Effects Of Leonurine On Immune And Antioxidation In Broilers And The Related Mechanisms

Object:To study the regulation mechanism of leonurine on growth performance,immune function,antioxidant activity and intestinal mucosal barrier protection in broiler chicks,and to provide theoretical basis for the application of leonurine in broilers.Methods:(1)A total of 600 1-day-old Ross 308 broilers were randomly allocated to five treatment groups consisting of eight pens of fifteen birds.Each treatment group was added with 0,15,30,60,120 mg/kg of leonurine in the basic diet.The feeding program was divided into two stages: the middle stage(0~21 days old)and the late stage(22~42).To explore the effects of different dietary levels of leonurine supplementation on growth performance,immune response,antibody titer,antioxidant capacity and serum lipid profiles in broiler chicks,determine the optimum addition level of leonurine.(2)Based on the results of the first part of study,the experiment was designed as a 2 × 2 factorial arrangement with leonurine(0 or 120 mg/kg)and LPS(injection of saline or 1.5 mg/kg body weight)levels as treatments.A total of 120 one-day-old male Ross broilers were randomly divided into 4 treatment groups with 6 replicates of 5 birds per cage.On days 14,16,18,and 20,half of the broilers in each treatment group were intraperitoneally injected with LPS,the remaining broilers were injected with the same dose of saline as the LPS injected group.To investigate explore the possibility of leonurine alleviate LPS-induced inflammatory response and oxidative stress,and provide experimental basis for subsequent studies,detecting the growth performance,organ weights,serum and spleen parameters of broiler chicken on days 21 and 28.(3)Based on the results of the second part of study,duodenal mucosa jejunal mucosa and ileum mucosa and their contents were collected from broiler chicken on days 21 and 28.The morphology of small intestinal mucosa,number of intestinal flora and intestinal antioxidant index were detected to observe the protective effect of leonurine on intestinal mucosal barrier injury induced by LPS.The m RNA expression levels of inflammatory factors and inflammatory mediators were detected by real-time fluorescence quantitative PCR to investigate the regulatory effect of leonurine on inflammatory response.The expression levels of tight junction proteins and MAPK/NF-?B signaling pathways-associated proteins were detected by Western blot to clarify the mechanism of leonurine alleviate intestinal inflammation and oxidative stress.Intestinal epithelial cells were isolated from SPF chicken embryos at the age of 14 to establish an in vitro culture model.The cell viability was measured by CCK-8 to determined the optimal concentration and time of leonurine and LPS.The m RNA expression levels of antioxidant enzymes and tight junction proteins were detected by real-time fluorescence quantitative PCR to investigate the protective effect of leonurine on LPS-induced intestinal epithelial cells inflammatory response.The protein expression of related signaling pathways was detected by Western Blot to determine whether the mechanism of leonurine on alleviating the damage of intestinal barrier function in vivo and in vitro is consistent.Results:The results indicate that leonurine did not alter the growth performance of broilers(P > 0.05).Supplementation of the basal diet with leonurine increased(P < 0.05)relative spleen index,newcastle disease virus antibody titer,serum immunoglobulin A(Ig A)and M(Ig M)concentrations,catalase(CAT),total superoxide dismutase(T-SOD),total antioxidant capacity(T-AOC)activities,low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)levels,while significant(P < 0.05)decreased serum malondialdehyde(MDA)activity,triglyceride(TC)levels and total cholesterol(CHOL)content.at d 21 and 42.In addition dietary leonurine supplementation significant(P < 0.05)increased the GSH activity in 42-day-old broilers.However,there was no significant effect on bursa and thymus index,infectious bursal disease antibody titer,serum Ig M,IL-1?,IL-6 and TNF-? concentrations(P > 0.05).Taken together,dietary leonurine supplementation was able to promote immune function and antioxidant capacity,and decrease blood lipid levels in broilers.(2)The results showed that broiler chicks fed diets supplemented with leonurine exhibited no significant difference in growth performance or immunoglobulin concentrations in the serum before and after LPS challenge(days 1-14;days 22-28).However,dietary leonurine prevented LPS-induced reductions in average daily gain(ADG)and average daily feed intake(ADFI)in the broilers on days 15–21 of the trial(P > 0.05).Dietary leonurine supplementation dramatically attenuated the LPS-induced increases in the spleen index,GSH activity(serum and liver)and T-SOD activity(serum and spleen)and significantly reduced MDA levels(serum,spleen and liver)on days 21 and 28(P < 0.05).Additionally,leonurine supplementation significantly mitigated the LPS-induced increases of interleukin(IL)-1?,IL-6,tumour necrosis factor(TNF)-? in serum and spleen,and significantly reduce the expression of nuclear factor(NF)-NF-?B m RNA in the spleen(P < 0.05).(3)LPS-challenge significantly destroy the morphology of duodenum and jejunum mucosa,increased in serum diamine oxidase(DAO)and D-lactic(D-LA)acid levels,increase the quantity of E.coli and salmonella in intestinal contents and the MDA content(P < 0.05),while reduced GSH content and T-SOD activity coupled with the quantity of lactobacillus(P < 0.05).However,dietary leonurine significantly alleviated the effect of LPS on the duodenum and jejunum(P < 0.05),but not effect of ileum(P > 0.05).Additionally,leonurine supplementation significantly downregulated the m RNA expression of NF-?B,COX-2,TNF-?,IL-1? and IL-6 and upregulated the m RNA expression of the ZO-1 and Occludin in the jejunum mucosa induced by LPS(P < 0.05).On the other hand,leonurine administration prevented the LPS-induced activation of the p38,ERK and JNK MAPKs and attenuated I?B? phosphorylation and nuclear translocation of NF-?B(p65)in the jejunum mucosa.(4)The results found that pretreatment with 40 ?M/m L leonurine for 3 h significantly attenuated LPS-induced apoptosis of intestinal epithelial cells.In addition,leonurine can reduce the expression of SOD1,GPX1,ZO-1 and Occludin,reduce the expression of i NOS,COX-2,TNF-? and IL-1? m RNA.Inhibit the activation of MAPK/ NF-?Bsignaling pathway,thereby alleviated the damage of LPS-challenge on intestinal epithelial cells and protect the integrity of cells.Conclusion:Dietary 120 mg/kg leonurine supplementation can improve immune function and antioxidant capacity,and alleviate LPS-induced intestinal inflammation response,oxidative stress and mucosal barrier function damage in broilers.The protective mechanism is through Inhibition of NF-?B nuclear translocation and phosphorylation of key molecules of the MAPK signaling pathway,impeding the expression of inflammatory factors and mediators,and improving the activity of antioxidant enzymee,and then intervening in the inflammatory response and oxidative stress caused by LPS.At the same time,it inhibits the transitional reproduction of intestinal pathogens,regulates the expression of tight junctions proteins,and maintains the stability and integrity of the intestinal mucosal barrier,to providing new theoretical evidence for the development of anti-stress new feed additives.