Study On The Effect And Mechanism Of Scutellaria Radix-Coptis Rhizoma Herbal Pair On Cognitive Dysfunction

Objectives:Explore the effect and mechanism of Scutellariae Radix-Coptidis Rhizoma herbal pair（SC）on improving cognitive dysfunction by mediating intestinal and brain immune inflammation.Methods:1.The animal model of cognitive dysfunction in C57BL/6J mice was established by sleep deprivation.The animals’learning and cognitive memory skills were assessed by the Y maze test（Y-maze）and the novel object recognition test（NOR）.Cerebral blood flow was collected with a laser scatter imaging system to evaluate the effect of modeling on cerebral blood flow in animals.The above evaluation indexes were combined to assess the performance of animal models of cognitive dysfunction.2.The mice for the experiment were randomly assigned to six groups:Control,SD,SC-L（1∶1）,SC-M（1∶1）,SC-H（1∶1）and Piracetam.The dosage for each dosing group was 1.2g/kg·d^-1of dried medicinal herbs in SC-L group,2.4 g/kg·d^-1in SC-M group,4.8 g/kg·d^-1in SC-H group,0.15 g/kg·d^-1in Piracetam group and 10 m L/kg·d^-1of saline in Control group and SD group until the end of the experiment.After the administration,behavioral and cerebral blood flow tests were conducted on each group of mice to evaluate the cognitive function of mice in each administration group.3.At the same dried medicinal herbs concentration as the SC-M（1∶1）group,we will use the compatibility ratio SC（2∶3）,SC（3∶1）,S and C.Behavioral and laser speckle were used to collect cerebral blood flow detection,and the pharmacodynamics of different proportions of SC and two herbs of S and C were compared with that of SC（1∶1）group.4.We used 16S r DNA to analyze the intestinal microflora of mice on intestinal contents.In this way,we explore the integral structural characteristics of intestinal contents flora between Control and SD,and the structure of the intestinal flora altered among each ration of SC or the single of them.5.We analyzed the transcriptomic data from the intestinal tissues of Control,SD and the different dose concentrations group of SC by RNA-seq comprehensively to screen out differentially co-expressed genes.At the same time,GO analysis and KEGG analysis were used to cluster and annotate the biological functions and signaling pathways involved in the common differential genes of the three groups.6.Based on intestinal flora and transcriptome results,the number of CD4⁺CD25⁺Tregs and the expression of Foxp3 gene in the brain and intestine were measured by flow cytometry and Real-time PCR.In addition,the inflammatory factors in the intestine and brain were detected.To explore the mechanism of SC regulating intestinal CD4⁺CD25⁺Tregs immunity and intervening cognitive dysfunction.7.Using LPS to induce BV2 cells,a neuroinflammatory model of cognitive dysfunction was constructed in vitro to explore the anti neuroinflammatory effect and mechanism of SC.Results:1.Establishment and evaluation of cognitive dysfunction model:The mice were deprived of sleep for 15 consecutive days,18 h per day,and given full day light stimulation.During this period,the body weight and cerebral blood flow decreased significantly,and the memory and discrimination ability were decreased in Y-maze and NOR,indicating that the cognitive dysfunction model of mice was successfully established by sleep deprivation method.2.The effect of SC on improving cognitive dysfunction:After administration,the weight of mice in the SC-L,SC-M,SC-H,and Piracetam groups increased compared to the SD group.The SC-M group was significantly higher than the SD group,the low concentration group had no significant change,and the high concentration group began to lose weight 11 days after administration.In Y-maze and NOR,the SC-L group,SC-M group and Piracetam group showed different degree of correction,and the improvement effect of SC-M group and Piracetam group was obvious,showing significant improvement in learning,spatial memory and discrimination ability.Cerebral blood flow imaging also showed that the cerebral blood flow of mice in the SC-M group was significantly increased compared with the SD group,and Piracetam and SC-L group could also increase the cerebral blood flow of rats,but there was no significant difference.According to various indexes,the SC-M group had a better effect on improving the cognitive ability of model mice,so the SC-M group was selected for follow-up experiments.3.Results of the effects of different combinations and single herbs on improving cognitive dysfunction:During the modeling period,the body weight of the three SC proportioned groups and the single herb group S stabilized after administration,among which,the body weight of the SC（1∶1）group and the SC（3∶1）group was significantly reversed,and the mice in group C were in a poor state and their body weight continued to decrease,which produced toxic effects on mice.Y-maze and NOR results showed that SC（1∶1）group,SC（3∶1）group and S group could improve the learning,recognition and memory ability and exploration desire of mice with cognitive dysfunction model,especially SC（1∶1）group.Compared with SC（1∶1）group,the effect of two single herb groups was significantly worse than that of two herbs combined.The cerebral blood flow test results showed that the three SC matching groups 1∶1,2∶3,3∶1 and two single herbs S group and C group could significantly increase the cerebral blood flow of SD mice compared with the SD group,and the SC（1∶1）group had the most significant effect.The results indicated that SC had obvious effect on improving cognitive dysfunction in group 1∶1 after compatibility.4.Analysis of 16S r DNA high-throughput sequencing results:The abundance and composition of intestinal microbiota in sleep deprived mice showed significant changes.After gavage,each administration group was able to alter the abundance and composition of intestinal microbiota in model mice.Bacteroidetes and Firmicutes are the main components of the intestinal microbiota in each group of mice.Compared to the Control group,the abundance of Bacteroidetes and Alistipes in the SD group increased,while the Firmicutes decreased.Compared with the SD group,the SC（1∶1）group can reduce the abundance of Bacteroidetes and Alistipes,while increasing the abundance of Firmicutes and Lactobacillus;The SC（2∶3）group decreased the abundance of Bacteroidetes and significantly increased the abundance of Verrucomimicrobia and Akkermansia.The SC（3∶1）group reduced Bacteroidetes and increased Firmicutes,Lactobacillus,and Akkermansia.Compared with SC（1∶1）group,with a significant increase in Bacteroidetes and a significant decrease in Firmicutes and Lactobacillus genera in S group.The number of Bacteroides and Firmicutes in Group C decreased,while the number of Firmicutes,Proteobacteria,Bacteroides,and Akkermansia significantly increased.5.Effect of SC on transcriptome of intestinal tissue in mice with cognitive dysfunction induced by SD:Compared with the Control group,the SD group up-regulated 474 genes and down-regulated 269 genes.Compared with the SD group,SC group increased 93 genes and decreased 197 genes.There are 46 differentially co-expressed genes in the three groups,which shows that the gene expression in the intestinal tissue of mice can be significantly changed after the sleep deprivation model and the treatment of SC.The results of GO enrichment analysis and KEGG pathway analysis suggest that the effect of SC on improving cognitive dysfunction is closely related to the intestinal inflammatory response,and mutually corroborates the results of antagonizing pathogenic bacteria that significantly increase the intestinal microflora of mice in the SC group,and maintaining the immune balance.6.The effect of SC on inflammation in the intestinal and brain of SD induced cognitive dysfunction model mice:The expression level of Foxp3 gene in the gut and brain of the model mice after sleep deprivation decreased significantly,and the number of CD4⁺CD25⁺Tregs was less than that of the Control group.At the same time,pro-inflammatory factor TNF-αin intestinal tissue and brain The content increased,and the anti-inflammatory factor IL-10 significantly decreased.After administration of SC,this phenomenon was reversed,which preliminarily showed that SD caused inflammatory reactions in the intestinal and brain tissues of mice.After administration of SC,by increasing the expression of Foxp3 gene,increasing the number of CD4⁺CD25⁺Tregs,inhibiting the occurrence of intestinal and brain inflammation,and finally improving the cognitive function of model mice.7.In vitro,the characteristics of SC anti-inflammation and its regulation of TLR4pathway showed that compared with Control group,Model cells induced by LPS were activated,the content of inflammatory factors was significantly increased,and NF-κB p65protein was shifted to the nucleus.The morphology of BV2 cells in the SC group was mostly recovered,and the pro-inflammatory factor was significantly decreased,while the nuclear translocation of NF-κB p65 was inhibited.Compared with SC group,the inhibitory effect of S or C alone on the production of inflammatory factors and nuclear translocation of NF-κB p65was significantly lower than that of the two herbs.Real-time PCR results showed that m RNA content of TLR4/My D88/NF-κB pathway protein was increased in Model group,and SC down-regulated the expression of the pathway protein.Single herb S or C also inhibited the expression of the pathway protein gene to varying degrees,but the regulation effect of the two herbs on TLR4-My D88-NF-κB signaling pathway was significantly stronger than that of single herb.After addition of CLI-095 and PDTC signaling pathway inhibitors,m RNA expression of TLR4/My D88/NF-κB pathway protein was significantly decreased,and NF-κB p65 was significantly inhibited,suggesting that this herb has an obvious anti-inflammatory effect on TLR4-my D88-NF-κB pathway through regulation.Conclusions:1.SC has a better effect on improving cognitive function in mice model of cognitive dysfunction,especially the medium dose concentration of S and C 1∶1 ratio has the best effect.2.SC is associated with improving insomnia related cognitive dysfunction and inhibiting inflammatory response.The mechanism of inhibiting inflammation include two aspects.On the one hand,it regulates the structure of intestine microbiota and inflammation pathways associated with CD4⁺T cells,and then increases the number of CD4⁺CD25⁺Tregs and Foxp3gene expression levels in the intestine and brain of model mice,thereby inhibiting the occurrence of inflammation in the intestine and brain.On the other hand,it inhibits the activation of TLR4 in BV2 cells and prevent the large expression of downstream My D88signal factor.So as to inhibit the activation of NF-κB reduces the inflammatory reaction of BV2 cells.So as to reduce the damage to the nervous system and improve cognitive dysfunction.