| Ambient temperature is the important environmental factor affecting poultry production performance.It can have various harmful effects on the physiology and production performance of poultry.Emerging evidence has revealed that heat stress can increase fat deposition in birds,but the underling mechanism is still unclear.AMPactivated protein kinase(AMPK),as an energy sensor in the body,plays an important role in lipid homeostasis of avian.Previous studies have shown that Sirtuin1(SIRT1)can activate AMPK by deacetyating LKB1.Several studies further confirmed the correlation between reactive oxygen species(ROS)and SIRT1,and the ROS produced by heat stress has actively induced the expression of various transcriptional coactivators.Among which,PGC1α plays a central role in mitochondrial biosynthesis and the activation of antioxidants through multiple signaling kinases(especially AMPK).However,the role of AMPK axis in the development of chronic heat stress of hens remains inconclusive,so in present study,we explored the mechanism of AMPK signaling pathway in liver lipid metabolism of CHS hens.In addition,accumulating studies have shown that heat stress can significantly damage the intestinal mucosa.Therefore,in the present study,we also investigated the role of AMPK in maintaining the intestinal mucosal integrity in heat-stressed hens.Previous studies have also revealed that damage to the intestinal mucosa is often with the changes in the intestinal microbiota,which may further cause lipid metabolism disorder in the host.Thus,this study also analyzed the contribution and potential relationship of heat stress-induced changes in chicken gut microbiota to host serum metabolism.Accumulated evidence has revealed that N-acetylcysteine(NAC)as an excellent antioxidant can relieve the negative effects of heat stress in poultry.In this study,we also explored the protective effect of NAC and its potential mechanism of heat-stressed growing hens from various perspectives.The main contents of present study are:1.Effect of CHS on physiological and biochemical indexes of growing hens and alleviative effect of NACThe 12-week-old laying hens with similar body weight(1.10±0.03kg)were randomly divided into four experimental groups: Thermoneutral group(TN,22℃),Heat Stress group(HS,36℃),Paired Feed group(PF,22℃,the feeding amount was consistent with the heat stress group)and Heat stress + NAC group(HSN,36℃).Three parallel replicates were set for each group with 10 chickens.Eight birds in each group were randomly sacrificed on the 7th,14 th and 21 th days.The experiment lasted for 5weeks,with the first 2 weeks serving as the adaptation period.The results showed that the average weight of the PF and HS groups was significantly lower at the three time points compared with the TN group(P<0.05).Furthermore,at the end of the experiment,the HSN group average weight was significantly higher compared with the HS group(P<0.05).Blood gas analysis results show that,compared with the TN group,HS group of blood gas index were all significantly decreased at three time points(P < 0.05)except the blood p H was significantly increased(P < 0.05).There was no significant difference were observed between PF and TN groups at each time point(P>0.05).At the end of the experiment,the blood gas parameters was significantly improved in the HSN group compared with the HS group(P<0.05).According to the serum biochemical analysis,there were no significant differences in levels of ALB and I-BIL among all groups at each time point.In addition,compared with TN group,the levels of TP,HDL,T-AOC and SOD in group HS were significantly(P < 0.05)decreased at all three points,whereas the levels of LDL,AST and MDA were considerably(P < 0.05)increased.Similarly,the levels of D-BIL,TC,TG,CREA-S,UA,γ-GT,ALT,LDH,CK and CKMB were significantly(P < 0.05)higher,while the levels of ALP,GLO,HDL and GSHPx were significantly lower in HS group for various periods of time(exposing day 14 and day 21)in hens relative to the TN group.Significant changes were not observed in all serum biochemistry and antioxidant status in this study except in level of TC and TG was significantly(P < 0.05)decreased in group PF compared with group TN on day 14 and day 21.All the biochemical and antioxidant parameters were significantly improved in the HSN group when compared to the HS group(P<0.05).All above results suggested that chronic heat stress can significantly affect the growth performance of growing hens causing respiratory alkalosis,then it also can induce the oxidative stress,and finally lead to the disorder of serum lipid metabolism.In addition,NAC can significantly inhibit the oxidative stress caused by CHS in growing hens and then alleviate the blood lipid metabolism disorders.2.Effect of chronic heat stress on liver lipid metabolism and SIRT1-AMPKPGC1α signaling pathway and the alleviative effect of NACThe experimental design of this experiment is the same as in Experiment 1.Histopathology analysis showed that the morphological structure of liver in HS group was appeared fat accumulation at the 14 d and 21 d,which reflect in significant increased lipid droplet staining in compared with the TN group.The morphological structure of liver was normal in both the PF and TN groups.Moreover,we observed less fat drops in the HSN group when compared to the HS group.The DHE fluorescence staining of liver showed that ROS content increased significantly in both HS and HSN groups compared with TN group(P<0.05).Compared with the TN group,we observed no difference of the ROS content in the PF group at each point(P>0.05).The ROS accumulation in the HSN group decreased significantly across all three time points when compare with HS group(P<0.05).Our results also showed that the expression level of mRNA and protein of AMPK gene in liver was significantly higher(P<0.05)at day7,but lower(P<0.05)at day14 and day21 in HS group when compare to the TN group,inversely,the expression levels of mRNA and protein of ACC,GAPT and SREBP1 c significantly decreased at day7(P<0.05),but increased significantly at day14 and day21(P<0.05).Meanwhile,the expression levels of mRNA and protein of the Drp1 gene increased significantly at each time point(P<0.05),but the mRNA and protein of SIRT1,Mfn2 and PGC1 decreased significantly(P<0.05).Compared with the TN group,the expression levels of mRNA and protein of AMPK gene in the PF group increased significantly across all three time points(P<0.05),while the mRNA and protein levels of ACC,GAPT and SREBP1 c gene decreased significantly(P<0.05),and the Drp1,SIRT1,Mfn2,and PGC1 were not significantly different at each time point(P>0.05).Furthermore,the mRNA and protein expression levels of AMPK and SIRT1 pathway-related genes were significantly improved in the HSN group when compared with the HS group(P<0.05).All above results suggested that CHS can induce abnormal lipid deposition and ROS accumulation in liver of growing hens,and AMPKSIRT1-PGC1α signaling pathway may be the potential underline mechanism of this phenomenon.We also found that the NAC can improve the activation status of AMPKSIRT1-PGC1α signaling axis by inhibiting ROS content,and subsequently alleviated hepatic lipid deposition.3.Effects of Heat stress on Lipid Metabolism and SIRT1-AMPK-PGC1αsignal Pathway in Hepatocytes and alleviative effect of NACThe hepatocytes from growing hens are randomly divided into three groups: The hepatocytes of Normal group(Con)was cultured in 37℃ cell incubator;and the Heat Stress group(HS)was transferred to 42℃ cell incubator;meanwhile,the Heat Stress +NAC group(HSN)was cultured with 1m M NAC medium in the 42℃ cell incubator too;the hepatocytes were collected at 1h,2h,3h,4h and 5h,respectively.The results of the Glycogen Periodic Acid Schiff(PAS)Staining and CK-18 immunofluorescence staining suggested our primary chicken hepatocytes were successful cultured in vitro.The results of hepatocytes proliferation index showed that compared with the Con group,the HS group decreased significantly with the prolonged heat exposure(P<0.05),inversely,it was higher in the HSN group during the whole heat exposure period than the HS group.The results of Oil red staining showed that red lipid droplets were observed in HS group across the all five time points and it increased with the prolonged heat exposure time.In addition,we found the less fat drops in the HSN group when compared to the HS group.The flow cytometry results showed that the ROS level and low membrane potential cell numbers in the HS group increased significantly at each time point(P<0.05)in compared with Con group.Additionally,in compared with HS group,the ROS level and low membrane potential cell numbers were significantly decreased in the HSN group(P<0.05).Our results showed that the expression level of mRNA and protein of AMPK gene were significantly higher in HS group at hour 1(P<0.05)in compared with Con group,but significantly decreased at 3h and 5h(P<0.05),inversely,the ACC,GAPT and SREBP1 c in HS group decreased significantly at hour 1(P<0.05)but increased significantly at hour 3 and 5(P<0.05).More importantly,compared with Con group,the mRNA and protein expression levels of Drp1 in HS group increased significantly at each time point(P<0.05),but the levels of SIRT1,Mfn2 and PGC1 decreased significantly(P<0.05).All above results suggested that heat stress can induce lipid metabolism disorders in chicken hepatocytes,leading to ROS accumulation and mitochondrial dysfunction in the hepatocytes.4.Effects of chronic heat stress on intestinal function and AMPK signaling pathway and the alleviative effect of NACThe experimental design of this experiment is the same as in Experiment 1.Histopathology analysis showed that the morphological structure of duodenum in HS group was appeared inflammatory cell infiltration and closure of the intestinal gland lumen,with congestion and disorder,and the HSN group showed alleviative injury compared to the HS group,meanwhile,we did not observe significantly difference between PF and TN group.The DHE fluorescence staining results showed that compared with the TN group,the ROS level in cecum increased significantly in both the HS and HSN groups across all time points(P<0.05),and PF group was not observed any difference(P>0.05),meanwhile,compared with the HS group,the ROS level in the HSN group was significantly decreased(P<0.05).Our results also showed that compared with TN group,the mRNA and protein expression levels of AMPK,ZO-1and Occludin genes in HS group were increased at day7(P>0.05)but reduced significantly at day14 and day21(P<0.05),and the mRNA and protein expression levels of the Nrf2,HO1,and NQO1 genes were significantly decreased at each time point(P<0.05),but the levels of the IκBα and NFκB genes increased significantly(P<0.05).The mRNA and protein expression levels of all above genes in PF group were not observed any significant difference except for AMPK gene increased significantly at each time point in compared to TN group(P<0.05).The expression levels of both mRNA and protein in the HSN compared to the HS group was significantly improved.All above results suggested CHS induced ROS accumulation in intestinal tissue of growing hens,resulting in intestinal mucosal injury and down-regulation of the expression of tight junction protein.AMPK-Nrf2-NFκB signaling pathway may play an important role in the CHS-induced intestinal injury.In addition,we also observed the NAC can inhibit the accumulation of ROS,improve the expression level of AMPKNrf2,and then subsequently inhibit the NFκB to relieve the intestinal inflammation.5.Effect of chronic heat stress on cecal microbiota of growing hens.The experimental design of this experiment is the same as in Experiment 1.No obvious histopathological and oxidative injury was observed between TN and PF groups,but the morphology of cecum in HS group showed a severe injury of intestinal,including shedding of the cecal mucosal epithelial cells into the intestinal cavity,shrunk the intestinal villi,and eventually damage the arrangement of intestinal villi on day 21.Alpha and Beta diversity analysis revealed that CHS has barely effected on alpha diversity of cecal microbiota in hens,but significantly altered the beta diversity over time,which was partially independent of decreased feed intake.Manhattan plots showed that these differential OTUs mainly belonged to four phyla,including Firmicutes,Bacteroidetes,Proteobacteria and Tenericutes(P<0.05).The taxonomic analysis showed CHS can affect cecal microbiota composition of growing hens through changing the abundance of OTUs,which are mainly from phyla Firmicutes and Bacteroidete.Correlation network analysis showed that CHS significantly simplified microbial associations loosened the inter-phyla interacted relationships of cecal bacterial.Mantel correlation analysis between gut microorganisms and physiological indicators showed that the phylum Firmicutes and Euryarchaeota have a potential interaction with serum lipid parameter.All above results suggested CHS could significantly change the composition,structure and interrelationship of intestinal microbial community,which was characterized by the increase of F/B ratio and the decrease of interaction among genera under different phylum.Additionally,there was a significant positive correlation between Firmicutes and blood lipid content in growing hens,suggesting that the change of its abundance induced by CHS may significantly affect the lipid metabolism of growing hens.6.Effect of chronic heat stress on serum metabolomics in growing chickensThe experimental design of this experiment is the same as in Experiment 1.After peak alignment and removal of missing values,1761 and 1337 valid peaks remained in POS and NEG scan modes,respectively.We then identified 440(POS)and 276(NEG)metabolites by matching these valid peaks with an in-house MS2 database.The PCA results showed a clear separation between the HS group and other groups in the positive and negative modes.As presented in the PCA score plots,in both POS and NEG modes,the samples from the chronic heat exposed hens were readily separable from the rest,whereas samples of PF hens also showed separation trend with TN hens.The significant differential metabolites(SDMs)analysis combined the ROS and AUC results finally confirmed 14,13 and 12 serum metabolites were selected as biomarker of CHS induced lipid disorder in each comparison pattern(HS and TN,PF and TN,HS and PF),respectively.The results of KEGG metabolic pathway enrichment analysis revealed that the cecal microbiota may influence the host serum metabolic profile by exerting physiological functions associated with amino acid metabolism.The results of Random forest and Spearman correlation analysis showed that several altered microbial genus affected the pattern of serum metabolism in heat-stressed hens.In particular,both genus Faecalibacterium and Methanobrevibacter are important factors for predicting serum lipid accumulation of CHS hens.All above results suggested that the altered serum metabolic profile of growing hens under CHS condition mainly involves an increase in protein decomposition rather than lipidolysis,which may be a special metabolic adaptation in poultry.Additionally,genus Faecalibacterium and Methanobrevibacter are important factors causing the disorder of lipid metabolism in CHS hens.To sum up,the main conclusions of this study are:(1)Our results suggested that chronic heat stress can significantly affect the growth performance of growing hens causing respiratory alkalosis,then it also can induce the oxidative stress,and finally lead to the disorder of serum lipid metabolism.In addition,NAC can significantly inhibit the oxidative stress caused by CHS in growing hens and then alleviate the blood lipid metabolism disorders.(2)Our findings suggested CHS can induce abnormal lipid deposition and ROS accumulation in liver of growing hens,and that AMPK-SIRT1-PGC1α signaling pathway may be the potential underline mechanism of this phenomenon.We also found that the NAC can improve the activation status of AMPK-SIRT1-PGC1α signaling axis by inhibiting ROS content,and subsequently alleviated hepatic lipid deposition.(3)The results of the experiment 4 suggested CHS induced ROS accumulation in intestinal tissue of growing hens,resulting in intestinal mucosal injury and downregulation of the expression of tight junction protein.AMPK-Nrf2-NFκB signaling pathway may play an important role in the CHS-induced intestinal injury.In addition,we also observed the NAC can inhibit the accumulation of ROS,improve the expression level of AMPK-Nrf2,and then subsequently inhibit the NFκB to relieve the intestinal inflammation.(4)The results of the experiment 6 suggested that the altered serum metabolic profile of growing hens under CHS condition mainly involves an increase in protein decomposition rather than lipidolysis,which may be a special metabolic adaptation in poultry.Additionally,genus Faecalibacterium and Methanobrevibacter are important factors causing the disorder of lipid metabolism in CHS hens. |
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