Preventive And Therapeutic Effect And Mechanism Of Xuefu Zhuyu Capsule On Post-stroke Depression Model Rats

Objective:To evaluate the efficacy of Xuefu Zhuyu Capsules on PSD,and to explore the effects of Xuefu Zhuyu Capsules on intestinal flora in PSD rats based on the brain-gut axis theory,as well as the effects on brain nerve inflammation and intestinal barrier function and related mechanism.Methods:Part ONE:Pharmacodynamics of Xuefu Zhuyu Capsule on PSD ratsA rat model of transient middle cerebral artery occlusion（t-MCAO）was established^[1].Neurobehavioral scores^[2]were used to screen out rats with impaired neurological function,and they were randomly divided into 6 groups according to body weight.,respectively,sham operation group（Sham）,model group（PSD）,Xuefu Zhuyu capsule low（XFL）,medium（XFM）,high（XFH）dose groups,fluoxetine group（Flu）,followed by chronic inoperable Predictive stress method（Chronic unpredictable mild stress,CUMS）^[3]stress model and given Xuefu Zhuyu capsules（0.22g/kg,0.43g/kg,0.86g/kg）,Sham and PSD groups were given the same volume of distilled water.After 28 days of stress,the neurobehavioral score,sugar water preference test,open field test and forced swimming test were used to evaluate the pharmacodynamics of the post-stroke depression model.Part Two:Effects of Xuefu Zhuyu Capsule on intestinal microflora diversity and short-chain fatty acids in PSD ratsA rat t-MCAO model and a pseudo-sterile model were established.Neurobehavioral scores were used to screen out rats with impaired neurological function.They were randomly divided into 5 groups according to body weight,namely the sham operation group（Sham）and the model group（PSD）.,Xuefu Zhuyu capsule group（XFM）,intestinal flora disorder group（PGF）,intestinal bacteria disorder+Xuefu Zhuyu capsule group（PGFX）,and then the chronic unpredictable stress method was used to establish a model and administer（0.43g/kg）,and the Sham and PSD groups were respectively gavaged with the same volume of distilled water.After 28 days of stress,the neurobehavioral score,sugar water preference test,open field test and forced swimming test were used to evaluate the pharmacodynamics of the intestinal flora imbalance model.16S r DNA sequencing and gas chromatography were used to detect the effects of Xuefu Zhuyu Capsules on the bacterial diversity of post-stroke depression rats and rats with intestinal dysbiosis.Combined with short-chain fatty acids,specific flora and metabolites were analyzed.Part Third:That is based on the brain-gut axis to explore the effects of Xuefu Zhuyu capsule on brain neuroinflammation and intestinal barrier function of post-stroke depression rats1.Immunofluorescence method was used to detect the positive expression of xuefu Zhuyu capsule on microglia in prefrontal cortex tissues of post-stroke depression rats.2.The expressions of TNF-α,IL-1 and IL-6 in serum of xuefu Zhuyu capsule on post-stroke depression rats were detected by Elisa kit.3.FITC marker was used to detect the intestinal permeability of Xuefu Zhuyu capsule in rats with post-stroke depression.4.HE staining method was used to observe the morphological structure and inflammatory infiltration of the colon of xuefu Zhuyu capsule in rats with post-stroke depression.5.Western blot was used to detect the expression levels of tight junction proteins Zonula Occludin-1（ZO-1）,Occludin and Claudin-1 in colon tissues of post-stroke depressed rats treated with Xuefu Zhuyu Capsule.Results:1.Xuefu Zhuyu capsule can significantly reduce the neurobehavioral score of post-stroke depression model rats;It can improve the rate of sugar water preference,improve the spontaneous activity behavior and shorten the immobility time of the rats with poststroke depression,and improve the poststroke depression-like symptoms.2.Xuefu Zhuyu capsule intervention did not improve depression-like symptoms（increased neurological function score,decreased sugar water preference rate,decreased spontaneous activity behavior and prolonged immobility time）in post-stroke depressed rats after intestinal microflora was destroyed by combined antibiotics.3.Effects of microflora on post-stroke depression model rats:Alpha analysis showed that the abundance and diversity of bacteria in the post-stroke depression model rats and intestinal disorder rats were significantly decreased,while the abundance and diversity of bacteria were significantly increased in the xuefu Zhuyu capsule intervention,while the abundance of bacteria in the PGFX group was not increased due to drug administration.According to the Beta diversity analysis,Sham,PSD,XFM group,PGF,PGFX group were obviously divided into two clusters,and the separation degree of the five groups was significant,indicating that the composition was more diverse and the differences between groups were obvious.Through the composition analysis of bacteria,it can be seen that proteobacteria,Campylobacter,Bacteroidetes,cyanobacteria,firmicutes,etc.are the main bacteria in phyla level.At the genus level,lactobacillus,Firmicute unclassified genus,HT002,Ruminococcus,Spironaceae,UCG-005,Clostridium,Klebsiella,Bacteroidetes and Akkermansia were the main species.LEf Se difference analysis of phylum,genus and among groups showed that compared with Sham group,the abundance of Ruminococcus and Clostridium in Firmicwall bacteria in PSD group and PGF group was significantly reduced.Under the intervention of Xuefu Zhuyu capsule,the abundance of microflora increased significantly,indicating that Xuefu Zhuyu capsule can improve the abundance of microflora,reduce inflammation,and protect the intestinal barrier function.4.Effects of short-chain fatty acids on post-stroke depression:Group compared with the Sham group,the PSD of acetic acid,propionic acid,butyric acid content is significantly reduced,under the capsule of therapeutic intervention,acetic acid,propionic acid,butyric acid content in XFM group increased significantly,combined with the flora analysis above,thick wall,clostridium,campylobacter bacteria produce short-chain fatty acids such as bacteria increased,the short chain fatty acids also will increase,consistent with the results of bacterial flora.The content of short-chain fatty acids in PGF and PGFX groups was lower than normal.5.Immunofluorescence staining showed that Xuefu Zhuyu capsule could reduce the activation of microglia and the inflammatory response in the prefrontal cortex to protect the cerebral nerve function.The expression level of microglia in PGF group and PGFX group was higher than that in PSD group.6.Elisa showed that Xuefu Zhuyu capsule could reduce the expression of TNF-α,IL-1βand IL-6 in serum,and alleviate the inflammatory response;Serum inflammatory factors were not significantly reduced in the PGFX group.7.FITC fluorescence marker was used to detect intestinal permeability.The intestinal permeability of PSD group and PGF group was large,but was significantly improved after administration,while that of PGFX group was not significantly improved.8.Colon HE staining showed that inflammatory infiltration was serious in PSD and PGF groups,and the intestinal gland and epithelium were destroyed.After the intervention of Xuefu Zhuyu capsule,inflammatory infiltration and glandular injury were alleviated.There was no significant improvement in injury in the PGFX group.9.Western blot analysis of colon tight junction related proteins,including ZO-1,Occludin and Claudin-1,showed that Xuefu Zhuyu capsule could significantly up-regulate the expression of tight junction protein,further suggesting that Xuefu Zhuyu capsule could improve intestinal barrier function.Tight junction proteins were not significantly upregulated in the PGFX group.Conclusion:1.Xuefu Zhuyu Capsule can improve the depression-like symptoms of post-stroke depression.2.Xuefu Zhuyu Capsule can increase the flora abundance of PSD rats,especially the flora abundance of short chain fatty acids produced by firmicum,Clostridium and Campylobacter,so as to increase the content of short chain fatty acids such as acetic acid,propionic acid and butyric acid,and improve the intestinal barrier function3.Xuefu Zhuyu Capsule can improve neuroinflammation in the brain by regulating intestinal flora and SCFAs,especially increasing the abundance of brain protective flora such as butyric acid,and finally achieve the goal of improving depression like symptoms.In conclusion,Xuefu Zhuyu capsule may regulate intestinal flora through the brain-gut axis,thus playing a role in the prevention and treatment of post-stroke depression.