyknotic,the nerve fiber structure was loose and grid-like,the glial cells were significantly increased,the inflammatory cells were scattered,and the capillaries were significantly expanded.Compared with the model group,the Chaihu Jia Longgu Muli Decoction group,Xiaoyao San group,and Tongqiao Huoxue Decoction combined with the Ditan Decoction group had different degrees of improvement.The nucleus pyknosis of neurons was significantly reduced,the morphology was more regular,the nerve fibers were closer,the number of glial cells and inflammatory cells was reduced,and the telangiectasia was reduced.Among them,the improvement of the Chaihu Jia Longgu Muli Decoction group was the most obvious,followed by the Xiaoyao San group and Tongqiao Huoxue Decoction combined with Ditan Decoction.6.Plasma Elisa:Compared with the normal group,the plasma TXB2 in the model group was significantly increased(P<0.01),and 6-keto-PGF1a was significantly decreased(P<0.01).Compared with the model group,Chaihu jia Longgu Muli Decoction group,Tongqiao Huoxue Decoction,and Ditan Decoction group rats plasma TXB2 decreased,6-keto-PGF1a increased,Xiaoyao San group compared with the model group,no statistical difference(P>0.05);the plasma TXB2 in the Chaihu jia Longgu Muli decoction group was slightly lower than that in the Tongqiao Huoxue decoction combined with Ditan decoction group,and there was no statistical difference between the two groups(P>0.05).The plasma 6-keto-PGF1a in the Chaihu jia Longgu Muli decoction group was slightly higher than that in the Tongqiao Huoxue decoction combined with the Ditan decoction group,and there was no statistical difference(P>0.05).7.Hippocampal Elisa:The DA content in the striatum of rats in the model group was significantly decreased(P<0.01).Compared with the model group,the DA content in the striatum of rats in the Chaihu jia Longgu Muli Decoction group,Xiaoyao San group,and Tongqiao Huoxue Decoction combined with Ditan Decoction group was significantly increased(P<0.01).Compared with the Chaihu jia Longgu Muli Decoction group,the content of DA in the striatum of the Xiaoyao San group and Tongqiao Huoxue Decoction combined with the Ditan Decoction group was significantly decreased.The DA content in the striatum of rats in the Xiaoyao San group was slightly higher than that in Tongqiao Huoxue Decoction combined with the Ditan Decoction group(P>0.05).8.Behavioral results:The weight of rats in the model group increased slowly,the preference rate of sucrose solution decreased,and the tail suspension time was prolonged.In the results of weight growth rate,compared with the model group,the weight of rats in the Chaihu jia Longgu Muli Decoction group,acupuncture group,and the acupuncture-medication group increased significantly(P<0.01),and the acupuncture-medication group was higher than the Chaihu jia Longgu Muli Decoction group and acupuncture group(P<0.01).The weight growth rate of rats in the acupuncture group was higher than that in the Chaihu jia Longgu Muli decoction group(P<0.01).In the sucrose solution preference experiment,the acupuncture-medication group was significantly higher than the Chaihu jia Longgu Muli decoction group and the acupuncture group(P<0.01).The sucrose solution preference of rats in the acupuncture group was slightly higher than that in the Chaihu jia Longgu Muli Decoction group,and there was no statistical difference between the two groups(P>0.05).In the rat tail suspension experiment,the rat tail suspension time of the acupuncture-medication group was significantly less than that of the Chaihu jia Longgu Muli Decoction group and the acupuncture group,and there was a statistical difference between the groups(P<0.05).The tail suspension time of rats in the acupuncture group was slightly less than that in the Chaihu Jia Longgu Muli decoction group,and there was no statistical difference between the groups(P>0.05).9.Substantia nigra Elisa:In the model group,MDA in the substantia nigra was significantly increased(P<0.01),and MLT,SIP,SOD,GSH,and GSH-Px were significantly decreased(P<0.01).Compared with the model group,the MDA in the substantia nigra of the rats in the acupuncture-medicine group,the acupuncture group,and the Chaihu Jia Longgu Muli Decoction group was significantly decreased,and the MLT、SIPSOD、GSH and GSH-Px were significantly increased.The MDA in the substantia nigra of the rats in the acupuncture-medication group was significantly lower than that in the acupuncture group and the Chaihu Jia Longgu Muli Decoction group.The MLT、SIP、SOD、GSH and GSH-Px in the substantia nigra of the rats in the acupuncture-medication group were significantly higher than those in the acupuncture group and the Chaihu Jia Longgu Muli Decoction group.The MDA in the substantia nigra of the rats in the acupuncture group was slightly lower than that in the Chaihu jia Longgu Muli decoction group,and the MLT、SIP、SOD、GSH and GSH-Px in the substantia nigra were slightly higher than those in the Chaihu jia Longgu Muli decoction group.There was no significant difference between the groups(P>0.05).10.Striatum Elisa:The T-AOC in the striatum of the model group was significantly decreased(P<0.01);compared with the model group,the T-AOC in the striatum of the Chaihu jia Longgu Muli Decoction group,the acupuncture group,and the acupuncture-medication group was significantly increased;compared with the acupuncture-medication group,the T-AOC in the striatum of the Chaihu jia Longgu Muli decoction group and the acupuncture group decreased(P<0.01).The T-AOC in the striatum of the acupuncture group was slightly higher than that of the Chaihu jia Longgu Muli Decoction group,and there was no statistical difference between the groups(P>0.05).Compared with the model group,the DA content in the striatum of the Chaihu plus Longgu Muli Decoction group,the acupuncture group,and the acupuncture-medication group was significantly increased(P<0.05).11.Immunohistochemistry of substantia nigra:The number of TH-positive cells in the substantia nigra of the model group was significantly reduced(P<0.01).Compared with the model group,The number of TH-positive cells in the striatum of the Chaihu jia Longgu Muli Decoction group,the acupuncture group,and the acupuncture-medication group was significantly increased(P<0.01).The acupuncture-medication group was significantly higher than the Chaihu jia Longgu Muli decoction group and the acupuncture group(P<0.01).The number of TH-positive cells in the striatum of the acupuncture group was slightly higher than that of the Chaihu Jia Longgu Muli Decoction group,and there was no statistical difference between the two groups(P>0.05).12.The results of protein detection in Keapl/Nrf2/HO-1 signaling pathway:The expression of Keapl,Nrf2,HO-1,and NQO1 protein in substantia nigra of rats in the model group was up-regulated(P<0.01),and the expression of SOD,GSH and GSH-PX protein was down-regulated(P<0.01).Compared with the model group,the expression of Keapl protein in the substantia nigra of the Chaihu jia Longgu Muli Decoction group,the acupuncture group,and the acupuncture-medication group was down-regulated,and the expression of Nrf2,HO-1,NQO1,SOD,GSH,and GSH-PX protein was up-regulated;compared with the Chaihu plus Longgu Muli Decoction group,the expression of Keapl,Nrf2,HO-1,NQO1,SOD and GSH-PX protein in the substantia nigra of the acupuncture group was significantly up-regulated,with statistical differences.The expression of SOD and GSH-PX protein in the acupuncture group was higher than that in the Chaihu plus Longgu Muli Decoction group(P<0.05).The expression of Keapl,Nrf2,HO-1,NQO1,SOD,GSH,and GSH-PX in the substantia nigra of the acupuncture group was higher than that of the acupuncture group.13.The results of gene detection in Keap 1/Nrf2/HO-1 signaling pathway:The expression of Keapl,Nrf2,HO-1,and NQO1 mRNA in the substantia nigra of the model group was up-regulated(P<0.01);compared with the model group,the expression of Keap 1 mRNA in substantia nigra was down-regulated,and the expression of Nrf2,HO-1 and NQO1 mRNA was up-regulated in Chaihu Jia Longgu Muli Decoction group,acupuncture group and acupuncture combined with medicine group.Compared with the acupuncture group,the expression of Keapl,Nrf2,HO-1,and NQO1 mRNA in the substantia nigra of the Chaihu plus Longgu Muli Decoction group and the acupuncture group was significantly decreased(P<0.01).The Nrf2,HO-1,and NQO1 mRNA in the substantia nigra of the rats in the acupuncture group were slightly higher than those in the Chaihu Jia Longgu Muli Decoction group,and there was no significant difference between the two groups(P>0.05).Conclusion:1.Unilateral striatum injection of 10ug/4u16-OHDA in 2-8 weeks can construct a stable PD precursor DA content decreased by 30%～40%rat model,and complicated with depression sleep disorders,depression,olfactory disorders PD precursor behavioral performance.2.Chaihu Jia Longgu Muli Decoction can significantly improve the general appearance and laboratory indexes of experimental rats.The rat model with 30%-40%reduction of DA content in the PD precursor period is in line with the characteristics of TCM syndromes of liver depression syndrome.3.The biological basis of liver depression syndrome in a rat model with a 30%～40%reduction of DA content in the PD precursor period may be related to the lesion of substantia nigra striatum,the decrease of monoamine neurotransmitter level in the hippocampus and the increase of plasma viscosity.4.Acupuncture,traditional Chinese medicine and acupuncture combined with medicine under the guidance of the theory of " Tiaoshen Changzhi " may improve the depression-like behavior of PD precursor rats by increasing the levels of antioxidant enzymes SOD,GSH,GSH-PX,MLT,SIP,T-AOC in striatum and DA in substantia nigra and striatum,reducing the accumulation of lipid peroxidation product MDA,and improving the depression-like behavior of PD precursor rats.5.The combination of acupuncture and medicine under the guidance of ’Tiaoshen Changzhi ’theory is superior to acupuncture group and Chaihu Jia Longgu Muli Decoction group in improving the antioxidant level of substantia nigra and striatum,DA content and improving depression-like behavior in rats with 30%-40%reduction of DA content in PD precursor period.6.The intervention of acupuncture and medicine under the guidance of ’Tiaoshen Changzhi ’theory can reduce the expression of Nrf2,NQO1,HO-1 protein and mRNA in substantia nigra of 30%-40%rats by up-regulating DA content in PD precursor period,down-regulate the expression of Keapl protein and mRNA,activate Keapl/Nrf2/HO-1 signaling pathway,and increase the levels of antioxidant enzymes SOD,GSH and GSH-PX. |
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