Effects Of Dihydromyricetin On Growth And Immune Function Of Chicks And Its Mitigation Of Liver And Spleen Injury Induced By LPS

Lipopolysaccharide(LPS)is one of the major components of the cell walls of gram-negative bacteria.Bacterium of negative of gut dissolves can release a large number of LPS into the blood or into the liver via portal vein.LPS can damage the liver and spleen tissue and destroy the immune system of chicken,and affect the growth performance and immune function,and even cause death,resulting in serious economic losses.Dihydromyricetin(DHM)is a flavonoid compound.DHM exists in many kinds of plants and its structure is clear.DHM has a variety of physiological activities such as scavenging oxygen free radicals,antioxidant and anti-inflammatory and has a high application prospect in the production of animal husbandry.In this experiment,different dose DHM were added into the blue sea chicks feed to study the effect of DHM on the growth performance and immune function,and the protective effect of DHM on liver and spleen injury induced by LPS,which provides the theoretical and scientific basis for the wide application of DHM in veterinary clinic.The experiments are designed as followed:(1)Study the effect of DHM on growth performance and immune function of chicks: The 1-day-old chicks were given adaptive feeding for 7 days,and the chickens were immunized with Newcastle disease vaccine at the age of 7 days.Then the chicks were divided into five groups and different dose DHM(0.025%,0.05%,0.1%,0.2%)was added to the feed for 35 days.Then the chickens were weighed on an empty stomach weekly to calculate the average daily weight gain(ADG)and average feed weight ratio(F/G).After weighing the chicks,blood samples were collected from heart to separate the serum,and the organs(heart,liver,kidney,spleen,thymus and bursa)were isolated and weighted to calculate the organ index.The content of serum total protein(TP),albumin(ALB),total cholesterol(TC),triglyceride(TG),glucose(GLU),direct bilirubin(DBIL),calcium(Ca),phosphorus(P),as well as the activity of aspertate aminotransferase(AST),alanine aminotransferase(ALT)and lactic dehydrogenase(LDH)were detected by automatic biochemical analyser.The concentration of serum Ig G,IL-2,IFN-γ were detected by ELISA.The antibody titer of Newcastle disease in serum were tested by hemagglutination inhibition(RPHI).The activities of total superoxide dismutase(T-SOD),glutathione peroxidase(GSH-Px),total antioxidant capacity(T-AOC)and malondialdehyde(MDA)in liver homogenate were tested with the kit.(2)Study the effect of DHM on the m RNA expression of TLR2,TLR4 and inflammatory factor in the liver and spleen of chicks: the 7-day-old chicks were divided into five groups,and dose DHM(0.025%,0.05%,0.1%,0.2%)was added to the feed for 35 days.At the age of 43 days,the chickens were killed by carotid artery bleeding and the liver and spleen tissues were separated for extraction of total RNA.And then m RNA expression of TLR2,TLR4 and inflammatory factors(IL-1β,IL-6,IL-8,IL-10 and TNF-α)in the chicks liver and spleen were detected with RT-PCR.(3)Study the protective effect of DHM on liver and spleen injury induced by LPS in chicks: the 7-day-old chicks were divided into five groups,dose DHM(0.025%,0.05%,0.1%,0.2%)was added to the feed for 14 days.At the age of 43 days,the chicks in the model group and DHM added group were intraperitoneally injected with LPS(60 mg/kg),and the chicks in the control group were intraperitoneally injected with equivalent normal saline.Intraperitoneal injection after 12 hours,blood was collected for testing ALT and AST activity and LDH and DBIL content and the liver and spleen tissue were collectedthe for extraction of total RNA and protein,making paraffin section and ultrafine slices.The m RNA expression of TLR2,TLR4,My D88,NF-κB p65 and inflammatory factors(IL-1β,IL-6,IL-8,IL-10 and TNF-α)in the liver and spleen were detected with RT-PCR.Western blot was performed to determine the phosphorylation level of NF-κB p65 protein in liver and spleen tissues.The test results are as follows:(1)The effect of DHM on the growth performance of chicks: compared with the control group,0.05% DHM significantly increased the weight of chicks at 28,35 and 42 days(p<0.05 or p<0.01),increased the ADG of chicks at 7-21 days and decreased the F/G of chicks at 22-42 days.In the 0.05% DHM group,the level of TC and TG in serum of 35 days chicks were significantly higher than that of control group(p<0.05).The serum GLU content of different day-old chicks were significantly higher than that in control group(p<0.05 or p<0.01).The LDH levels in serum of 14,21 and 28 days chicks and the DBIL content in serum of 14 days chicks were lower than that in control group and the difference were significant(p<0.05 or p< 0.01).(2)The effect of DHM on the immune function of chickens: in the 0.05% DHM group,the spleen index,thymus index and bursa index of chickens at 14 days and the spleen index of chickens at 42 days were higher than those in the control group with significant differences(p<0.05 or p<0.01).Compared with the control group,0.025%,0.05% and 0.1% DHM increased the spleen index,thymus index and bursa index of 21 days chicks respectively with significant differences(p<0.05 or p<0.01).In the 0.025%,0.05% and 0.1% DHM group,the serum levels of IL-2,IFN-γ and Ig G in chicks at 14 days were significantly higher than those in the control group(p<0.01).In the 0.05% DHM group,the serum levels of IL-2,IFN-γ and Ig G in chicks at 21 days were significantly higher than those in the control group(p<0.01).The Newcastle disease antibody titers of 14,28 and 42 days chicks in the 0.05%,0.1% and 0.2% DHM group were significantly higher than those in the control group(p<0.05 or p<0.01).In the 0.025% and 0.05% DHM group,GSH-Px activity and A-AOC in the liver of 21 and 42-day-old chicks were significantly higher than those of the control group(p<0.05 or p<0.01),while 0.05% DHM increased SOD activity in the liver of 21 and 42-day-old chicks,which was significantly different from that of the control group(p<0.05).(3)RT-PCR results: compared to the control group,0.05% and 0.1% DHM promoted the m RNA expression of TLR4,IL-1β,IL-6,IL-8,IL-10 and inhibited the m RNA expression of TLR2,TNF-α in the liver(p<0.01).DHM(0.05% and 0.1%)can promote the m RNA expression of TLR2,TLR4,TNF-α and inhibit m RNA expression of IL-1β,IL-6,IL-8 and IL-10 in the spleen(p<0.01).(4)After intraperitoneal injection of LPS,the activity of AST,ALT and LDH as well as the content of DBIL and TG in chicks serum were significantly higher than those in the control group(p<0.05 or p<0.01).0.05% DHM significantly reduced the activities of LDH,AST and ALT,and the levels of DBIL and TG in the serum of chicks(p<0.01).(5)After intraperitoneal injection of LPS,compare with control group,TLR2 m RNA expressions was significantly increased in the liver and spleen(p<0.05 or p<0.01).Compare with control group,TLR4 m RNA expression was not significantly changed in the liver(p<0.01).The m RNA expression of My D88 and NF-κB p65 were sgnificantly lower in liver than that of control group and siginficantly higher in spleen than that of control group(p<0.01).Moreover,the phosphorylation level of NF-κB p65 was not significantly changed in the liver and decreased significantly in the spleen(p<0.01).Compared to control group,the m RNA expression of IL-1β and IL-8 was significantly increased in the liver and spleen(p<0.05 or p<0.01),while the m RNA expression of IL-6 was siginficantly decreased in the liver(p<0.01).The m RNA expression of IL-10 in the liver and spleen was sinificantly lower than that in the control group(p<0.01),while the m RNA expression of TNF-α in liver were significantly higher than that in the control group(p<0.01).There was no significant change in the phosphorylation level of NF-κB p65 in the liver,but it was significantly decreased in the spleen(p<0.01).Compared with model group,0.05% and 0.1% DHM significantly reduced the m RNA expression of TLR2,IL-1β,IL-8 and TNF-α,(p<0.01),and significantly increased the m RNA expression of TLR4,My D88、IL-6 and IL-10 in the liver(p<0.05 or p<0.01).DHM had no significant effect on the m RNA expression of NF-κB p65 and the phosphorylation level of NF-κB p65 protein in the liver.Meanwhile,0.025% DHM can inhibit TLR2 expression and promote IL-10 expression in the spleen with significantly difference(p<0.05 or p<0.01).0.05% DHM could significantly increase the expression of TLR4,IL-6 and IL-10 expression(p<0.01).0.1% DHM reduced the m RNA expression of My D88,IL-1β and IL-8 in the spleen with significantly difference(p<0.05 or p<0.01).In the group of DHM(0.05% and 0.1%),the m RNA of NF-κB p65 was significantly reduced(p<0.01),and the phosphorylation level of NF-κB p65 protein was significantly increased in spleen(p<0.01).(6)Morphological examination results showed that after intritonesl injection of LPS,hepatocytes were swollen,hepatic cords were disorganized with the inflmma tory cell infiltration,and the boundaries between white pulp and red pulp in the spleen tissue were blurred.The ultrastructural results showed a large number of vacuoles were formed in the endoplasmic reticulum of liver cells,while a small number of vacuoles were formed in the endoplasmic reticulum of spleen cells.Adding 0.05% and 0.1% DHM can alleviate the damage of liver and spleen cells of chicks.The results showed that adding DHM into the feed of helan white chicks could promote the weight gain of chicks,improve the biochemical indexes of serum,increase the content of antibodies and cytokines in serum,and regulate the m RNA expression of TLR2,TLR4 and inflammatory factors(IL-1β,IL-6,IL-8,IL-10 and TNF-α)in the liver and spleen of chicks.DHM can improve the degree of injury of chick liver and spleen by regulating the m RNA expression of TLR2,TLR4,My D88,NF-κB p65 and inflammatory factor,and the phosphorylation level of NF-κB p65 protein in the liver and spleen tissue.In conclusion,0.05% DHM can improve the growth performance and immune function of chicks,alleviate the liver and spleen damage caused by LPS.