| Objective:The pathogenesis of schizophrenia and the therapeutic mechanism of Bupleurum plus Dragon Bone and Oyster Shell decoction were studied by using Illumina MiSeq high-throughput sequencing platform and metabolomics technique.Further reveal the characteristics and rules of Bupleurum plus Dragon Bone and Oyster Shell decoction in the prevention and treatment of schizophrenia.Methods:1.The schizophrenia model rats were induced by didroxepine maleate(MK-801),and their general behavioral characteristics were observed.Space exploration and navigation were carried out through stereotypical reaction,ataxia,open field experiment and water maze test.To study the behavioral characteristics of Bupleurum plus Dragon Bone and Oyster Shell decoction on schizophrenia model rats.2.The serum levels of SOD,MDA,IL-2,IL-6,IFN-y,IgG,IgM,TNF-α,NO and PKC were measured by enzyme-linked immunosorbent assay(Elisa),and the thymus and spleen gland indexes were calculated to explore the effect of Bupleurum plus Dragon Bone and Oyster Shell decoction on the immune level of schizophrenic rats.3.The contents of DA,NE and 5-HT were determined by Western Blotting analysis and enzyme-linked immunosorbent assay(Elisa).Meanwhile,Western Blot was used to detect the neurofactors of PKC,P38-MAPK,ErbB4,C-fos,S100-β,NRG1,GFAP and Cx43 in hippocampus of rats.The expression of related proteins was detected.4.High throughput sequencing technique was used to determine the 16s rRNA V4 region of bacteria in the intestinal samples of rats using Illumina Miseq as the platform.The total DNA and total RNA of intestinal samples of rats with schizophrenia and Bupleurum plus Dragon Bone and Oyster Shell decoction treated and analyzed.To analyze the abundance,distribution and Alpha diversity of species,and the diversity of species abundance,KEGG analysis and metabolic function annotation of community.5.UPLC-QTOF-MS combined with pattern recognition technique was used to explore the metabolic pathway of Bupleurum plus Dragon Bone and Oyster Shell decoction intervention in schizophrenia model rats.Results:1.Bupleurum plus Dragon Bone and Oyster Shell decoction could significantly improve the abnormal activities of space exploration and navigation in schizophrenia model rats,such as stereotype reaction,ataxia,open field test and water maze test(P<0.05 or P<0.01),and promote serum SOD,MDA,IL-2 in rats.,IL-6,IFN-y,IgG,IgM,TNF-α,no and PKC contents in normal controls(P<0.05 or P<0.01),and increased the thymus and splenic gland index(P<0.05 or P<0.01),and regulated the levels of PKC,P38-MAPK,ErbB4,C-fos,S100-β,NRG1,GFAP and Cx43 in hippocampus of schizophrenic rats.2.By analyzing the diversity of intestinal flora,it was found that Bupleurum plus Dragon Bone and Oyster Shell decoction could significantly improve the number of OTUs,the abundance and diversity index of intestinal flora,the distribution of species and the diversity of Alpha in schizophrenia model rats.At the same time,Beta diversity analysis also showed that the diversity structure of intestinal communities in the schizophrenic model rats was also affected,and the differences among the groups were statistically significant(P<0.05 or P<0.01).3.The results of serum metabonomics showed that 26 potential biomarkers with significant difference were obtained from the analysis of serum metabolites from Bupleurum plus Dragon Bone and Oyster Shell decoction in schizophrenic rats.24 markers were up-regulated and 2 were down-regulated in schizophrenia model rats.After administration of drugs,all 26 biomarkers were reversed,and the trend of callback was observed.Conclusions:1.This study found that Bupleurum plus Dragon Bone and Oyster Shell decoction could significantly improve the ability of space exploration and localization analysis in the ataxia,stereotype reaction and water maze test of the model rats,and improve the abnormal behavior of the rats with schizophrenia.To promote their return to normal behavior.2.Bupleurum plus Dragon Bone and Oyster Shell decoction could significantly regulate the contents of interleukin,interferon,immunoglobulin and superoxide dismutase in the serum of schizophrenic rats,and promote the stability of immune factors.To improve the immune function of schizophrenic rats.3.The results showed that Bupleurum plus Dragon Bone and Oyster Shell decoction could significantly improve the levels of DA,NE and 5-HT in hippocampus of schizophrenic rats,and regulate the levels of NRG1,ErB4,Cx43,PKC,P38-MAPK,C-fos,GFAP,S-100β.Promote the smooth and normalization of the content of neurocytokines in the brain of schizophrenic rats,so as to play a therapeutic and protective role on the injured hippocampus.4.It was found that Bupleurum plus Dragon Bone and Oyster Shell decoction could significantly improve the number of OTUs,intestinal flora richness and diversity index,species distribution and Alpha diversity of the rat model of schizophrenia.At the same time,the diversity structure of intestinal community in rats was affected to some extent,which indicated that Bupleurum plus Dragon Bone and Oyster Shell decoction could prevent and control the diversity of intestinal flora in schizophrenic rats.5.The combined analysis of serum metabolomics and intestinal flora diversity showed that,The primary bile acid biosynthesis folate biosynthesis sphingolipid metabolism and glycerol phospholipid metabolism were the most important metabolic pathways of Bupleurum plus Dragon Bone and Oyster Shell decoction. |
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