| Objective:Through in vivo and in vitro experiments,the effect and mechanism of Zuogui Jiangtang Jieyu decoction on regulating the Wnt3a and Shh double signals,improving the proliferation and differentiation of neural stem cells in the dentate gyrus of the hippocampus of diabetes patients with depression,enhancing the ability of adult hippocampal neurogenesis,and thus regulating the depression like behavior and cognitive function of animals were studied in depth.Methods:This study consists of six parts,the first three parts are animal experiments,and the last three parts are cell experiments.The research methods of each part are described as follows.1.This part aims to clarify the intervention effect of Zuogui Jiangtang Jieyu decoction on hippocampal neurogenesis in rats with diabetes and depression.Six groups were set up in the experiment,including normal group,diabetes and depression model group,positive drug(metformin+fluoxetine)group,and Zuogui Jiangtang Jieyu decoction 2,1,0.5 times(20.52,10.26,5.13 g/kg)group.After administration,the animals’ depressive behavior and cognitive function were evaluated by open field test,forced swimming test and water maze test.The contents of peripheral blood glucose,blood insulin,cholesterol,triglyceride,high-density lipoprotein and low-density lipoprotein were measured by ELISA and the insulin resistance index was calculated.HE staining,Nissan staining and Golgi staining were used to detect the morphology of neurons and dendrites in the dentate gyrus of hippocampus.The expression of Nestin,Brdu,DCX,NeuN and Tuj-1 proteins in the dentate gyrus of hippocampus was detected by immunofluorescence method.2.This part aims to study the effect of Zuogui Jiangtang Jieyu decoction on regulating Wnt3 a signal to improve the differentiation function of hippocampal neural stem cells in rats.The experiment is divided into five groups,including normal group,diabetes complicated depression model group,positive drug(metformin+fluoxetine)group,Zuogui Jiangtang Jieyu decoction group,Zuogui Jiangtang Jieyu decoction+DKK-1 group,In this part,the molecular mechanism of Zuogui Jiangtang Jieyu decoction was studied at 20.52g/kg.After administration,the animals’ depressive behavior and cognitive function were evaluated by open field test,forced swimming test and water maze test.The content of fasting blood glucose was measured by ELISA.Western blot was used to detect the expression of Wnt3a,Dvl-1,APC protein and β-Nuclear expression of catenin.Western blot was used to detect the expression of the regulatory factors of neural stem cell differentiation,Ngn2 and NeuroD1,in the dentate gyrus of rats.The fluorescence expression of Nestin,DCX and NeuN in the dentate gyrus of hippocampus was detected by immunofluorescence method.3.This part aims to study the effect of Zuogui Jiangtang Jieyu decoction on improving the proliferation of neural stem cells in the hippocampus of rats by regulating Shh signal.The experiment is divided into five groups,including normal group,diabetes complicated with depression model group,positive drug(metformin+fluoxetine)group,Zuogui Jiangtang Jieyu decoction group,Zuogui Jiangtang Jieyu decoction+cycloparamine group.The dosage of Zuogui Jiangtang Jieyu decoction in this part is 20.52g/kg,which is the same as the second part.After administration,the animals’ depressive behavior and cognitive function were evaluated by open field test,forced swimming test and water maze test.The content of fasting blood glucose was measured by ELISA.Western blot method was used to detect the expression of Shh,Ptch 1,Smo protein and Gli-1 protein in the dentate gyrus of the rat hippocampus.Western blot was used to detect the expression of cyclinD1 and SOX2 proteins,which regulate the proliferation of neural stem cells in the dentate gyrus of rats.The fluorescence expression of Brdu in rat hippocampal dentate gyrus was detected by immunofluorescence method.4.This part aims to verify the regulatory effect and molecular mechanism of Zuogui Jiangtang Jieyu Formula on the function of neural stem cells in vitro through cell experiments.The first is to clarify the intervention effect of Zuogui Jiangtang Jieyu Recipe containing serum on the proliferation and differentiation of neural stem cells in vitro under the simulated brain environment of diabetes complicated with depression.The experiment was divided into four groups,including the normal group,the model group,the blank serum group,and the Zuogui Jiangtang Jieyu Formula drug-containing serum group.Among them,the dosage of Zuogui Jiangtang Jieyu formula involved in the cell experiment was 30.78g/kg.The stem cell culture medium was added with glucose and corticosterone to simulate the brain environment of diabetes complicated with depression for modeling.The blank serum group and Zuogui Jiangtang Jieyu Decoction containing serum group were given different serum at the same time of modeling.After 18 hours of intervention,WST-1 was used to detect the viability of stem cells,and the fluorescence expression of Brdu,DCX,NeuN,GFAP in cells was detected by immunofluorescence combined with high content cell imaging technology.The second is to study the mechanism of Zuogui Jiangtang Jieyu Prescription containing serum interfering with Wnt3a signal regulating the differentiation function of neural stem cells in vitro.The experiment was divided into five groups,including normal group,model group,blank serum group,Zuogui Jiangtang Jieyu Formula containing serum group,and XAV-93 9 group.Among them,the dosage of Zuogui Jiangtang Jieyu Formula was 30.78g/kg,and the cells of XAV-93 9 group were simultaneously added with 3 0.7 8g/kg Zuogui Jiangtang Jieyu Formula drug-containing serum and XAV-939.After cell intervention,WST-1 was used to detect stem cell viability.The fluorescence expression of DCX,NeuN and GFAP in cells was detected by immunofluorescence combined with high content cell imaging technology.Western blot was used to detect Wnt3a,LRP5,LRP6 β-Catenin protein expression was detected by qRT-PCR.Finally,to study the mechanism of Zuogui Jiangtang Jieyu Prescription containing serum interfering with Shh signal regulating the proliferation of neural stem cells in vitro.The experiment was divided into five groups,including the normal group,the model group,the blank serum group,the Zuogui Jiangtang Jieyu Formula medicated serum group,and the cyclophosphamide group.Among them,the dosage of Zuogui Ji angtang Jieyu Formula is 30.78g/kg,and 30.78g/kg Zuogui Jiangtang Jieyu Formula drug-containing serum and cyclopamide are added to the cells of the cyclopamide group.After cell intervention,WST-1 was used to detect stem cell viability.The fluorescence expression of Brdu in cells was detected by immunofluorescence combined with high content cell imaging technology.Western blot was used to detect the expression of Shh,Ptch 1,Smo protein and the nuclear expression of Gli-1 protein in each group,and the expression of CyclinDl and SOX2 gene was detected by qRT-PCRResults:1.The behavioral results showed that Zuogui Jiangtang Jieyu decoction could significantly increase the number of activities of rats in the open field experiment,significantly shorten the static time of animals,shorten the immobility time of rats in the forced swimming experiment,shorten the time spent in finding the underwater platform in the water maze experiment,extend the time and distance in the target quadrant,and increase the number of times through the platform.The biochemical test results showed that Zuogui Jiangtang Jieyu decoction could significantly reduce the fasting blood glucose and blood insulin content of rats,improve the insulin resistance index,reduce the content of cholesterol,triglyceride,low density lipoprotein,and increase the content of high density lipoprotein.The results of pathological staining showed that Zuogui Jiangtang Jieyu decoction could significantly improve the pathological damage of hippocampal dentate gyrus neurons in the model rats,and increase the number of Nissan bodies and the density of dendritic spines in the cells.The results of fluorescence analysis showed that Zuogui Jiangtang Jieyu decoction could significantly increase the expression of Nestin,Brdu,DCX,NeuN and Tuj-1 in the dentate gyrus.2.DKK-1 is a classic inhibitor of Wnt3a signal.The study found that compared with Zuogui Jiangtang Jieyu decoction group,the number of activities in the open field experiment was significantly reduced,the time of rest was significantly prolonged,the time of immobility in the forced swimming experiment was significantly increased,the latency of escape in the water maze experiment was significantly prolonged,and the number of crossing the original platform was significantly reduced.At the same time,the expression of Wnt3 a,Dvl-1,Ngn2,NeuroD1 protein in rat hippocampus was significantly decreased β-Catenin expression was significantly reduced.The results of immunofluorescence test showed that DKK-1 could significantly inhibit the effect of Zuogui Jiangtang Jieyu decoction on enhancing the expression of nestin,DCX and NeuN in hippocampus.3.Ciclopramide is a classical inhibitor of Shh signal.The study found that compared with Zuogui Jiangtang Jieyu decoction group,the rats’ immobility time in forced swimming test was significantly increased,the escape latency in water maze test was significantly prolonged,and the swimming time and distance in the target quadrant were significantly reduced after the Zuogui Jiangtang Jieyu decoction and Ciclopramide were given at the same time.At the same time,the expression of Shh,Ptch 1,Smo,CyclinD1,SOX2 protein and Gli-1 protein nucleus in the hippocampus of rats were significantly decreased,while the expression of Gli-3 protein was significantly increased.The results of immunofluorescence test showed that cyclophosphamide could significantly inhibit the effect of Zuogui Jiangtang Jieyu decoction on enhancing the expression of Brdu in hippocampus.4.Cell experiment research found that Zuogui Jiangtang Jieyu Formula can significantly increase the activity of neural stem cells in the damaged hippocampus,increase the expression of Brdu,DCX,GFAP,NeuN,and enhance their proliferation and differentiation ability.The mechanism study found that Zuogui Jiangtang Jieyu Formula can significantly increase the Wnt3a,LRP5,LRP6,total βCatenin,nuclear β-The expression of Catenin protein activates Wnt3a signal and increases β-The accumulation and entry of catenin promote the transcription of downstream Ngn2 target gene.XAV-93 9 can be added β-The degradation of Catenin reduces its entry into the nucleus and the mRN A transcription of downstream Ngn2,thus reducing the differentiation ability of stem cells.In addition,Zuogui Jiangtang Jieyu Formula can significantly increase the expression of Shh,Ptch 1,Smo and nuclear Gli-1 proteins in neural stem cells,activate Shh signal and promote the transcription of downstream cyclinD1 and SOX2 target genes.Cyclopazide can reduce the mRNA transcription of cyclinDl and SOX2 downstream by inhibiting Shh signal,thus reducing the proliferation ability of stem cells.Conclusion:The results of animal experiments and cell experiments show that Zuogui Jiangtang Jieyu Recipe can effectively improve the disorder of glucose and lipid metabolism and depression like behavior in rats with diabetes complicated with depression.It may increase the expression of Ngn2,NeuroD1,CyclinDl and SOX2 genes and proteins by activating Wnt3a and Shh double signals,thus promoting the process of adult hippocampal neurogenesis in the hippocampus dentate gyrus,Play the role of improving the depressive behavior and cognitive function of diabetes depression model rats. |
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