| Poria cocos(Schw.)Wolf,a traditional Chinese medicine,is the dry sclerotia of Poria cocos,which belongs to the family Polyporaceae.As a traditional Chinese medicine with the same origin as medicine and food,Poria cocos are widely used in medicine,food and health products.The main active ingredient in Poria cocos is Poria cocos polysaccharide(PCP),which has strong immune activity,but it is difficult to be absorbed,how to exert the immune activity remains to be explored.In this study,PCP,the main active component of Poria cocos,was extracted and purified,and the process of extraction and purification of PCP was established,and the structure and components of purified PCP were characterized and analyzed,and then the model of spleen deficiency was used to explore the relationship between the immune regulation of PCP on spleen deficiency rats and intestinal flora,in order to provide a new possibility for the immune regulation mechanism of PCP on spleen deficiency rats.Objective:To extract and purify PCP by ultrasonic extraction and macroporous resin purification,and to replicate the model of spleen deficiency by two compound modeling methods of overwork and uncontrolled diet.To explore the relationship between the immunomodulatory effect of pachymaran on spleen deficiency rats and intestinal flora,in order to provide a new possibility for the immune regulation mechanism of pachymaran on spleen deficiency rats.Methods:(1)Taking the extraction rate of PCP as the index,the optimum technology of ultrasonic extraction of PCP was optimized by orthogonal experiment.(2)Taking protein removal rate,pigment removal rate and polysaccharide retention rate as comprehensive indexes,the optimum process of purification of PCP by macroporous resin was optimized by orthogonal experiment,and the structure and components of purified polysaccharides were characterized by infrared chromatography(IR)and gas chromatography(GC).(3)The model of spleen deficiency was established by overwork and uncontrolled diet.The rats were treated with low dose of pachymaran(75 mg/kg)and high dose of medium(150 mg/kg)and high dose(300 mg/kg)for 21 days.The rat model of spleen deficiency was evaluated from the general state,pathological index(H&E),physiological index(body weight,body temperature),intestinal propulsion rate,fecal water content and so on.The number of peripheral white blood cells was detected by blood cell analyzer,the contents of Immunoglobulin G(Ig G),Immunoglobulin M(Ig M),Interleukin-10(IL-10),Interferon-gamma(TNF-?),Interleukin-6(IL-6)in serum and Secretory immunoglobulin A(Slg A)in small intestinal contents were detected by Elisa kit.The number of CD3~+T cells and CD19~+B cells in spleen and thymus and the expression of Immunoglobulin A(Ig A)and zonula occludens-1(ZO-1)protein in small intestine were detected by immunohistochemical method to investigate the effect of Poria cocos on the immune function of rats with spleen deficiency.(4)The spleen deficiency model was established by overwork method and uncontrolled diet method.The colon feces of rats were collected,and the 16S Ribosomal RNA(r RNA)gene sequencing technique was used to extract and detect the Deoxyribo Nucleic Acid(DNA)of the related bacteria in the samples.After the detection was qualified,a series of PCR primers designed by Jin Weizhi were used to amplify two highly variable regions of prokaryote 16S r RNA gene,including V3 and V4.Then Meta Vx TM library construction,Ilumina Mi Seq sequencing,sequence alignment and species identification analysis were carried out.Then the relative abundance,diversity and distribution of intestinal flora were analyzed.Results:(1)The best extraction process of PCP was as follows:Poria cocos powder sifted through 80 mesh,ratio of material to liquid 1:20,ultrasonic temperature at room temperature,ultrasonic power 240 watt(w),ultrasonic time 5 minute(min),and the extraction rate of PCP was 3.4%.(2)The best purification process of pachymaran is AB-8 macroporous adsorption resin,solution p H is 5,shaker speed is 180 r/min,shaking time is 2 hours(h).The pachymaran is mainly composed of arabinose(Ara),mannose(Man),galactose(Gal)and glucose(Glc),the retention time is 8.552,11.813,11.973 and 12.100 min,the molar ratio is 0.091:0.116:0.154:0.298.(3)On the 21st day,the model rats showed obvious physiological state of yellow hair,tiredness,lazy movement and squinting;the body weight of the model rats increased slowly,and the body temperature had no obvious change;in the digestive tract,the digestive tract of the model rats advanced faster and had loose stools;in the pathological changes,the thymus tissue of the model rats showed obvious pathological changes,while the spleen tissue did not show obvious pathological changes.After the intervention of PCP,this situation can be improved to a certain extent;in the detection of immune indicators,the results showed that the contents of white blood cells and lymphocytes increased significantly after the intervention of PCP,and the difference was statistically significant.Low dose of PCP had little effect on leukocytes,lymphocytes,neutrophils and monocytes,and there was no statistical significance.Low,middle and high PCP groups could increase the expression of Ig G,Ig M and IL-10,and significantly reduce the expression of IL-6and TNF-?.PCP can increase the number of CD3~+T cells and CD19~+B cells in spleen and thymus of spleen deficiency rats,and enhance the expression of s Ig A in small intestinal fluid and Ig A and ZO-1 protein in small intestinal tissue of spleen deficiency rats.(4)16S r RNA sequencing analysis of colonic faeces of rats showed that the number of intestinal microflora in the blank control group was the highest at different classification levels(phylum,class,order,family,genus,species).Compared with the control group,the abundance of intestinal flora in the spleen deficiency model group decreased significantly and was in a state of imbalance.At the level of door,class and eye,the regulatory effect of PCP on intestinal flora in rats with spleen deficiency was not obvious.At the family level,compared with the model group,the low,middle and high dose groups of PCP could significantly increase the abundance of intestinal flora.At the genus and species level,the middle and high dose of PCP can significantly increase the abundance of intestinal flora.It is worth noting that at the gate level,the homeostasis of intestinal flora in the spleen deficiency model group was destroyed,which was manifested by a significant increase in the relative abundance ratio(F&B)of Firmicutes to Bacteroidetes.After the intervention of PCP,F&B gradually recovered and showed dose dependence.At the genus level,a large number of Lactobacillus,were found in the spleen deficiency model group,while the abundance of Prevotella,Muribaculaceae and Prevotella?UCG-003 decreased significantly in the spleen deficiency model group.After the intervention of PCP,the abundance and structure of intestinal flora in rats with spleen deficiency gradually returned to the level of the control group,but the abundance of Prevotella?UCG-003 was still lower than that of the control group.At the species level,the abundance of Lactobacillus increased greatly in the spleen deficiency model group,and became the most abundant dominant species.The abundance of Prevotella?UCG-003,Alloprevotella,[Eubact-erium]-coprostanoli-genes?group and Lachnospiraceae?NK4A136?group decreased in the spleen deficiency model group.After the intervention of PCP,the abundance of Lactobacillus gradually returned to the normal level,and showed dose-dependent.Conclusion:The immune function of spleen deficiency rats is low and the intestinal flora is in a state of imbalance.PCP can enhance the immune function and restore the homeostasis of intestinal flora in spleen deficiency rats.PCP may play a role in enhancing immunity by regulating intestinal flora. |
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