| Diseases caused by Gram-negative bacteria are common and highly prevalent in poultry farming,seriously threatening the healthy development of poultry farming as well as public health and food safety.Endotoxin,also known as lipopolysaccharides（LPS）,is a component unique to the surface of Gram-negative bacteria,which is released into the liver with the lysis of the bacterium,causing oxidative stress and causing liver damage.Silent information regulator of transcription 1（SIRT1）,a class III histone deacetylase,is highly expressed in the liver and is involved in regulating various physiological processes,such as cell proliferation,oxidative stress,and inflammation.MicroRNAs（miRNAs）are a class of endogenous small non-coding RNAs that can regulate gene expression by binding to the 3′UTR region of target genes and play an important role in a variety of biological processes.It was found that miR-138-5p can bind to the 3′UTR region of SIRT1 and regulate the expression of SIRT1,but the regulatory mechanism of miR-138-5p on SIRT1 is not well understood in avian diseases.Dihydromyricetin（DHM）,a natural flavonoid with high content extracted from snake grapes,has various pharmacological effects such as anti-inflammatory and antioxidant,but the specific mechanism of action is complex and unclear.Therefore,the study was conducted on chicken primary hepatocytes and chicks to investigate the role of SIRT1 in chicken liver oxidative stress injury based on LPS-induced oxidative stress injury,to clarify the molecular mechanism of miR-138-5p regulating SIRT1,and then to elucidate the intervention effect and potential protective mechanism of DHM on chicken liver oxidative stress injury.The study contents and results of this experiment are as follows:（1）Study on the role of DHM in alleviating LPS-induced oxidative stress injury in chicken hepatocytes:DHM treatment was given in LPS-induced oxidative stress injury in chicken hepatocytes to clarify the protective effect of DHM on LPS-induced oxidative stress injury in chicken hepatocytes.The results showed that compared with the LPS group,DHM highly significantly reduced ALT,AST and LDH activities in the supernatant,and highly significantly altered cellular SOD,CAT,GSH-Px,GSH,MDA and ROS levels,as well as highly significantly reduced IL-1β,IL-6 and TNF-αexpression and elevated SIRT1 expression（p<0.01）.These results suggest that DHM could improve cellular antioxidant capacity,scavenge ROS,inhibit inflammatory factor release,alleviate LPS-induced oxidative stress damage in hepatocytes,and promote SIRT1expression.（2）Study on the role of SIRT1 in LPS-induced oxidative stress injury in hepatocytes:firstly,the changes of oxidative stress injury-related indexes in chicken hepatocytes and the expression of transcription factors NOX4 and PPARαdownstream of SIRT1 were detected after overexpression of SIRT1.The results showed that compared with the LPS group,overexpression of SIRT1 could significantly reduce ALT,AST and LDH activities in the supernatant,increase cellular SOD,CAT,GSH-Px activity,GSH content and PPARαexpression,and reduce cellular MDA and ROS content and IL-1β,IL-6,TNF-αand NOX4 expression（p<0.01）,indicating that SIRT1 high expression could alleviate LPS-induced oxidative stress damage in hepatocytes,inhibit ROS production and affect the expression of downstream transcription factors.To further clarify whether SIRT1 affects ROS production dependent on downstream transcription factors,we applied NOX4 inhibit or（GKT137831）and PPARαagonist（WY-14643）.The results showed that inhibition of NOX4 or activation of PPARαexpression both significantly reduced ROS levels.The above results indicated that SIRT1 was involved in LPS-induced oxidative stress injury in chicken hepatocytes,and overexpression of SIRT1 could inhibit ROS production by regulating NOX4 and PPARαexpression,enhance cellular antioxidant capacity and reduce oxidative stress injury.（3）Molecular mechanism of miR-138-5p regulation of SIRT1 in oxidative stress injury in chicken hepatocytes:first,the non-coding RNA（miR-138-5p）that binds to the 3’UTR region of SIRT1 was predicted and selected,and the expression of miR-138-5p under the action of LPS was examined;then the binding of miR-138-5p to SIRT1 was then verified by constructing a dual luciferase reporter gene plasmid,then apply miR-138-5p inhibitor（inhibitor）and mimic（mimic）and negative control to transfected chicken hepatocytes to detect the expression of miR-138-5p and SIRT1,and analyze the regulatory effect of miR-138-5p on SIRT1.The results showed that miR-138-5p was highly expressed in the LPS group compared with the control group;miR-138-5p mimic could highly significantly reduce the luciferase activity of SIRT1 wild-type reporter gene（p<0.01）,while there was no significant regulation of luciferase activity of mutant reporter gene,indicating that miR-138-5p could directly bind to SIRT.Application of miR-138-5p inhibitor inhibited the expression of miR-138-5p resulted in a highly significant increase in SIRT1 expression（p<0.01）,and application of miR-138-5p mimic overexpressed miR-138-5p resulted in a highly significant decrease in SIRT1 expression（p<0.01）,indicating that miR-138-5p can target negatively regulate the expression of SITR1.To further investigate the mechanism of miR-138-5p regulation of SIRT1in oxidative stress injury,miR-138-5p inhibitor and SIRT1 inhibitor（EX527）were applied to co-treated cells with LPS to detect the changes of SIRT1 expression and oxidative stress injury indicators in chicken liver cells.The results showed that inhibition of miR-138-5p expression upregulated SIRT1 expression,decreased ALT,AST and LDH activities in the supernatant,increased cellular SOD,CAT,GSH-Px activity and GSH content,and decreased cellular MDA,ROS content and IL-1β,IL-6 and TNF-αexpression（p<0.01）;while inhibition of miR-138-5p expression was given after EX527 treatment,cellular antioxidant enzyme activity decreased,ROS level increased,inflammatory factor release increased,and the degree of cellular damage increased,indicating that the intervention effect of miR-138-5p inhibitor on cells could be inhibited by EX527.The above results suggest that both miR-138-5p and SIRT1 are involved in LPS-induced oxidative stress injury in chicken hepatocytes and miR-138-5p has a negative regulatory effect on SIRT1.（4）Mechanisms by which DHM alleviates oxidative stress injury in chicken hepatocytes through miR-138-5p/SIRT1:DHM-treated cells were first applied followed by LPS,and the expression of miR-138-5p was detected to analyze the effect of DHM on miR-138-5p expression.The results showed that miR-138-5p expression was highly significantly decreased after administration of DHM compared with the LPS group（p<0.01）,indicating that DHM could down-regulate miR-138-5p expression.Then miR-138-5p inhibitor,miR-138-5p mimic and DHM were applied to give cells to explore whether the effect of DHM in alleviating oxidative stress damage was dependent on miR-138-5p.The results showed that inhibition of miR-138-5p expression resulted in a highly significant decrease in supernatant ALT,AST and LDH activities,and cellular SOD,CAT,GSH-Px activity and GSH content were highly significantly increased,and MDA,ROS content and expression of IL-1β,IL-6 and TNF-αwere highly significantly decreased（p<0.01）;on the basis of this administration of DHM,none of the above indicators changed significantly.After overexpression of miR-138-5p,the activity of cellular antioxidant enzymes decreased,the level of ROS increased,the release of inflammatory factors increased,and the degree of cellular damage increased;applying DHM on top of overexpression of miR-138-5p significantly reduced the changes of the above indices due to overexpression of miR-138-5p.These results suggest that DHM can alleviate LPS-induced oxidative stress injury in hepatocytes by inhibiting miR-138-5p expression.To further clarify the mechanism of DHM in alleviating hepatocyte oxidative stress injury through miR-138-5p/SIRT1,miR-138-5p and SIRT1 were overexpressed,and then DHM treatment was given to detect SIRT1 expression as well as oxidative stress injury in chicken hepatocytes.The results showed that compared with the LPS+DHM group,overexpression o f miR-138-5p followed by DHM administration resulted in highly significant increases in supernatant ALT,AST and LDH activities,highly significant decreases in cellular SOD,CAT,GSH-Px activities and GSH content,and highly significant increases in MDA,ROS content and expression of IL-1β,IL-6 and TNF-α（p<0.01）,while SIRT1 expression was highly significantly decreased（p<0.01）,indicating that the protective effect of DHM on cell injury and the upregulation of SIRT1 was blocked by miR-138-5p overexpression,and thus DHM attenuated cell injury by inhibiting miR-138-5p expression.Further,on this basis,overexpression of miR-138-5p and SIRT1 followed by administration of DHM resulted in a highly significant increase in SIRT1 expression,a highly sign ificant decrease in supernatant ALT,AST and LDH activities,a highly significant increase in antioxidant enzyme activities,and a highly significant decrease in ROS levels and inflammatory factor expression（p<0.01）,indicating that SIRT1 overexpression reversed the effect of DHM by miR-138-5p overexpression,and the protective effect of DHM inhibition of miR-138-5p on hepatocyte injury was dependent on the involvement of SIRT1.The above results suggest that DHM alleviates LPS-induced oxidative stress injury in hepatocytes by inhibiting miR-138-5p and thus upregulating SIRT1 expression.（5）DHM alleviates LPS induced liver oxidative stress injury in chicks:The protective effect of DHM on LPS induced liver injury in chicks was clarified by detecting the expression of oxidative stress injury related indicators,inflammatory factors,mir-138-5p and SIRT1 and histopathological observation.The results showed that serum ALT,AST and LDH activities were highly significantly increased in the LPS group,the levels of liver SOD,CAT,GSH-Px,GSH,MDA,H2O2 and NO were highly significantly changed,and the expression of IL-1β,IL-6 and TNF-α,miR-138-5p were highly significantly increased and the expression of SIRT1 was highly significantly decreased in the LPS group（p<0.01）.DHM treatment reduced the extent of hepatocyte injury,increased antioxidant enzyme activity,inhibited inflammatory factors and miR-138-5p expression,as well as elevated SIRT1 expression.Pathological histological examination showed that th e hepatic cords in the LPS group were disorganized with obvious inflammatory cell infiltration and accompanied by hemorrhagic spots;DHM treatment showed intact hepatic cords with fewer inflammatory cells and hemorrhagic spots.The results of transmission electron microscopy showed that the nucleus of the LPS group had an enlarged nuclear membrane gap,a large number of vacuoles in the endoplasmic reticulum and mitochondria,and a decrease in intracellular glycogen,while the vacuoles in the endoplasmic reticulum and mitochondria were reduced in the DHM treatment,and the hepatocyte structure was normalized.The above study showed that DHM could enhance the antioxidant capacity of liver,inhibit the inflammatory response,suppress miR-138-5p expression and promote SIRT1 expression,thus improving LPS-induced oxidative stress damage in chick liver.In summary,SIRT1 plays an important role in LPS induced oxidative stress injury of chicken hepatocytes,and overexpression of SIRT1 could inhibit ROS production an d enhance cellular antioxidant capacity by regulating the expression of NOX4 and PPARα.miR-138-5p and SIRT1were involved in the mechanism of LPS-induced oxidative stress injury in chick hepatocytes and miR-138-5p had a negative effect on SIRT1 miR-138-5p and SIRT1 were involved in the mechanism of LPS-induced oxidative stress injury in chicken hepatocytes and miR-138-5p had a negative regulatory effect on SIRT1.This study was the first to investigate the molecular mechanism of miR-138-5p regulation of SIRT1 in chicken liver oxidative stress injury,and to explore the potential mechanism of DHM intervention in chicken liver oxidative stress injury caused by LPS,providing a theoretical basis and scientific basis for the clinical application of DHM in veter inary medicine,and providing new targets and new ideas for the development of new drugs. |
PDF Full Text Request