Effects Of Chronic Lead Exposure On Cecal Microbial Diversity And AMPK-mediated Hepatic Lipid Metabolism In Female Japanese Quail

Lead(Pb)is one of the most toxic heavy metals to human and wildlife.It had multiple negative influences on birds with physical,neurological and hematological clinical signs.However,the impacts of lead on bird intestinal microbiota and liver lipid metabolism are still unclear.In this study,we used female Japanese quails(Coturnix japonica)to examine the effects of chronic lead exposure on cecum microbial communities diversity,immune function,liver oxidative stress and AMPK(AMP-activated protein kinase)based lipid metabolism.Our results are divided into two parts.The first part:We used female Japanese quails to examine the effects of chronic lead exposure(0,50 ppm and 1000 ppm)on cecal histology,microbial communities and immune function.The results showed 50 ppm lead exposure caused subtle damages of cecum microstructure including fewer Lieberkuhn glands.However,1000 ppm lead exposure caused severe cecum histopathological changes characterized by mucosa abscission,Lieberkuhn glands destruction and lymphocyte proliferation.Furthermore,There was a significant reduction of ratio of villi height/crypt depth in 50 ppm Pb group quails and 1000 ppm Pb group quails.Both lead concentrations(50ppm and 1000ppm)induced cecum ultrastructural damages featured by nucleus deformation,mitochondrial aggregation and vacuolation and chromatin condensation.Meanwhile,1000 ppm lead exposure caused nucleus pyknosis,nucleolar peripherization,expansion of smooth endoplasmic reticulum and shortening of microvilli.Through MiSeq sequencing of the 16S rRNA,1,483 OTUs at 97%identity were obtained by Usearch at control group and different Pb treatments.The result of alpha-diversity analysis in intestinal microbiota revealed that Chao1 index and Shannon index of cecal microbial community in 1000 ppm Pb group quails tended to increase compared with that in control and 50 ppm Pb group quails with no significant difference.Beta-diversity of the microbial communities in intestines from control and Pb exposure groups were analyzed.On the basis of unweighted UniFrac distance PCoA,microbial communities of cecal samples from control,50 ppm and 1000 ppm Pb group were divided into three clear groups.The hierarchical clustering tree showed that 3 samples from control group and 4 samples of 50 ppm Pb group clustered together into one group by phylogenetic composition,and 4 samples of 1000 ppm Pb group and 1 sample from control group clustered together into another group by similarity.The result of taxonomic analysis showed that 50 and 1000ppm lead exposure could significantly decrease the proportion of Firmicutes,while the abundance of Bacteroidetes was significantly increased in 50 and 1000 ppm Pb group compared with control group at the phylum level.The abundance of Bacteroidaceae and Veillonellaceae significantly increased in 1000 ppm Pb group compared with control group at the family level.We found that 1000 ppm Pb group significantly decrease the abundance of Faecalibacterium and Bifidobacteria,and significantly increase the abundance of Bacteroides compared with control group at the genus level.Furthermore,the mRNA relative expression of immunity-related genes such as interleukin 2(IL-2)and gamma interferon(IFN-y)was significantly downregulated while that of interleukin 6(IL-6),nuclear factor kappa-B(NF-?B)and tumor necrosis factor ?(TNF-?)was significantly upregulated in the cecum of 50 and 1000 ppm lead exposure groups.We concluded that lead exposure may cause gut health impairment of female Japanese quails by inducing cecal histopathological changes,microbiota dysbiosis and cecal immune disorder.The second part:Based on the three concentrations(0,50 and 1000ppm Pb)in the previous experiment,we set 5 concentration gradients(0,50 ppm,250ppm,500ppm and 1000 ppm Pb)to examine the effects of chronic lead exposure on female Japanese quails liver histology,oxidative stress and AMPK(AMP-activated protein kinase)based lipid metabolism.The result showed that exposure to 250,500 and 1 000 ppm Pb induced severe histopathological damages characterized by liver lipid vacuoles and accumulation,hepatic cytoplasmic hyalinization and vacuolization,hepatocytes necrosis,hepatic sinusoid congestion,and it also caused ultrastructural alterations featured by swelling and vacuolar mitochondria,the depolymerization of polyribosome,and lipid droplets accumulation.Moreover,significant decrease of activities of GPx(glutathione peroxidase),SOD(superoxide dismutase),CAT(catalase)and level of T-AOC(total antioxidant capacity)while significant increase of MDA(malondialdehyde)content were found in livers of all Pb exposure groups.The transcript level of five lipogenesis-related transcription factors showed different patterns under lead exposure.Transcript level of AMPK?1(AMP-activated protein kinase-al)and LXR?(liver X receptor ?)were significantly down regulated in Japanese quails exposed to 50,250,500 and 1 000 ppm Pb groups,while transcript level of SREBP-lc(sterol regulatory element binding protein-1 c)and ChREBP(carbohydrate response element binding protein)was significantly up regulated in 250,500 and 1 000 ppm Pb groups compared to that in the control.Moreover,exposure to 250 ppm Pb group significantly up regulated transcript level of Spot 14?.For five lipogenesis related genes.The mRNA expression of ACL(ATP-citrate lyase)was not significantly affected after Pb exposure.The mRNA expression of FAS(fatty acid synthase)was significantly up-regulated in 50,250,500 and 1 000 ppm Pb groups.SCD-1(stearoyl-CoA desaturase-1)mRNA levels showed significantly upregulated in 250 ppm Pb group.The mRNA expression of ACC(acetyl-CoA carboxylase)and ME(malic enzyme)in 250,500 and 1 000 ppm Pb groups were significantly higher than that in control group.whereas mRNA expression of MCD(malonyl-CoA decarboxylase)was significantly down-regulated in 50,250,500 and 1 000 ppm Pb groups.For fatty acid ?-oxidation,significant down-regulation in CPT-1(carnitine palmitoyl-CoA transferase-1)and CPT-2(carnitine palmitoyl-CoA transferase-2)mRNA levels was detected in 250 ppm Pb group,whereas their expression exhibited a significantly higher transcription in 50,500 and 1 000 ppm Pb groups compared to that in control.Our results indicated that lead exposure does causes bird health damages through inducing liver microstructural and ultrastructural injury,oxidative damages and lipid metabolism disorder.