| Based on the abundant material resources in modern society,the high-calorie diet structure characterized by eutrophication is becoming more and more common;combined with the physiological characteristics of delicate viscera and spleen and stomach of children,the intake of eutrophic diet,i.e.overeating,fat and sweet taste,and children are always with rapid growth and development,strong nutritional needs,parents are difficult to control and correct the eutrophic diet structure in a short period of time.Therefore,many chronic and refractory diseases in children are easily seen in clinic,and there is still no effective preventive treatment in clinic.High-calorie diet is closely related to the imbalance of intestinal microecology.High-calorie diet can cause intestinal flora imbalance,which can lead to short-chain fatty acid metabolism disorder,trigger the start of metabolic downstream mechanism.GPR43,as an important link in the downstream pathway,affects the expression of IL-18,which may affect the integrity of intestinal epithelial cells,resulting in damage to the mechanical barrier and mucosal permeability.Change.This study explored the effects of high calorie diet on intestinal flora-SCFAs-GPR43-IL-18 pathway and the role of fecal bacteria transplantation on the pathway.To provide evidence and ideas for the prevention and treatment of pediatric diseases caused by eutrophic diet.Experiment 1ObjectiveThe aim of this study was to research the effects of fecal bacteria on “intestinal flora--short chain fatty acid--GPR43--IL-18” pathway in rats fed with high-calorie diet.MethodsForty SPF SD rats were randomly divided into 5 groups.They are divided into normal group 1(NC1),normal group 2(NC2),high-calorie diet model group(M1),high-calorie diet fecal microbiota transplantation group(M2),normal fecal microbiota transplantation group(M3).Antibiotic intervention was given to simulate the state of antibiotics-treated rats and special feed was used to replicate the high-calorie diet model.The normal dietary bacterium solution,high calorie dietary fecal bacterium solution and saline enema were givenrespectively.The changes of liver index,spleen index and thymus index in rats were observed.The morphological changes of colonic tissues were observed under optical microscope,and the changes of intestinal flora were detected by 16 s rDNA.The contents of acetic acid,propionic acid and butyric acid in short chain fatty acids(SCFAs)were detected by gas chromatography-mass spectrometry(GC-MS),the expression of GPR43 in intestinal tissue was detected by immunohistochemistry(IHC),and the content of IL-18 in serum of rats was determined by Elisa method.Results1.Basic characterizationRat characterization:During the 1-6 days of the experiment,compared with NC1 group,there was no significant difference in the appearance of the other five groups,but only yellowing and shaping of feces.During the 7-12 days of the experiment,rats in the normal group were characterized by good mental state,flexible movement,smooth and lustrous fur.The stools are brown and black,spindle-shaped,with large quantity and thick quality.The rats in M1 and M2 groups had worse mental state than those in NC1 and NC2 group.They were curled up and bundled,and their activity was decreased.The gloss of the fur is weakened,the stools are sticky and shapeless and even loose stools appeared.The same characterization could be observed in rats after fecal bacteria intervention with high-calorie diet.Weight: During the 1-6 day of the experiment,the body weight of NC1 rats increased.Compared with NC1 group,the body weight of rats in other groups decreased in varying degrees(P<0.05).During the 7-12 day of the experiment,the body mass of rats in NC1 group increased with time,but there was no significant difference between NC2 group and NC1 group.Compared with NC1 and NC2 groups,the body weight of M1 group decreased significantly(P<0.05).Compared with M1 groups,the body weight of M2 and M3 group increased significantly(P<0.05).Abdominal circumference: The abdominal circumferences of NC1 rats increased gradually during the first to sixth days of the experiment.Compared with NC1 group,the abdominal circumferences of rats in other groups decreased in varying degrees(P<0.05).The abdominal circumferences of rats in NC1 group gradually increased and finally stabilized during the 7-12 days of the experiment.Compared with NC1 and NC2 groups,the abdominalcircumferences of rats in M1 group were significantly lower(P<0.05).Compared with M1 group,the abdominal circumferences of rats in M2 and M3 groups increased to a certain extent(P<0.05).2.Visceral organ indexIn the thymus index,there was no significant difference between each groups(P>0.05).Compared with NC1,NC2 groups,the spleen index in M1 group decreased significantly(P<0.05);Compared with M1,the spleen index in M3 group increased significantly(P<0.05).In the liver index,Compared with NC1,NC2 groups,liver index of rats in M2,M3 groups decreased(P>0.05).3.Histological observation of colon:Observation under 200-fold light microscope showed that the intestinal structure of rats in NC1,NC2,M3 groups was clear and complete.The structure of mucosal epithelium in the M1,M2 groups change was not obvious,compared with that in the normal group.It could be seen that cells arranged irregularly,goblet cell aggregation as well.4.Intestinal microecology:4.1 Microbial community structure-PCA,PCoA,NMDS,PLS-DAThe microflora structure of the M1 and the M2 groups were more similar,which were significantly different from that of the NC1,NC2 and M3 groups.Compared with the NC2 group,NC1 and NC3 groups had more tendency in bacterial flora structure.4.2 Microbial Alpha Diversity-Sob,Shannon,Chao 1,AceThe abundance and diversity of fecal bacteria in M2 and M3 groups were lower than those in normal rats(P<0.05).4.3 Species diversity(family level)On Family level,there was no significant difference in flora structure between NC1 and M3 groups(P>0.05).Compared with NC1 group,Verrucomicrobiaceae in NC2 group increased.Compared with NC2 group,S24-7,Lactobacillaceae decreased and Bacteroidaceae,Prevotellaceae,Verrucomicrobiaceae increased in M2 group(P<0.05).AND compared with NC2 group,S24-7,Lactobacillaceae,Ruminococcaceae decreased and Bacteroidaceae,Verrucomicrobiaceae increased in M1 group(P<0.05).5.Short Chain Fatty AcidsAcetic acid: Compared with NC1 group,acetic acid in NC2,M1 and M2 group decreased significantly(P<0.05);compared with NC2 group,acetic acid in M1 and M2 group decreased significantly(P<0.05);compared with M1 and M2 group,acetic acid in M3 group increased significantly(P<0.05).Propionic acid: Compared with NC1 group,propionic acid in M1 and M2 group decreased significantly(P<0.05);compared with NC2 group,propionic acid in M1 group decreased significantly(P<0.05),while propionic acid in M2 group decreased,but there was no significant difference(P>0.05);compared with M1 and M2 group,propionic acid in M3 group increased significantly(P<0.05).Butyric acid: Compared with NC1 group,butyric acid in NC2,M1,M2 and M3 groups decreased significantly(P<0.05);compared with NC2 group,butyric acid in M1 and M2 groups decreased,but there was no significant difference(P>0.05);compared with M1 and M2,butyric acid in M3 group increased significantly(P<0.05).6.Expression of GPR43GPR43 could be expressed in colon tissues of rats in each group.The expression level of M1 and M2 was significantly lower than that of NC1,NC2 and M3.7.Serum IL-18 levelCompared with NC1 and NC2 groups,the levels of IL-18 in serum in M1 group increased significantly(P<0.05).Compared with M1 group,the levels of IL-18 in serum in M3 group decreased significantly(P<0.05).Conclusions1.The pseudo-sterile rat model was successfully replicated.The intestinal flora of rats could be restored by antibiotic intervention through normal bacterial fluid transplantation.2.The rat model of high calorie diet was successfully replicated and accorded with the clinical characteristics.During the modeling period,the activity of rats fed with high calorie diet was poor,their body weight and abdominal circumference showed a decreasing trend,their organ index was abnormal,and the colon tissues showed corresponding pathological changes.3.High-calorie dietary factors cause a series of pathological reactions by affecting intestinal flora-short-chain fatty acid-GPR43-IL-18 pathway.It causes intestinal flora disorder,short-chain fatty acid metabolism disorder and affects the expression of GPR43.The expression of IL-18 in serum was increased.Experiment 2ObjectiveThe aim of this study was to research the effects of fecal microbiota transplantation combined with Xiao Er Hua Shi Pills on “intestinal flora--short chain fatty acid--GPR43--IL-18” pathway in rats fed with and high-calorie diet were also studied.To provide direction for the prevention of intestinal flora disorders related diseases in the future.MethodsForty-eight SPF SD rats were randomly divided into 6 groups.They are divided into normal group 2(NC2),high-calorie diet model group(M1),MMA group(MMA),Xiao Er Hua Shi Pills group(HSW),combined group(LH),normal fecal microbiota transplantation group(ZFYZ).Antibiotic intervention was given to simulate the state of antibiotics-treated rats and special feed was used to replicate the high-calorie diet model.The latter four groups were treated with MMA,Xiaoer Huashi Pill,normal diet fecal bacteria combined with Xiaoer Huashi Pill and normal dietary bacteria solution respectively.The observation index is the same as experiment 1.Results1.Basic characterizationRat characterization: During the 1-6 days of the experiment,there was no significant difference in the performance of rats in each group.During the 12 days of the experiment,the rats in M1 group had worse mental state than those in NC2 group.They were curled up and bundled,and their activity was decreased.The gloss of the fur is weakened,the stools are sticky and shapeless and even loose stools appeared.After MMA,Xiao Er Hua Shi Pill,normal fecal microbiota transplantation,normal fecal microbiota transplantation combined with Xiao Er Hua Shi Pill treatment,rats showed different degrees of improvement in mental state.Weight: During the first to sixth day of the experiment,the body weight of the antibiotic rats in each group was not identical(P<0.05).During the 7-12 days of the experiment,the body weight of rats in NC2 group increased with time.Compared with NC2 group,the bodyweight of MMA,LH,ZFYZ,HSW and M1 groups decreased significantly(P<0.05).Abdominal circumference: During the first to sixth day of the experiment,the abdominal circumference of the antibiotic rats in each group was not identical(P<0.05).During the 7-12 days of the experiment,the abdominal circumference of rats in NC2 group increased with time.Compared with NC2 groups,the abdominal circumferences of rats in MMA,HSW,ZFYZ,LH and M1 groups decreased significantly(P<0.05).2.Visceral organ indexCompared with M1 group,the thymus index of rats in ZFYZ group increased(P<0.05).Compared with ZFYZ group,the thymus index of rats in HSW group decreased(P<0.05).In the liver index,there was no significant difference between each groups(P>0.05).Compared with NC2 group,the spleen index of rats in M1,MMA,LH groups decreased(P<0.05).Compared with M1 group,spleen index of ZFYZ group increased(P<0.05),while that of HSW,MMA and LH groups increased to some extent,but there was no significant difference(P>0.05).3.Histological observation of colon:Observation under 200-fold light microscope showed that the intestinal structure of rats in NC2 groups was clear and complete.The structure of mucosal epithelium in the M1 group change was not obvious,compared with that in the NC2 group.It could be seen that cells arranged irregularly,goblet cell aggregation as well.The pathological changes of colon tissue in ZFYZ,HSW,MMA,LH groups were significantly less than those in M1 group.4.Intestinal microecology:4.1 Microbial community structure-PCA,PCoA,NMDS,PLS-DAAfter the intervention of different factors,there were significant differences between M1 and MMA,HSW,ZFYZ,LH groups.NC2 and LH groups were more similar in bacterial flora structure.4.2 Microbial Alpha Diversity-Sob,Shannon,Chao 1,AceCompared with NC2 group,the abundance and diversity of M1 group decreased significantly(P<0.05).Compared with NC2 and LH groups,the abundance and diversity of MMA,HSW and ZFYZ groups decreased significantly(P<0.05).4.3 Species diversity(family level)On Family level,compared with NC2 group,S24-7,Lactobacillaceae,Ruminococcaceae decreased and Bacteroidaceae,Verrucomicrobiaceae increased in M1 group(P<0.05).Compared with M1 group,lachnospiraceae,Lactobacillus,Ruminococcaceae,Christensenellaceae increased in LH group,while Verrucomicrobiaceae and Bacteroidaceae decreased(P<0.05);ZFYZ group showed an increase in lachnospiraceae,Lactobacillus,Christensenellaceae(P<0.05),while Verrucomicrobiaceae and Bacteroidaceae decreased(P<0.05);HSW group showed a decrease in Prevotellaceae(P<0.05);while MMA group showed a decrease in Verrucomicrobiaceae(P<0.05).5.Short Chain Fatty AcidsAcetic acid: Compared with NC2 group,the levels of acetic acid in M1,HSW,ZFYZ and MMA group were significantly lower,while the levels of acetic acid in LH group were significantly higher(P<0.05).Compared with M1 group,acetic acid increased in ZFYZ,HSW,MMA and LH group(P<0.05).Propionic acid: Compared with NC2 group,propionic acid levels in M1,HSW and ZFYZ groups were significantly lower(P<0.05).Compared with M1 group,propionic acid increased in LH group(P<0.05).Butyric acid: Compared with NC2 group,the levels of butyric acid in HSW and ZFYZ group were significantly lower(P<0.05).Compared with ZFYZ and HSW groups,butyric acid increased in LH group(P<0.05).6.Expression of GPR43GPR43 could be expressed in colon tissues of rats in each group.The expression level of M1 lower than that of NC2.Compared with M1,the expression level of GPR43 in LH,ZFYZ,HSW and MMA groups increased to a certain extent.7.Serum IL-18 levelCompared with NC2 group,the levels of IL-18 in serum in M1 group increased significantly(P<0.05).Compared with M1 group,the levels of IL-18 in LH,HSW and ZFYZ groups were significantly lower(P<0.05).ConclusionsHSW,MMA,ZFYZ and Fecal microbiota transplantation therapy combined with Chinese patent medicine Xiao Er Hua Shi Pill may have protective effects on high-calorie dietrats by affecting intestinal flora-short-chain fatty acid-GPR43-IL-18 pathway.And fecal microbiota transplantation therapy combined with Chinese patent medicine may be better than others interventions. |
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