Study On The Pathological Mechanism Of Damp-heat Pathogen Throug Intestinal Microecology And Mucosal Immunity In Damp-heat Environment Feeded Mice Infected With Influenza Virus

OBJECTIVE : Damp-heat pathogen is often manifested as excretion of viscous,unpleasant,stinky,hiding fever,lingering refractory and so on.This study intends to explore the pathological mechanism of damp-heat pathogenicity through the influence of damp heat on intestinal microecology,intestinal mucosal immune system and its regulatory pathways.METHOD:1.Artificial climate chambers were used to simulate normal and damp heat conditions.20% LD50 mouse adapted strain of influenza virus was used to establish influenza mouse model in a normal environment and moist heat environment.1,the survival rate and pathological changes and microstructure of the lung tissue was observed,RT-PCR and HE staning was used to verify the success of the mouse model or not.2.16 S rDNA sequencing technology was used to detect the intestinal microbiota of the mouse.The mRNA expression of intestinal permeability-related proteins and short-chain fatty acid receptors in mice was detected to elucidate the characteristics of dampness-induced excrement stickiness.3.To compare the changes of immune recognition pathways of NLRs in influenza mice in two different environments.RT-PCR was used to detect the gene expression of key nodes of NLRs immune recognition pathway in mice,such as NOD1,NOD2,RIP2,NF-kB;4.Detection of B-cell-associated intestinal mucosal immune system poly-immunoglobulin receptors pIgR(polymeric immunoglobulin receptor)and immunoglobulin SIgA,IgA and other changes,elucidate the damp heat on natural immune influences.5.To compare the changes of helper T-lymphocyte subsets in influenza mice in normal and hot-humid environment.Flow cytometry was used to detect the ratio of Th1/Th2 and Th17/Treg cells in mouse.T-cell related cytokines was detected byLuminex MagPix suspension chip.6.Treating influenza virus infected mice in Damp-heat environment by using Gegen Qilian Decoction.Fecal transplant was used to verifies that the nature of the damp-heat pathogenicity.RESULT:1.There was no significant change in the activity of mice raised in normal environment.No reduction in body weight,slow growth,smooth coat,normal stool color,no abnormal smell.In a hot and humid environment,mice gained weight slowly,and their activity,diet,and drinking water decreased.Mice liked the body to contract,the coat was uneven,the anus prolapsed,the response was dull,the eyes were sluggish,the stool became thin,and the smell was smelly.After intranasal injection of the FM1 influenza virus,the body weight of the viral model control group was significantly reduced,the mice activity was extremely reduced,the response was dull,diet and drinking water decreased,and a small number of mice died after infection(controllable range);feeding in a hot and humid environment In comparison with the virus model control group,the mice had occasional sneezing,body contracture,clumping,anal prolapse,sluggish eyes,and smelly stool.2.The intestinal microflora of mice raised in hot and humid environment changed,and the number of OTUs in the intestinal microbiota of mice was less in hot and humid environment,and the distribution of OTU was also more different.By analysis of Beta diversity analysis,we used PCA principal component analysis and NMDS analysis to find that there are significant differences in the composition of the intestinal microbiota between normal and hot and humid environments.And the multivariate statistical method PCoA analysis also showed that the evolutionary similarity of microorganisms in the feces of normal and hot and humid environments was smaller and the differences were greater.The analysis of intestinal flora community structure and the results of OTU and species abundance distribution Heatmap also proved that the moist heat environment can significantly cause the imbalance of intestinal microflora in mice,causing changes in their structure and composition.And we also found a differential flora between normal and hot andhumid environments.Moreover,we found that the expression of Occludin and ZO-1in the intestine of mice was down-regulated in hot and humid environment(P<0.05).After infection with the virus,the expression of Occludin and ZO-1 was significantly down-regulated(P<0.05).Compared with normal environment mice,the expression of metabolite receptors FFAR3 and FFAR2 receptors increased and the expression of HCAR3(Gpr109a)was downregulated(P<0.05).3.After the damp-heat environment causes dysbacteriosis,the bacteria translocating into the blood activate pattern recognition receptors.The expression of NOD1,RIP2 and NF-kB was significantly increased(P<0.05);the expression of IgA and IgA decreased.After infection with the virus in the normal environment,the expression of NOD1,NOD2,RIP2,and NF-kB all increased.After infection with influenza virus in a hot and humid environment,NOD1,NOD2,RIP2,and NF-kB were up-regulated compared to normal influenza virus(P<0.05).In humid environment,IFN-?,IL-1,IL-17,IL-6 and IL-22 increased,and IL-10 decreased(P<0.05).In the damp heat environment + virus infection group,IL-2 increased,IL-6,IL-10 and IL-22 decreased(P<0.05).4.The ratio of Th17/Treg cells in the damp environment was decreased compared with the normal environment(P<0.05).There was an increase in viral infection relative to the hot and humid environment group(P<0.05),which may be due to inflammation of mice caused by viral infection.Compared with the normal environment group,there were IL-2,IL-6,levels in the heat-and-heat environment group,and IFN-? and IL-17 A were decreased in the expression level.After viral infection,the expression of IL-2 and IL-10 was elevated in the damp-heat environment + viral infection group compared with the normal environment + virus infection group,while IL-17 A and IFN-? were decreased in expression.5.After the treatment of bacterial colonies after treatment with Qingre Lishui Recipe,we found that the general condition of the mice was improved,and the abundance and diversity of the intestinal flora was increased compared with the mice without treatment groups,and the feces compared with the hot and humid environment.The expressions of ZO-1,Occludin,and Claudin-5 in the intestine ofmice transplanted with hot and humid environment and Kudzu Qinlian Decoction were increased(P<0.05).After infection with the virus,the expression of Occludin and Claudin-5 in the damp-heat environment + Gegen Qilian Decoction +virus-infected mice was significantly higher than that in the feces transplanted mice +virus infection group(P<0.05).The expression of FFAR3,FFAR2,and HCAR3 receptors in the intestine metabolites of mice transplanted with environment and Gegen Qilian Decoction was decreased(P<0.05),but the expression of NOD1 and RIP2 decreased after transplantation of Gegen Qilian Decoction.The expression of pIgR was increased(P<0.05).The expression of NOD2 decreased(P>0.05).After infection,the expression of NOD1,NOD2,NF-kB and pIgR increased.The proportion of Th17 cells decreased and the proportion of Treg cells increased after treatment with Gegen Qinlian Decoction in the bacterial group transplantation group,which was significantly different from that of mice not treated with Gegen Qinlian Decoction(P<0.05).There was no significant difference in changes in Th1 and Th2.After infection with the virus,the proportion of Th1 was significantly higher than that of mice without virus infection(P<0.05).After treatment with Gegen Qinlian Decoction,the ratio of Th1/Th2 and Th17/Treg in mice could be decreased(P<0.05).After the virus was infected,mice in the Gegenqinlian decoction group were lower than those in the untreated group.The ratio of Th17/Treg decreased after viral infection.Compared with mice in the humid and heat environment transplantation group,the ratio of Th17/Treg in the mice after transplantation of Gegen Qinlian Decoction decreased significantly(P<0.05).CONCLUSION:1.Exogenous moist heat environment can affect the body's intestinal micro-ecology,leading to the imbalance of composition,structure and flora abundance and diversity of intestinal microflora,resulting in changes in metabolites that change the intestinal short-chain fatty acid receptors and terpenoids,etc.The expression of metabolite receptors is elevated and can increase the permeability of the intestinal mucosa,leading to symptoms such as stool stickiness and skunk caused by exogenous damp heat.2.The hot and humid environment can affect the immune system of the intestinal mucosa,leading to the decreased expression of pIgR,sIgA,and IgA.This makes it impossible for the body to remove the local bacterial flora and associated pathogens in the intestinal mucosa.In addition,the imbalance of intestinal flora caused by the hot and humid environment can also increase the expression of genes related to NLRs immunorecognition signaling pathways in the intestine,leading to increased inflammatory factors in the intestinal tract,aggravating local inflammation,and increasing intestinal permeability.More serious translocations.3.The hot and humid environment can affect the expression of inflammatory cytokines in the body,destroy the balance of T lymphocyte subsets in Th/Treg immunity,and then affect the body's immune response,so that the body can not effectively identify and resist foreign microorganisms,thus making the infection After lingering,the course of disease becomes longer.