| Background:The symbiotic relationship between host and microorganism is common in mammals,including human beings.Emerging evidence indicates that there are a large number of microorganisms in human body which are distributed in different parts of human body according to the diversity of microbial community.The intestinal flora occupies the main part of human microorganism and coexists with the host.From the initial colonization in the neonatal period to the individual specific differentiation in the growing process,the intestinal flora and the host are in a dynamic balance and harmony,helping the host to resist the invasion of pathogens,strengthen the intestinal epithelial barrier,enhance digestion and metabolism,promote nutrition absorption and guide the maturity of the host immune system.Intestinal flora plays an important role in regulating human behavior.A large number of clinical reports and experimental studies have confirmed that intestinal flora disorders can lead to behavioral abnormalities including depression and anxiety.As a representative of traditional Chinese medicine,Chinese herbal medicine is highly appreciated for its important contribution to human survival and reproduction for its remedy and preventive management of diseases.There is interaction between Chinese herbal medicine and intestinal flora.The research shows that intestinal flora plays a key role in Chinese herbal medicine therapy through complex interaction with components.Xiaoyaosan,a compound formula in traditional Chinese medicine(TCM),was first described in Taiping Huimin Heji Jufang,a Chinese materia medica officially compiled in the Song Dynasty of China(960-1127 AD).Modern clinical research shows that Xiaoyaosan can effectively improve depression.Our previous study found that Xiaoyaosan has a good regulatory effect on depression-like behavior in mice,but its specific mechanism is still unclear,especially whether the intestinal flora participates in its improvement mechanism remains to be studied.Therefore,to explore whether Xiaoyaosan can improve the behavior abnormality of mental diseases through intestinal flora can provide pharmacological basis for it.Objective:The purpose of this study is to investigate the regulatory effect of Xiaoyaosan on the abnormal behaviors of mice with intestinal flora disorder and its underlying mechanism.Methods:(1)Sixty specific-pathogen-free healthy male C57BL/6 mice were randomly divided into four groups(control,model,Xiaoyaosan and probiotics).The latter three groups were subjected to 14 days of ampicillin to create a model of intestinal flora imbalance.Mice in the Xiaoyaosan and probiotics groups received intragastric administration of Xiaoyaosan and probiotics,respectively.Behavioral changes in the experimental mices were observed after 14 days.At the end of the experimental period,samples of serum,fecal and colon were collected and ELISA used to determine the relative levels of LPS and IL-1? inflammation indicators.Pathological changes in the colon tissue were investigated by hematoxylin and eosin staining.The levels of NLRP3,ASC and Caspase-1 were detected by immunohistochemistry as well as polymerase chain reaction(PCR)and Western blotting.(2)NCM460 cells were divided into blank control group(control),LPS induced endotoxin injury model group(LPS),LPS+NLRP3 knockout group(LPS+Si NLRP3),LPS+low dose Xiaoyaosan containing serum group(15%XYS),LPS+medium dose Xiaoyaosan containing serum group(20%XYS),LPS+high dose Xiaoyaosan containing serum group(25%XYS).LPS(1mg/L)was used for the cells to simulate the endotoxin damage model caused by flora imbalance.Low,medium and high doses of Xiaoyaosan containing serum were intervened.CCK8 was used to detect cell viability,IL-1 ? level in cell culture medium was measured by ELISA,cell status was observed by HE staining,and expression of NLRP3 inflammatory bodies was detected by Western blotting.Results:(1)Before the experiment,the mental state of mice in each group was good,the fur was glossy,the movement was flexible,and the feces were spherical.On the 14th day of the experiment,the mental state of the mice in the model group was poor,the hair was disordered,the activity was reduced,the feces were soft and not spherical.The mice in Xiaoyaosan group and probiotics group had good mental state,sensitive movement and no abnormal feces.The weight of mice in each group showed that on the 14 th day,compared with the normal group,the weight of mice in the model group was lower than that in the normal group,the difference was statistically significant(P<0.01).Open field experiment showed that on the 14 th day,compared with the normal group,the total distance of movement in the model group was significantly lower than that in the normal group(P<0.001),while Xiaoyaosan group and probiotic group was significantly higher than that of model group(P<0.001).Elevated maze experiment showed that on the 14 th day,the distance of open arm movement in the model group was significantly lower than that in the normal group(P<0.01).The movement distance between Xiaoyaosan and probiotic group was significantly higher than that of model group(P<0.05).The results of tail suspension test showed that: on the 14 th day,compared with the normal group,the immobility time in the model group was significantly higher than that in the normal group,the difference was statistically significant(P<0.01).The immobility time in Xiaoyaosan and probiotic group was significantly lower than that in model group(P<0.01).The 16 S analysis of in each group showed that compared with the normal group,on the species level,the intestinal microorganisms in the model group were lower than those in the normal group,while those in the Xiaoyaosan and probiotic group were higher than those in the model group.(2)The pathological results of colon stained by he showed that on the 14 th day of model establishment,compared with the normal group,the colonic tissue of the model group showed obvious inflammatory infiltration pathological changes,including the thinning of colonic wall,disorder of arrangement and vacuolation.Xiaoyaosan group and probiotics group have different degrees of improvement.The results of LPS expression in feces,colon homogenate and serum of mice in each group showed that the LPS level in feces,colon homogenate and serum of mice in the model group was significantly higher than that in the normal group on the 14 th day of modeling(feces: P<0.001;colon homogenate: P<0.01;serum: P<0.001).The expression of LPS in feces,colon homogenate and serum of Xiaoyaosan and probiotic group was significantly lower than that of model group(feces: P <0.001;colon homogenate: P<0.05;serum: P<0.01).(3)The expression of NLRP3?ASC?and CASPASE-1 m RNA in the colon of mice in each group showed that the expression of NLRP3 gene in the colon of mice in the model group increased on the 14 th day(P<0.01).The expression of NLRP3 gene in colon of Xiaoyaosan and probiotics group was significantly lower than that of model group(P<0.05;P<0.05).Similarly,compared with the normal group,the expression of ASC gene in colon of model group increased significantly(P< 0.001),as well as the expression of ASC gene in colon of Xiaoyaosan and probiotics group was significantly lower than that of model group(P<0.05;P<0.001).On the 14 th day,compared with the normal group,the expression of CASPASE-1 gene in colon of model group increased significantly(P<0.01).The expression of CASPASE-1 gene in Xiaoyaosan and probiotics group was significantly lower than that in model group(P<0.01;P<0.01).The results of immunohistochemistry of NLRP3,ASC and CASPASE-1 in colon of mice in each group showed that the average optical density of NLRP3 in colon of model group was significantly higher than that in normal group on the 14 th day of modeling(P<0.001).The average optical density of NLRP3 in Xiaoyaosan and probiotic group was significantly lower than that in model group(P<0.01;P<0.001).On the 14 th day,compared with the normal group,the average optical density of ASC in colon of the model group was higher than that of the normal group(P<0.05).The average optical density of ASC in Xiaoyaosan and probiotic group was significantly lower than that in model group(P<0.05;P<0.05).On the 14 th day,compared with the normal group,the average optical density of CASPASE-1 in the model group was higher than that in the normal group,the difference was statistically significant(P<0.01).The average optical density of the two groups was significantly lower than that of the model group(P<0.05;P<0.05).The results of NLRP3,ASC and CASPASE-1 Western blot showed that the protein expression of NLRP3 in colon of model group was significantly higher than that of normal group on the 14 th day(P <0.05).The protein expression of NLRP3 in the colon of Xiaoyaosan and probiotic group was lower than that of the model group,the difference was statistically significant(P<0.05;P<0.05).On the 14 th day,compared with the normal group,the protein expression of ASC in colon of the model group was higher than that of the normal group(P<0.01).The protein expression of ASC in colon of Xiaoyaosan and probiotic group was lower than that of model group,the difference was statistically significant(P<0.05;P<0.05).On the 14 th day,compared with the normal group,the protein expression of CASPASE-1 in colon of the model group was higher than that of the normal group,the difference was statistically significant(P<0.05).The protein expression of caspase-1 in Xiaoyaosan and probiotic group was lower than that in model group,the difference was statistically significant(P<0.05;P<0.05).(4)The results of cell viability test showed that the cell viability of LPS model group was significantly lower than that of normal group(P<0.001).The activity of cells of Xiaoyaosan drug-containing serum group was significantly higher than that in model group(P<0.001;P<0.001).ELISA showed that the level of IL-1 ? in the model group was significantly higher than that in the normal group,and the difference was statistically significant.The level of IL-1 ? in Si+NLRP3 group was significantly lower than that of model group(P<0.001).HE staining showed that in LPS model group,there were obvious phenomena of large amount of cytoplasm,broken body,fuzzy structure,dissolution and pyknosis of nucleus.Cells of Xiaoyaosan drug-containing serum group was improved in varying degrees.The expression results of NLRP3,ASC,and CASPASE-1 protein in the cells of each group showed that the expression of NLRP3 protein in the model group increased,and the difference was statistically significant compared with the normal group(P<0.001).The expression of NLRP3 protein in Xiaoyaosan drug-containing serum group and Si+NLRP3 group was significantly lower than that in model group,the difference was statistically significant(P<0.001;P<0.001).The expression of ASC protein in the model group increased,and the difference was statistically significant(P <0.001).Theexpression of ASC protein in Xiaoyaosan drug-containing serum group and Si+NLRP3 group was significantly lower than that in the model group,and the difference was statistically significant(P<0.001;P<0.001).The expression of CASPASE-1 protein in the model group increased,and the difference was statistically significant(P<0.001).The expression of CASPASE-1 protein in Xiaoyaosan-containing serum group and Si+NLRP3 group was significantly lower than that in the model group,and the difference was statistically significant(P<0.001;P<0.001).Conclusion:(1)Xiaoyaosan can improve the depression-like behavior and anxiety-like behavior of mice with intestinal flora disorder induced by ampicillin.(2)The depression-like behavior induced by ampicillin is related to the imbalance of intestinal flora and the increase of LPS.(3)The increase of LPS can induce the excessive activation of NLRP3-ASCCASPASE-1 pathway and release a lot of IL-1?.(4)The mechanism of Xiaoyaosan to improve the abnormal behaviors may be related to the improvement of intestinal flora imbalance,the reduction of LPS,the inhibition of NLRP3-ASC-CASPASE-1 pathway,and the reduction of IL-1?. |
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