Study On The Effect Of Damp Environment On Blood Glucose,Blood Lipid And Intestinal Microflora

ObjectiveCorrespondence between nature and human is the basic content of holism.Different climate and environment will cause corresponding changes in human physiology and pathology.Three causes and its rules are the basic principles followed by TCM on the treatment and heath maintenance.Environmental health preservation is one of the basic treatment of health maintenance based on TCM.Dampness is closely related to many diseases.A number of studies have found that phlegm-dampness constitution has abnormal metabolism of glucose and lipid,as well as intestinal flora.However,the relationship of damp environment with glycolipid metabolism and intestinal flora has not been reported.Thus,the purpose of this research is to explore the influence of high-humidity environment on glucose,lipid and intestinal.As a result,our study may provide research basis for environmental health preservation.MethodsExperiment 1.Balb/c mice were randomly divided into the normal group(C)and damp group(D).These mice were fed in normal SPF environment and damp environment of artificial climate box for 2 weeks.Fasting blood glucose(FBG)was detected by biochemical instrument.The related hormones(insulin,glucagon-like peptide 1,et al)were detected by ELISA kit.The change of intestinal flora was analyzed by 16S rDNA sequencing.The content of short chain fatty acids was detected by gas chromatography/mass spectrometry(GC/MS).The level of protein expression was detected by western blot.Experiment 2.Balb/c mice were randomly divided into the normal group(C)and damp group(D).These mice were fed in normal SPF environment and damp environment of artificial climate box for 8 weeks.Blood lipid were detected by biochemical instrument.The related hormones were detected by ELISA kit.The change of intestinal flora was analyzed by 16S rDNA sequencing-The expression level of lipogenesis related genes was analyzed by PCR.The change of proteins were studied by proteomics.The change of intestinal structure was studied by hematoxylin-eosin staining.Experiment 3.The germ-free Balb/c mice were divided into the NF group received the bacteria from normal group(N)and DF group received the bacteria from damp group(D).The feces of mice fed in normal environment and humid environment were prepared into the bacterial solution,and the two bacterial solutions were separately transplanted into the germ free mice of NF and DF group.After 2 weeks of fecal bacteria transplantation,blood glucose and lipid were detected by biochemical instrument,the related hormones were detected by ELISA kit,the change of intestinal flora was analyzed by 16S rDNA sequencing.So as to study whether the germ-free mice would exhibit similar glycolipid metabolism and intestinal microflora disorder caused by damp environment after fecal microbial transplantation.Results1.Results after 1 week of molding of experiment 1.The weight gain of group D was significantly lower than group C(P=0.028),and there was no difference in fasting blood glucose between the two groups(P=0.988).The plasma insulin(INS)concentration(P=0.346),glucagon like peptide 1(GLP-1)concentration(P=0.310),ghrelin(GHRL)concentration(P=0.316)in group D were lower than those in group C,but none of the differences were statistically significant.The concentration of glucagon(GC)in group D was significantly lower than group C(P=0.029).The concentrations of GIP(P=0.005)and PYY(P=0.002)in group D were significantly higher than those in group C.There was no significant difference in sobs(P=0.485),chao(P=0.562),shannon(P=0.572)and simpson(P=0.589)indexes between the two groups.Analysis of ? diversity of intestinal flora showed that there was no significant difference in community structure between the two groups.The RDA linear model analysis showed that the correlation between intestinal flora and blood glucose was high(R2=0.429,P=0.090).The phenotype related genus was studied by Spearman correlation analysis.Ruminococcaceae_UCG-013(R=0.806,P=0.002),Lachnospiraceae_FCS020_group(R=0.658,P=0.020)were positive related with fasting blood glucose.Ruminococcaceae_UCG-013(R=0.832,P=0.001),Desulfovibrio(R=0.720,P=0.008)and Lachnospiraceae_FCS020_group(R=0.643,P= 0.024)were positive related with INS,while Bacteroides(R=-0.629,P=0.028)and Prevotellaceae_UCG-001(R=-0.657,P=0.020)were negative related with INS.Ruminococcaceae_UCG-013(R=0.706,P=0.010)was positive related with GLP-1,while Prevotellaceae_UCG-001(R=-0.622,P=0.031)was negative related with GLP-1.2.Results after 2 weeks of molding of experiment 1.The weight gain of group D was lower compared with group C,but the difference was not statistically significant(P=0.337).The fastingblood glucose concentration in group D was significantly increased compared with group C(P=0.002).The concentrations of INS(P=0.010),GLP-1(P=.001),GC(P=0.010)and GHRL(P= 0.004)in group D were significantly lower than those in group C.There was no significant difference in GIP concentration between the two groups(P=0.786).PYY concentration in group D was higher compared with group C,but the difference was not statistically significant(P=0.172).The Analysis of a diversity of intestinal flora showed that the sobs(P=0.100),chao(P=0.090),shannon(P=0.079)index of group D was lower compared with group C,and simpson(P=0.074)index of group D was higher,while the changes of these indexes were not statistically significant.The Analysis of ? diversity of intestinal flora showed that there were significant differences in community structure between the two groups.The RDA linear model analysis showed that intestinal flora was highly correlated with INS(R2=0.420,P=0.080)and GLP-1(R2=0.425,P=0.097).The phenotype related genus level was studied by Spearman correlation analysis.Oscillibacter(R=0.746,P-0.005)was positive related with FBG,while Ruminococcaceae_UCG-014(R--0.725,P=0.008)was negative related with FBG.Eubacterium]_xylanophilum_group(R=0.651,P=0.022)was positive related with INS,while Lachnospiraceae_UCG-004(R=-0.832,P=0.001),Prevotellaceae_UCG-001(R=-0.671,P=0.017)and Butyricicoccus(R=-0.664,P=0.018)were negative related with INS.Ruminococcaceae_NK4A214_group(R=0.606,P=0.037)was positive related with GLP-1,while Prevotellaceae_UCG-001(R=-0.594,P=0.042)and Lachnospiraceae_UCG-004(R=-0.580,P=0.048)were negative related with GLP-1.The content of short chain fatty acids in group D was generally lower than that in group C,especially butyric acid(P=0.001)and valeric acid(P=0.005).3.Results after 4 weeks of molding of experiment 2.The weight gain of group D was lower compared with group C(P=0.078).There was no significant difference in cholesterol(CHOL)concentration(P=0.210),triglyceride(TG)concentration(P=0.222),high density lipoprotein(HDL)concentration(P=0.566)and low density lipoprotein(LDL)concentration(P=0.583)between the two groups.GHRL concentration in group D was significantly lower than group C(P=0.046).There was no significant difference in GIP(P=0.394)and PYY(P=0.976)between the two groups.Analysis of ? diversity showed that there was no significant difference in sobs(P=0.217)and chao(P=0.560)index between the two groups,shannon index in group D was significantly lower compared with group C(P=0.019),simpson(P=0.050)index in group D was higher compared with group C.Analysis of ? diversity of intestinal flora showed that the differences of community structure between the two groups were obvious.The relative abundance of f_Prevotellaceae(P=0.020),g_Alloprevotella(P=0.045),s_Bacteroides_acidifaciens(P=0.045)of group D were significantly increased compared with group C,while the s_Helicobacter_ganmani was significantly decreased(P=0.013).The RDA linear model analysis of the relationship between gut flora and lipids showed that TG(R2= 0.495,P=0.048),CHOL(R2=0.485,P=0.048)and LDL(R2=0.542,P=0.030)were correlated with intestinal flora.Study on the relationship between genus and lipid by Spearman correlation analysis.Tyzzerella₃(P=0.091)was positively correlated with TG,while the difference was not statistically significant.Ruminiclostridium₅(P=0.020).Ruminococcaceae_UCG-014(P=0.026)were negatively correlated with TG.Anaerotruncus(P=0.012)and Lachnospiraceae=UCG-001(P=0.021)were positively correlated with CHOL,while Butyricicoccus(P=0.013)was negatively correlated with CHOL.Anaerotruncus(P=0.014)and Lachnospiraceae UCG-001(P=0.021)were positively correlated with HDL,while Tyzzerella₃(P=0.046)was negatively correlated with HDL.Anaerotruncus(P=0.008)and Lachnospiraceae_UCG-001(P=0.033)were positively correlated with LDL.Tyzzerella₃(P=0.053)was negatively correlated with LDL.4.Results after 6 weeks of molding of experiment 2.The weight gain of group D was significantly lower compared with group C(P=0.039).The concentrations of CHOL(P=0.135),TG(P=0.180),HDL(P=0.065)and LDL(P=0.180)in group D were higher than group C,but they were not statistically significant.There was no significant difference in GHRL(P=0.885),GIP(P=0.786)and PYY(P=0.812)concentrations between the two groups.There was no significant difference in sobs(P=0.368),chao(P=0.818),shannon(P=0.186)and simpson(P=0.214)index between the two groups.The analysis of ? diversity of intestinal flora showed there was no significant difference in the community structure between the two groups.The relative abundance of s_Mucispirillum_schaedleri_ASF457(P=0.031)and s_Helicobacter_hepaticus(P=0.045)of group D were decreased when compared with group C.The RDA linear model analysis of the relationship between the gut flora and blood lipid showed that LDL(R2=0.576,P=0.022)was correlated with the intestinal flora.The genus related to lipid was studied by Spearman correlation analysis,as a result,the correlation between the top 50 genus and TG was not statistically significant.Lachnospiraceae_UCG-001(P=0.012)was positively related with CHOL,while Oscillibacter(P=0.000),Blautia(P=0.041),Mucispirillum(P=0.014),Intestinimonas(P=0.023)and Ruminiclostridium(P=0.042)were negatively related with CHOL.Lachnospiraceae_UCG-001(P=0.006)was positively related with HDL,while Mucispirillum(P=0.000),Ruminiclostridium(P=0.004),Rikenellaceae-RC9_gut_group(P=0.022),Intestinimonas(P=0.005),Desulfovibrio(P=0.045)and A2(P=0.026)were negatively related with HDL.Lachnospiraceae_UCG-001(P=0.009)and Ruminococcaceae_UCG-013(P=0.043)were positively related with LDL,while Rikenellaceae_RC9_gut_group(P=0.000),Oscillibacter(P=0.002),Eubacterium]_xylanophilum_group(P=0.030),Alistipes(P=0.036),Ruminiclostridium(P=0.041),Intestinimonas(P=0.041),Mucispirillum(P=0.046),Rikenella(P=0.046)and Helicobacter(P=0.050)were negatively related with LDL.5.Results after 8 weeks of molding of experiment 2.The weight gain of group D was lower than group C,but there was no statistical significance(P=0.265).The concentration of CHOL(P=0.811),HDL(P=0.812)and LDL(P=0.247)were not different between the two groups,but the concentration of TG in group D was significantly higher than group C(P=0.009).The concentration of GHRL in group D was lower than group C,but it was not statistically significant(P=0.092).The concentration of GIP(P=0.017)and PYY(P=0.033)in group D were significantly higher than those in group C.The analysis of a diversity showed that there was no significant difference in sobs(P=0.174),chao(P=0.157),shannon(P=0.374)and simpson(P=1.000)indexes between the two groups.The analysis of ? diversity showed that the difference of community structure between the two groups was obvious.The relative abundance of f_Prevotellaceae(P=0.020),g_Alloprevotella(P=0.005),s_Bacteroides_acidifaciens(P=0.045)of group D were significantly increased compared with group C,while s_Helicobacter_ganmani(P=0.031),s_Mucispirillum_schaedleri_ASF457(P=0.005)and s_Helicobacter_hepaticus(P=0.030)were significantly decreased.The RDA linear model analysis of the relationship between gut flora and lipids showed TG(R2=0.516,P=0.048),CHOL(R2=0.628,P=0.013),HDL(R2=0.728,P=0.004)and LDL(R2=0.522,P=0.038)were all correlated with intestinal flora.The lipid related genus was studied by Spearman correlation analysis.The Tyzzerella₃(P=0.006),Alloprevotella(P=0.010)and Prevotellaceae_UCG-001(P=0.027)were positive related with TG,while Ruminiclostridium₅(P=0.002),Ruminococcaceae_UCG-014(P=0.015),Mucispirillum(P=0.000),Lachnospiraceae_UCG-006(P=0.010),Helicobacter(P=0.015),Desulfovibrio(P=0.018),Rikenella(p=0.018)and Roseburia(P=0.023)were negative related with TG.Butyricicoccus(P=0.010)and A2(P=0.040)was negatively related with CHOL.Butyricicoccus(P=0.007)and A2(P=0.016)were negatively related with HDL.Ruminiclostridium(P=0.045)was positively related with LDL,while Butyricicoccus(P=0.003)and Marvinbryantia(P=0.005)were negatively related with LDL.The expression of ACC1(P=0.003),ACLY(P=0.034)mRNA of group C were significantly increased compared with group C,while there was no significant difference between group C and group D on the expression of PDHB(P=0.247),MTTP(P=0.214),DGAT2(P=0.678),FAS(P=0.565),and apo A-?(P=0.759)mRNA.Gene Ontology(Go)analysis of differentially expressed proteins of ileum samples showed that these proteins were enriched in lipid homeostasis,cholesterol homeostasis and other biological processes.Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis of differentially expressed proteins showed that these proteins were enriched in biosynthesis of unsaturated fatty acids signaling pathway,PPAR signaling pathway and other signaling pathway.6.Results after fecal microbiota transplantation of Experiment 3.There was no significant difference in body weight gain between NF and DF group(P=0.648).The TG(P=0.072)and FBG(P=0.211)in DF group were higher than NF group,but not statistically significant.The concentration of CHOL(P=0.045)and LDL(P=0.010)in DF group were significantly lower than NF group.The HDL(P=0.169)in DF group was lower compared with NF group,but there was no statistical significance.There was no significant difference in the concentration of INS(P=0.803),GC(P=0.226)and GHRL(P=0.218)between the two groups.The concentration of GLP-1 in DF group was higher compared with NF group(P=0.055),but it was not statistically significant.The concentration of GIP(P=0.067)and PYY(P=0.061)in DF group were lower than NF group,but not statistically significant.The a diversity analysis of intestinal flora showed that sobs index of DF group was significantly lower compared with NF group(P=0.004),and the chao(P=0.138)and shannon(P=0.078)index of DF group were lower compared with NF group,but not statistically significant,moreover,the simpson(P=0.257)index of DF group was higher compared with NF group,but not statistically significant.The ?diversity analysis of intestinal flora showed obvious difference of the community structure between NF and DF group.The principal co-ordinates(PCoA)and species composition analysis of intestinal flora of donor and recipient showed that there was significant difference between group N(donor)and NF(recipient),as well as between group D(donor)and DF(recipient).Conclusion1.Damp environment may cause the increase of the blood glucose concentration of Balb/c mice,which may be related with the decrease of SCFAs,GLP-1 and INS concentration caused by intestinal flora disorder.2.Damp environment may cause the increase of blood triglyceride concentration of Balb/c mice,which may be related with the disturbance of intestinal flora,the increase of lipogenic gene expression and the change of proteins in lipid metabolism.3.On the germ-free Balb/c mice,there was no similar intestinal flora change and glycolipid metabolism disorder as the donor caused by damp environment after the fecal microbiota transplantation.