| 1.Clinical ResearchObjectiveObserve the effect of Xingpi Jieyu Recipe on intestinal flora,intestinal mucosa permeability and serum inflammatory factors in patients with depression of liver stagnation and spleen deficiency,and initially reveal the effect mechanism of Xingpi Jieyu Recipe.MethodPatients with depression of liver stagnation and spleen deficiency and healthy volunteers were included.Patients with depression of liver stagnation and spleen deficiency were treated with Chinese medicine Xingpi Jieyu Recipe granules.The course of treatment was 6 weeks.Safety testing was carried out during the treatment.Before and after the treatment,Hamilton Depression Scale(HAMD-17)and patient health questionnaire(PHQ-15)were carried out.Patients with depression of liver stagnation and spleen deficiency and healthy volunteers were collected blood and feces before and after treatment,16SrRNA high-throughput sequencing was used to detect fecal intestinal flora,and enzyme-linked immunosorbent assay(Elisa)was used to detect serum intestinal mucosal barrier function indicators and inflammation Cytokines,include:lipopolysaccharide(LPS),D-lactic acid,diamine oxidase(DAO),interleukin-6(IL-6),and tumor necrosis factor-?(TNF-?).Result(1)Curative effect scale score:Compared with before treatment,the HAMD-17 score and PHQ-15 score of the liver stagnation and spleen deficiency depression group were significantly reduced after treatment,and the difference was statistically significant(P<0.01).(2)Sequencing results of intestinal flora:?? diversity:there was no significantdifference in Shannon index,Simpson index,Chao1 index and ACE index between the healthy control group and the depression group of liver stagnation and spleen deficiency,as well as before and after treatment in the depression group of liver stangation and spleen deficiency(P>0.05).?? diversity:PCoA and NMDS showed that the liver stangation and spleen deficiency depression group(before treatment)and the healthy control group were clustered separately.There was a difference in the microbial composition of the samples.The samples of liver depression of spleen deficiency depression group overlapped before and after treatment,after treatment Sample clustering is more obvious.?Species composition analysis:Firmicutes,Bacteroidetes,Proteobacteria and Actinobacteria were the absolute dominant bacteria in the healthy control group and the liver stangation and spleen deficiency depression group before and after treatment,but the proportion was different in each group.There are different genus of bacteria in each classification level.?Species difference analysis:Compared with the healthy control group,T test showed that Verrucomicrobia,Clostridia,Verrucomicrobiae,Clostridiales,Verrucomicrobiales,Akkermansiaceae,Faecalibacterium,phascolarctobacterium,Akkermansia relative abundance decreased compared with healthy control group(P<0.05),The relative abundance of Corynebacteriales,Nocardiaceae,Roseburia,Butyricicoccus,Rhodococcus,and Turicibacter increased(P<0.05).Compared with before treatment,the relative abundance of Negativicutes,Coriobacteriia,Selenomonadales,Coriobacteriales,veillionellaceae,Coriobacteriaceae,Clostridiales,Dialister,Collinsella,Clostridiales,and unidentified families in the Clostridiales and Turicibacter decreased after treatment(P<0.05),the relative abundance of Corynebacteriales increased(P<0.05).LefSe analysis showed that the healthy control group was enriched in Clostridia,Clostridiales,Faecalibacterium,Blautia,Bacteroides.stercoris;the liver stangation and spleen deficiency depression group was enriched in Actinobacteria and Ruminococcus.Compared with before and after treatment,the most predominant flora before treatment is Selenomonadales class,Negativicutes order,Veillonellaceae families.(3)Changes in serum LPS,D-lactic acid and DAO levels:Compared with the healthy control group,the levels of serum LPS,D-lactic acid and DAO in liver stangation and spleen deficiency depression group(before treatment)were significantly increased(P<0.01);Compared with the liver stangation and spleen deficiency depression group before treatment,the serum levels of LPS,D-lactic acid and DAO were significantly reduced after treatment(P<0.01).(4)Changes in serum levels of inflammatory cytokines IL-6 and TNF-?:Compared with healthy controls,the levels of serum IL-6 and TNF-? in liver stangation and spleen deficiency(before treatment)were significantly increased(P<0.01);compared with before treatment of liver stangation and spleen deficiency depression group,serum IL-6 and TNF-?levels of patients after treatment were significantly reduced(P<0.01).ConclusionPatients with liver depression and spleen deficiency depression have intestinal flora disorders,intestinal mucosal permeability changes and increased peripheral blood inflammatory factors.Xingpi Jieyu Recipe can play its antidepressant role by reducing the level of pathogenic bacteria,improving the permeability of intestinal mucosa and reducing the peripheral inflammatory response.2.Experimental researchObjectiveTo further verify the regulatory effect of Xingpi Jieyu Recipe on the intestinal microecology of the depression of liver depression and spleen deficiency,and to explore the specific regulatory mechanism of Xingpi Jieyu Recipe on the inflammatory response mediated by intestinal microecology imbalance through the detection of the key protein of TLR4/NF-?B pathway in the gut and brain,and to add the western medicine positive control,to reveal the different effect mechanisms of Chinese and Western medicine.MethodEight-week-old SPF male SD rats were randomly divided into a blank control group,a model group,a traditional Chinese medicine group,and a western medicine group.The blank control group and the model group each had 7 rats,and the traditional Chinese medicine group and the western medicine group each had 8 rats.The rat model of liver stangation and spleen deficiency was replicated by chronic restraint,and the model was given by intragastric administration,and the model was evaluated after continuous stress for 21 days.Fecal samples were collected and colon and brain tissues were separated 16SrRNA high-throughput sequencing was used to detect fecal intestinal flora,Western blot and Q-PCR were used to detect colon ZO-1,Occludin protein and mRNA expression,colon and prefrontal lobe TLR4,NF-?B protein and mRNA expression,ELISA was used to detect the expression of LPS,IL-6 and TNF-? in colon and prefrontal cortex.Result(1)Model evaluation:At the late stage of modeling,the model group had significantly slower weight gain,mental burnout,huddling or curling,no obvious resistance to restraint,and dry and yellow hair;the preference rate of sugar water was significantly lower than that of the blank control group(P<0.01),the immobility time of forced swimming was significantly longer than that of the blank control group(P<0.01).In addition,the Xingpi Jieyu prescription again to verify the model,indicating that the rats with liver stangation and spleen deficiency were successfully modeled(2)Sequencing results of intestinal flora:?? diversity analysis:There was no significant difference between the Shannon index and Simpson index of rats in each group(P>0.05).Compared with the blank control group,the model group's Chaol index and ACE index were significantly decreased(P<0.01);compared with the model group,the Chinese medicine group's Chaol index and ACE index were significantly increased(P<0.01),The western medicine group had an upward trend,but the diffcrence was not statistically significant(P>0.05);Compared with the western medicine group,the Chaol index and ACE index in the Chinese medicine group were significantly higher(P<0.01).?? diversity analysis:PCoA and NMDS showed a significant difference in the microbial composition between the blank control group and the model group.The Chinese herbal medicine group and the blank control group had similar bacterial flora,and the western medicine group had a similar bacterial flora to the model group.?Species composition analysis:Firmicutes and Bacteroidetes are the absolute dominant bacteria in each group,but the proportions are different,and there are different genera at each classification level.? Species difference analysis:The difference between the model group and the blank control group is mainly Proteobacteria,Chloroflexi,Deltaproteobacteria,Desulfovibrionales,Lachnospiraceae,Desulfovibrionaceae,Gammaproteobacteria,Roseburia,Oscillibacter abundance decreased(P<0.05),Peptostreptococcaceae,Romboutsia abundance increased(P<0.05).The difference between the model group and the group after the intervention of traditional Chinese medicine was that the abundance of Actinobacteria,Lachnospiraceae,Anaerostipes,Lachnoclostridium increased(P<0.05),and the abundance of Peptostreptococcaceae,Clostridiales,Romboutsia decreased(P<0.05).The difference between western medicine and model group was Bacilli,Lactobacillales,Lachnospiraceae,Lactobacillus and Roseburia increased(P<0.05),while the abundance of Tenericutes,Mollicutes,Muribaculaceae,Peptostreptococcaceae,Romboutsia,Dubosiella Clostridialesand decreased.(P<0.05)(3)Intestinal tight junction protein expression:Western blot results showed that compared with the blank control group,the ZO-1 and Occludin protein expression of the model group were significantly reduced(P<0.01);compared with the model group,the Western medicine group Occludin protein expression Elevated,there was a significant difference(P<0.01),the expression of ZO-1 protein had a tendency to increase,the difference was not statistically significant,the expression of ZO-1 and Occludin protein in the Chinese medicine group were significantly increased(P<0.05,P<0.01);Compared with the western medicine group,the expression of ZO-1 protein in the traditional Chinese medicine group increased,but the difference was not statistically significant,the expression of ZO-1 protein in the traditional Chinese medicine group increased,and the difference was statistically significant(P<0.01).Q-PCR results showed that compared with the blank control group,the expression of ZO-1 and Occludin mRNA in the model group were significantly reduced(P<0.01);compared with the model group,the expression of ZO-1 and Occludin mRNA in the western medicine group were significantly increased(P<0.05,P<0.01),the expression of ZO-1 and Occludin mRNA in the Chinese medicine group were significantly increased(P<0.01);Compared with the western medicine group,the expression of ZO-1 mRNA in the traditional Chinese medicine group increased,and the difference was statistically significant(P<0.01),the expression of Occludin mRNA in the traditional Chinese medicine group increased,but the difference was not statistically significant.(4)Contents of LPS,IL-6,and TNF-? in the colon and prefrontal lobe:?Colon:Compared with the blank control group,the contents of LPS,IL-6,and TNF-? in the model group were significantly increased(P<0.01);Compared with the model group,the contents of LPS,IL-6 and TNF-? in the Chinese medicine group were significantly reduced(P<0.01),and the contents of LPS and IL-6 in the Western medicine group were significantly reduced(P<0.01,P<0.05),The content of TNF-? has a tendency to decrease,and the difference is not statistically significant;Compared with the western medicine group,the content of LPS,IL-6,TNF-? in the Chinese medicine group decreased significantly(P<0.01).?Prefrontal lobe:Compared with the blank control group,the content of LPS,IL-6,and TNF-? in the model group were significantly increased(P<0.01);compared with the model group,the LPS,IL-6,and TNF-? in the traditional Chinese medicine group The contents were significantly reduced(P<0.01),the LPS and IL-6 contents of the western medicine group were significantly reduced(P<0.01),and the TNF-? content showed a decreasing trend,and the difference was not statistically significant;Compared with the western medicine group,the content of LPS and IL-6 in the Chinese medicine group decreased significantly(P<0.01).(5)The expression of key proteins in the TLR4/NF-?B signaling pathway in the colon and prefrontal lobe:? Colon:Western blot results showed that,compared with the blank control group,the expression of TLR4 and NF-?B protein in the model group were significantly increased(P<0.01);Compared with the model group,the colon TLR4 protein expression in the Western medicine group has a tendency to decrease,the difference is not statistically significant,the expression of NF-?B protein is significantly reduced(P<0.01),and the expression of TLR4 and NF-?B protein in the Chinese medicine group is significantly reduced(P<0.01);Compared with western medicine group,the expression of TLR4 Protein in Chinese medicine group decreased significantly(P<0.01);Q-PCR results showed that compared with the blank control group,the expression of TLR4 and NF-?B mRNA in the colon of the model group were significantly increased(P<0.01);compared with the model group,the expression of TLR4 mRNA in the colon of the western medicine group decreased significantly(P<0.05),the expression of TLR4 and NF-?B mRNA in the Chinese medicine group decreased significantly(P<0.01);Compared with the western medicine group,the expression of TLR4 mRNA and NF-?B mRNA in the Chinese medicine group decreased significantly(P<0.01).? Prefrontal lobe:Western blot results showed that compared with the blank control group,the expression of TLR4 and NF-?B protein in the prefrontal lobe of the model group were significantly increased(P<0.01);compared with the model group,the TLR4 and NF-?B protein expression of the prefrontal lobe of the Western medicine group had a decreasing trend,and the difference was not statistically significant.The TLR4 protein expression in the Chinese medicine group was significantly reduced(P<0.01),and the NF-?B protein expression had a decreasing trend,with no statistically significant difference;There was no significant difference between Chinese and Western medicine groups.Q-PCR results showed that compared with the blank control group,the expression of TLR4 and NF-?B mRNA in the prefrontal lobe of the model group were significantly increased(P<0.01);compared with the model group,the expression of TLR4 and NF-?B mRNA in the western medicine group was significantly decreased(P<0.05,P<0.01),and the expression of TLR4 and NF-?B mRNA in the traditional Chinese medicine group was significantly decreased(P<0.01);Compared with the western medicine group,the expression of TLR4 mRNA and NF-?B mRNA in the prefrontal cortex of the Chinese medicine group were significantly reduced.Conclusion(1)There are intestinal flora disorder,intestinal mucosa barrier damage,intestinal and prefrontal cortex inflammatory reaction in rats with depression of liver depression and spleen deficiency.Xingpi Jieyu recipe can increase the abundance of intestinal flora,adjust the intestinal flora structure of rats with depression of liver depression and spleen deficiency to close to normal state,increase the intestinal tight junction protein,reduce the inflammatory reaction of colon and prefrontal cortex,and is better than escitalopram to a certain extent.(2)Xingpi Jieyu Recipe can significantly reduce the expression of key proteins in TLR4/NF-?B signaling pathway while improving the intestinal microecology,suggesting that the effect mechanism of Xingpi Jieyu Recipe may be through regulating the intestinal microccology and inhibiting TLR4/NF-?B signaling pathway,Thereby improving the body's low-grade inflammation and exerting an antidepressant effect. |
PDF Full Text Request