| Lipopolysaccharide（LPS）is one of the main components of the cell wall of Gram-negative bacteria such as Escherichia coli.Its mass production can induce intestinal injury.LPS can damage the intestinal barrier function and LPS will be further absorbed and transferred,therefore,it is essential to ensure a good intestinal condition.Reactive oxygen species（ROS）is a highly active small molecule widely present in various cells,which is closely related to many physiological or pathological processes and ROS has been proved to be related to the cell injury,oxidative stress,and apoptosis.ROS is also involved in inducing the activation of NLRP3 inflammasomes as an upstream signal,and promotes maturation and secretion of IL-1βand IL-18.Dihydromyricetin（DHM）,a flavonoid compound extracted from rattan tea,has antioxidant,anti-inflammatory and anti-tumor activities.At present,most research on DHM focuses on mammals such as humans and mice,and there are few studies on livestock and poultry.Moreover,the study on the mechanisms of DHM protecting poultry from intestinal injury has not been reported.Therefore,based on chicks and chicken primary intestinal epithelial cells（IECs）,this study carried out the intervention effects of DHM on LPS-induced intestinal injury.Moreover,this study explored whether ROS/NLRP3 inflammasomes were involved in LPS-induced intestinal injury and DHM intervened LPS-induced intestinal injury in chickens by regulating ROS/NLRP3 inflammasomes,which will provide a theoretical and scientific basis for application and research of DHM in veterinary.The test contents and results are as follows:（1）Intervention effects of DHM on chicken intestinal injury induced by LPS:Chicks were randomly divided into six groups,namely control group,LPS group,0.025%DHM+LPS group,0.05%DHM+LPS group,0.1%DHM+LPS group and 0.1%DHM group.At 22 days of age,the chickens in LPS groups were injected with 60 mg/kg LPS into the abdominal cavity.Blood and intestinal tissue were collected 12hours later to detect related indicators.The results of HE staining of intestinal tissue showed that in LPS group,the intestinal villi were severely fragmented,a large number of inflammatory cells were infiltrated,the structure of blood vessels was blurred,and there were sloughed tissues in the lumen;the preventive effects of 0.05%and 0.1%DHM were better,the mucosal morphology and structure were relatively normal,there was only a small amount of inflammatory cell infiltration.Scanning electron microscopic observation showed that in LPS group,the intestinal villi were severely damaged,the surface was rough and uneven,the structure was chaotic,and there was no normal finger shape;After 0.05%and 0.1%DHM prevention,the intestinal villi structure basically returned to normal,there was only a small amount of damage.Compared to control group,the plasma and intestinal tissue DAO activity,intestinal tissue ROS substances（H2O2 and NO）,oxidative stress indicators（MDA,T-SOD,GSH and GSH-Px）,tight junctions protein（ZO-1,occludin and claudin-1）,apoptosis-related factors（bcl-2,bax and caspase-3）,inflammation-related factors（TLR4,p-p65/p65,IL-6,IL-8,TNF-αand IL-10）and NLRP3inflammasome-related factors（NLRP3,caspase-1,IL-1βand IL-18）have been significantly changed（p<0.01）.Compared to LPS group,0.05%and 0.1%DHM significantly reduced（p<0.01）plasma DAO activity and intestinal tissue H2O2,NO and MDA contents,and significantly increased（p<0.01）intestinal tissue GSH content,T-SOD and GSH-Px activities to interfere with intestinal mucosal damage and oxidative stress;DHM significantly increased（p<0.01）the protein expression of ZO-1,occludin and claudin-1 to maintain the intestinal tissue barrier function;DHM significantly increased（p<0.01）the expression of bcl-2,and obviously reduced（p<0.01）the expression of bax and caspase-3 to inhibit apoptosis caused by LPS;DHM also significantly reduced（p<0.01）intestinal tissue TLR4 protein expression,activation of NF-κB p65,IL-6,IL-8 and TNF-αm RNA expression,and significantly increased（p<0.01）IL-10 m RNA expression for anti-inflammation;Importantly,DHM significantly reduced（p<0.01）the m RNA and protein expression of NLRP3 and caspase-1 in intestinal tissues,thereby significantly inhibiting（p<0.01）the m RNA expression and contents of IL-1βand IL-18.（2）Establishment of LPS-induced IEC injury model:the primary IECs were isolated by type I collagenase in 14-day-old SPF chicken embryo,purified by adherence time difference method and identified by immunofluorescence.The results showed that the isolated IECs were composed of intact small intestinal crypt cluster cells and a small number of single cells.The cells were flat polygonal or elliptical,with clear cell boundaries and no overlap with each other.After culturing for 24-48 h,IECs can adhere to the wall and spread in a single layer on cell culture flasks or culture plates.Different concentrations of LPS（60,80,100,120,150μg/m L）were treated to the cells for 8,10,and 12 h,and the cell viability was measured by MTT method.The results showed that after different concentrations of LPS treated on cells,cell viability decreased in a dose-and time-dependent manner.Based on the concentration and time of LPS that caused cell damage,100μg/m L LPS was finally used to treat the cells for 12 h to induce primary IEC injury model.（3）ROS/NLRP3 inflammasome participates in the mechanisms of LPS-induced chicken primary IEC injury:NAC is a broad-spectrum ROS inhibitor.In this experiment,1.25,2.5,and 5 mmol/L NAC was used to interfere with LPS-induced primary IEC injury,and cell viability was measured to screen the optimal concentration of NAC,namely 2.5 mmol/L.Subsequently,the cells were divided into four groups,namely control group,LPS group,NAC+LPS group,and NAC group.After being pretreated with NAC for 2 h,the cells were treated with LPS for 12 h and the supernatant and cells were collected.The results showed that compared to LPS group,NAC significantly reduced（p<0.01）the overproduction of ROS induced by LPS;inhibiting ROS significantly reduced（p<0.01）MDA content,and significantly increased（p<0.01）GSH content,T-SOD and GSH-Px activities;inhibition of ROS also maintained the integrity of the barrier function of IECs by significantly increasing（p<0.01）the protein expression of ZO-1,occludin and claudin-1;moreover,inhibition of ROS reduced apoptosis,which significantly increased（p<0.01）the expression of bcl-2,and significantly reduced（p<0.01）the expression and activation of bax and caspase-3;inhibition of ROS significantly reduced（p<0.01）the m RNA expression of IL-6,IL-8 and TNF-α,and further increased（p<0.01）the m RNA expression of IL-10;importantly,inhibiting ROS significantly reduced（p<0.01）the m RNA expression of NLRP3,caspase-1,IL-1βand IL-18,and significantly reduced（p<0.01）the expression and activation of NLRP3 and caspase-1,and the secretion of IL-1β,IL-18 and LDH.To further determine whether the generation of ROS could induce the secretion of IL-1βand IL-18by regulating the activation of NLRP3 inflammasomes in LPS-induced IEC injury,the NLRP3 specific inhibitor MCC950 was used to intervene the damage in this experiment.The cells were divided into four groups,namely control group,LPS group,MCC950（10μmol/L）+LPS group and MCC950（10μmol/L）group.After pretreatment with MCC950 for 2 h,the cells were treated with LPS for 12 h and the supernatant and cells were collected.The results showed that compared to LPS group,MCC950significantly reduced（p<0.01）the m RNA expression of NLRP3 and caspase-1,and significantly inhibited（p<0.01）the protein expression and activation of NLRP3 and caspase-1,but it has no effect on IL-1βand IL-18 m RNA expression.Moreover,inhibiting NLRP3 inflammasome activation significantly reduced（p<0.01）the secretion of IL-1βand IL-18 and the release of LDH.The above results indicate that ROS/NLRP3 inflammasome is involved in the LPS-induced chicken primary IEC injury.（4）The mechanisms of DHM intervening chicken primary IEC injury by regulating ROS/NLRP3inflammasome:Different concentrations（10,20,40,80,160,320,640μmol/L）of DHM were used to treat IECs to screen the safe concentration range and concentration of DHM in this test.The cells were divided into six groups,namely control group,LPS group,DHM（10μmol/L）+LPS group,DHM（20μmol/L）+LPS group,DHM（40μmol/L）+LPS group and DHM（40μmol/L）group.After pretreatment with DHM for 2 h,the cells were treated with LPS for 12 h and the supernatant and cells were collected.After screening,40-640μmol/L DHM significantly increased（p<0.01）the viability of IECs,indicating that DHM has no obvious cytotoxicity within this concentration range.Compared to LPS group,20-320μmol/L DHM intervention significantly increased（p<0.01）cell viability.Therefore,10,20,and 40μmol/L DHM were selected to the subsequent test.The results showed that DHM interfered with the oxidative stress of IECs,significantly reduced（p<0.01）MDA content,and significantly increased（p<0.01）GSH content,T-SOD and GSH-Px activities;DHM significantly increased（p<0.01）the protein expression of ZO-1,occludin and claudin-1 to maintain the barrier function of IECs;DHM also significantly increased（p<0.01）the expression of bcl-2,and significantly decreased（p<0.01）the expression and activation of bax and caspase-3,indicating that it had a obvious inhibitory effect on apoptosis;DHM significantly inhibited the protein expression of TLR4,the activation of NF-κB p65 and the m RNA expression of IL-6,IL-8 and TNF-α,and further increased（p<0.01）the m RNA expression of IL-10 for anti-inflammation.To explore whether DHM achived its effects by regulating ROS/NLRP3inflammasome,this experiment measured the ROS content.The results showed that DHM significantly reduced（p<0.01）the generation of ROS,and further significantly reduced（p<0.01）the m RNA expression of NLRP3,caspase-1,IL-1βand IL-18,and significantly inhibited（p<0.01）the protein expression and activation of NLRP3 and caspase-1,which ultimately significantly inhibited（p<0.01）the secretion of IL-1βand IL-18 and the release of LDH.The above results indicate that DHM interferes with LPS-induced chicken primary IEC injury by regulating ROS/NLRP3 inflammasome.In summary,DHM intervenes intestinal injury,oxidative stress and apoptosis caused by LPS in chickens,maintains the intestinal epithelial barrier function,and inhibits the inflammation and the activation of NLRP3 inflammasome.Chicken primary IECs were isolated,purified and identified.Moreover,the IEC injury model was induced by 100μg/m L LPS for 12 h.Inhibition of ROS interferes with LPS-induced IEC damage,oxidative stress and apoptosis;The elimination of ROS inhibits the inflammation and reduces the release of IL-1βand IL-18 and the membrane permeability by inhibiting the activation of NLRP3 inflammasomes.DHM promotes the proliferation of IECs and interferes with cell injury,oxidative stress,apoptosis and inflammation caused by LPS;DHM also maintains the barrier function of IECs;Importantly,DHM inhibits the activation of NLRP3 inflammasomes by inhibiting the generation of ROS,which ultimately reduces the release of IL-1βand IL-18 and cell membrane permeability.Therefore,DHM can alleviate LPS-induced chicken intestinal injury and primary IEC injury by regulating ROS/NLRP3 inflammasome. |
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