Effects Of Flaxseed Meal And Flaxseed Oil On Regulating Liver Inflammation And Intestinal Microflora In Type 1 Diabetic Mice

Objective: The mouse model of Type 1 diabetes mellitus(T1DM)induced by streptozotocin(STZ)was used to study the effects of flaxseed meal(FM)and faxseed oil(FO)on liver inflammation and intestinal flora.Moreover,the lipopolysaccharide(LPS)/toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-?B)signaling pathway was examinzed as a target to preliminarily explore the possible molecular mechanisms,providing a scientific basis for the application of FM and FO in T1 DM dietary management.Methods: Male C57BL/6J mice aged 4-6 weeks were fed adaptively for one week.After that,12 randomly selected mice were included in CON group(AIN-93 G Purified Diet),and the rest mice who were fasted but allowed to drink water lasting for 12 hours were injected intraperitoneally with a large dose of 170.00 mg/kg STZ.Fasting blood glucose(FBG)?16.7mmol/L were considered as the mouse model of T1 DM when combination with the symptoms of polydipsia,polyuria,polyphagia and weight loss.Afterwards,60 T1 DM model mice were randomly divided into MOD group(AIN-93 G Purified Diet),FM1 group(25.00% FM added),FM2 group(16.70% FM added),FO1 group(10.50% FO added)and FO2 group(7.01% FO added)according to the levels of FBG and weight,then the intervention of different groups continued for 6 weeks.Three days before the end of the experiment,the oral glucose tolerance test(OGTT)was conducted.Finally,all mice were scarified in order to collect the blood and tissue samples for further analysis.Using the colorimetric method to determine the contents of hepatic glycogen,and the ELISA method was used to measure LPS levels in the mouse serum.The level of tumor necrosis factor-?(TNF-?),interleukin-1?(IL-1?),interleukin-6(IL-6)and LPS in the liver were also detected by the ELISA method.The changes of intestinal flora in mice were analyzed by 16 S r DNA high-throughput sequencing technology.Quantitative realtime polymerase chain reaction(RT-q PCR)was used to determinate the m RNA expression levels of TLR4,myeloid differentiation factor 88(My D88),tumor necrosis factor receptor associated factor 6(TRAF6)and NF-?B and Western Blot(WB)was used to measure the expression levels of the key proteins.Results:(1)Effects on glucose metabolism and liver inflammation: Compared with the CON group,the body weight and liver glycogen content in the MOD group were significantly reduced(P<0.05),while the levels of FBG,AUC,TNF-? and IL-1? were significantly increased(P<0.05).In the MOD group,the islet structure was severely damaged with irregular and atrophic shape.And the number of islets was decreased and the boundary was blurred.Besides,the amount of ? cells was very relatively small with the exhibition of the compensatory cytoplasmic swelling.After 6 weeks of dietary intervention,FM and FO prevented the continuous weight loss of T1 DM mice to a certain extent and the difference between FM1 and MOD groups was significant(P<0.05).Compared with the MOD group,FBG levels in the FM2 and FO1 groups were declined,AUC was decreased and the liver glycogen content was increased significantly(P<0.05);The intervention of FM1 showed a trend of reducing FBG and increasing the liver glycogen content,but the differences have not be statistically significant(P=0.08,P=0.09);The FBG level in the FM2 group was the lowest among the intervention groups,and the difference was statistically significant compared with the FO2 group(P<0.05).At 400 times light,the structure of islet in the FM and FO group has improved,but there was still obvious atrophy and a decrease in the number.The boundary was relatively blurry as well.The number of ? cells has increased compared with the MOD group.In addition,the FM and FO groups could also change the level of liver inflammation in T1 DM mice,with the significant reduction of TNF-? levels in each intervention group(P<0.05),and the levels of IL-1? were also significantly down-regulated in the FM1 and FO2 groups(P<0.05);The IL-1? levels in the FM2 group was higher than the FO2 group(P<0.05),proving that the relatively stronger ability of FO2 on regulation the liver inflammation.(2)Effect on intestinal flora: Compared with the CON group,the diversity reduction of intestinal microbiota was statistically significant(P<0.05)in the MOD group,and the flora structure was changed obviously.At the level of phylum,the relative abundance of Firmicutes and Bacteroidetes decreased significantly(P<0.05),and the proportion of Proteobacteria was increased significantly(P<0.05),which became the leading bacteria in MOD group.The changes of the main dominant phylums in abundance led to the increase of G-bacteria proportion in the MOD group,but no statistical difference was found(P>0.05).The proportion of Lactobacillus,uncultured?bacterium?f?Muribaculaceae and Escherichia-Shigella was also changed obviously(P<0.05),while the abundance of Faecalibaculum between the two groups was not significantly different(P>0.05).Compared with the MOD group,the bacterial diversity increased significantly in the FM group(P<0.05)and returned to normal levels.FM and FO intervention could also restore the composition in the intestinal microbiota to varying degrees,so that the microflora structure tended to be normal.In the FM group,the relative abundance of Firmicutes and Bacteroides were increased and the proportion of Proteobacteria was decreased significantly(P<0.05).The ranking of three dominant phylums was the same as that in the CON group,the specific proportions in the FM1 group also returned to be normal;FO intervention reduced the proportion of Proteobacteria(P<0.05),and FO2 intervention could also reverse the decrease of Firmicutes in T1 DM mice,so that there was no significant difference between CON group and FO2 group(P>0.05).In addition,the abundance of Gbacteria in each intervention group decreased to different degrees,but the difference has not been statistically significant(P>0.05).At the level of phylum,the improvement effect of FM was relatively better,mainly manifested as the highest abundance of Bacteroidetes and Firmicutes,as well as the lowest abundance of Proteobacteria and G-bacteria in FM1 and FM2 groups respectively.According to the results of variance analysis,compared with the MOD group,FM1 intervention could increase the relative abundance of Lactobacillus,uncultured?bacterium?f?Muribaculaceae and Faecalibaculum(P<0.05),the proportion of Escherichia-Shigella decreased significantly in the FM1 and FM2 group(P<0.05),FO1 intervention significantly increased the relative abundance of uncultured?bacterium?f?Muribaculaceae and Faecalibaculum(P<0.05);In the FO2 intervention group,the proportion of Escherichia-Shigella was significantly reduced(P<0.05);At the genus level,the effect of FM1 intervention were relatively better,which was mainly reflected in the abundance of the four major dominant genus,among them,uncultured?bacterium?f?Muribaculaceae and Escherichia-Shigella returned to normal levels;(3)Effect on LPS/TLR4/NF-?B signaling pathway: Compared MOD group with CON group,the levels of LPS in serum and liver were significantly increased(P<0.05).While compared with the MOD group,FM1 intervention had a reduction effect on them(P=0.09,P<0.05)and in the FO2 group,both levels were significantly lowered(P<0.05).RT-q PCR results showed that TLR4,TRAF6 and NF-?B m RNA expression levels in the MOD group were higher than that in the CON group(P<0.05);And Compared with the MOD group,the expression levels of TRAF6 and NF-?B were significantly down-regulated in the FM and FO groups(P<0.05),while the expression of TLR4 and My D88 were only significantly reduced in the FM1 and FO2 groups(P<0.05).It can be seen from the WB results that the expression levels of TLR4 and NF-?B proteins expression levels in the liver of the MOD group were significantly increased(P<0.05),indicating that the TLR4/NF-?B signaling pathway was activated.Compared with the MOD group,TLR4 and NF-?B levels were significantly down-regulated in each intervention group(P<0.05),while My D88 protein expression was only significantly decreased after the intervention of FM1 and FO2(P<0.05),which was consistent with the results at the gene level.Conclusion:(1)The dietary intervention of FM and FO can regulate the glucose metabolism of T1 DM mice and restore liver inflammation.Among them,the dietary adding16.70% FM has a better hypoglycemic effect,while the dietary adding 7.01% FO has stronger ability of regulating the liver inflammation,and adding 25.00% FM can play a beneficial regulatory role in both hypoglycemic and anti-inflammatory;(2)FM and FO interventions(especially the dietary adding 25.00% FM)can increase the diversity of intestinal flora to varying degrees.Meanwhile,the relative abundance of Firmicutes and Bacteroides was increased and the proportion of Proteobacteria and G-bacteria was reduced in FM groups and FO groups.Besides,they can also restore the structure of the flora,increase the abundance of the beneficial bacteria(Lactobacillus,uncultured?bacterium?f?Muribaculaceae and Faecalibaculum)and reduce the proportion of harmful bacteria(Escherichia-Shigella)to improve intestinal health,and finally delay the progress of T1DM;(3)The improvement effect of FM and FO(especially the dietary adding 25.00% FM or7.01% FO)on liver inflammation may be related to the regulation of intestinal flora,which may regulate the expression levels of TLR4,My D88 and NF-?B genes and proteins in the liver by inhibiting the abnormal increase of pathogenic bacteria LPS,thus further exert the antiinflammatory effects.