Mechanism Of MiR-27b And MiR-223 On Polysaccharide Of Atractylodes Macrocephala Koidz Alleviate Lipopolysaccharide-stimulated Liver Inflammation Injury Of Goslings

During the breeding and production of livestock and poultry,the infection of bacteria and endotoxins will lead to inflammatory reactions in animals,which will lead to an increase in the incidence of livestock and poultry and a decrease in production performance.Therefore,alleviating the inflammatory response caused by pathogens is a scientific issue which is worth for more further research.Polysaccharide of Atractylodes macrocephala Koidz(PAMK)is the main active ingredient of Atractylodes macrocephala which has anti-aging,immune regulation,antioxidant and other effects.been confirmed to shown to have the ability to antiaging,anti-oxidation,and regulate the immune function.Previous studies have shown that PAMK could protect liver from inflammation caused by LPS in mice.However,weather PAMK could alleviate liver inflammatory injury in goslings induced by LPS and the specific molecular mechanism is still unknown.For evaluating whether PAMK could alleviate liver inflammatory injury induced by LPS in goslings,a total of 120 healthy one-day old Magang goslings were randomly divided into6 groups: control group,PAMK group,LPS group,PAMK+LPS group,Dexamethasone(DXMS)group and DXMS+LPS group with 4 replicates per group and 5 goslings per replicate in a single-factor completely randomized experimental design.Goslings in PAMK group and PAMK+LPS group were fed basal diet supplemented with 400 mg/kg PAMK,and other groups were fed with basal diet to the end of trail.On 24 days of age,goslings in the control group and PAMK group were intraperitoneal injected 0.5 m L normal saline,goslings in LPS and PAMK+LPS groups were intraperitoneal injected with LPS at 5 mg/kg BW,goslings in DXMS group were intraperitoneal injected with DXMS at 5 mg/kg BW,and goslings in DXMS+LPS group were intraperitoneal injected with LPS at 5 mg/kg BW and DXMS at 5 mg/kg BW.The serum and liver samples were collected for further analysis after treatment of LPS at 6,12,24,and 48 h.The liver injury was evaluated by histopathology and biochemical indexes.The results showed that: compared with DXMS group,PAMK could alleviate the phenomenon of hepatic steatosis,inflammatory cell infiltration and fibrous hyperplasia after LPS treatment more efficiently.And LPS-induced liver injury increased serum ALT and AST level,which decreased by PAMK pretreatment in the PAMK+LPS group.LPS could stimulate TLR2 receptor on cell membrane and activate the TLR2 cell signaling pathway.Besides,bioinformatics predicted that miR-27 b could potentially target and regulate TLR2 gene.In order to verify whether miR-27 b can target and regulate TLR2 and the specific role of miR-27b/TLR2 in the process of PAMK alleviating inflammatory liver damage induced by LPS in goslings,this study used the samples of control group,PAMK group,LPS group and PAMK + LPS group in the above experimental groups for following experiment,and gosling liver cells were isolated for follow-up detection.The cells in the PAMK and PAMK+LPS groups were pretreated with 40 μg/m L PAMK for 12 h.After the medium was changed,200 μg/m L LPS was added to the LPS and PAMK+LPS groups on the basis of retaining the original PAMK treatment.The results showed that: The sequencing results predicted that TLR2 was the target gene of miR-27 b.PAMK can up-regulate the expression of miR-27 b gene in gosling liver 24 h after LPS treatment,and decrease m RNA and protein expression of TLR2 and NF-κB in goslings liver and hepatocytes,and reduce TNF-α and IL-4 level in serum of goslings at the same time.LPS infection could activate the inflammatory pathway in host cells and induce the production of NLRP3.Bioinformatics predicts that miR-223 potentially targets and regulates NLRP3 gene.In order to verify whether miR-223 can target and regulate NLRP3 and the specific role of miR-223/NLRP3 in the process of PAMK alleviating inflammatory liver damage induced by LPS in goslings,this study used the samples of control group,PAMK group,LPS group and PAMK + LPS group in the above experimental groups for following experiment,and gosling liver cells were isolated for follow-up detection.The cells in the PAMK and PAMK+LPS groups were pretreated with 40 μg/m L PAMK for 12 h.After the medium was changed,200 μg/m L LPS was added to the LPS and PAMK+LPS groups on the basis of retaining the original PAMK treatment.The results showed that: LPS decreased expression levels of miR-223 and increased protein expression levels of NLRP3,Caspase1 and cleaved-Caspase1,up-regulated cytokines IL-1β and IL-18 levels.PAMK pretreatment could reduce the IL-1β and IL-18 levels,inhibit the expression of NLRP3,Caspase1 and cleaved-Caspase1,and increase the expression of miR-223.Based on the establishment of miR-223 overexpression / inhibition model of hepatocytes,the expression levels of NLRP3,Caspase-1 and cleaved Caspase-1 decreased in LPS induced hepatocytes after overexpression of miR-223.Inhibiting the expression of miR-223 could reverse the effect of PAMK on LPS induced goslings embryonic hepatocytes and up regulate the expression levels of NLRP3 and cleaved Caspase-1.The results above indicated that PAMK could alleviate LPS-induced liver pathological damage and abnormal secretion of AST and ALT in goslings.At the same time,PAMK could reduce the expression of TLR2 gene by increasing miR-27 b in the late stage of LPS infection of goslings,thereby inhibiting the TLR2/NF-κB pathway and further reducing the secretion of TNF-α and IL-4.In addition,PAMK could also reduce the expression of NLRP3 gene by increasing miR-223 in the late stage of LPS infection of goslings,thereby inhibiting the NLRP3/Caspase-1 pathway and further reducing the secretion of IL-1β and IL-18.Ultimately,this study proved that PAMK could play a protective role in inflammatory liver injury in goslings,and laid a foundation for the development and application of green feed additives in the animal husbandry industry.