Study Of The Regulatory Effect Of Ampelopsin On Acute Phase Response Of Weaned Piglets

This thesis aims to study the effects of diet supplemented with Ampelopsin (APS) on substance metabolism, the function of liver mitochondria, the redox status, and the acute phase protein in the acute phase reaction of weaned piglets. Three experiments were conducted to examine the effects of APS on inflammatory cytokines, acute reactive protein, and oxidative stress in acute phase reaction induced by LPS.1.The antioxidant capacity of Ampelopsin in vitro. The main purpose of this experiment is to study the APS antioxidant in vitro and protective effects on erythrocyte oxidative stress induced by AAPH. Firstly evaluated the effects of APS as an antioxidant by using several antioxidants assays in vitro, including scavenging radical activity on DPPH, ABTS, hydrogen peroxide, and superoxide anion radical, in addition the Ferric-reducing antioxidant power (FRAP) assay. Secondly, we evaluated the antioxidative effects of APS in the oxidative stress of pig erythrocytes, induced by 2,2’-azobis-2-amidinopropane dihydrochloride (AAPH), to test the markers of oxidative stress, including hemolysis, lipid peroxidation, total superoxide dismutase enzyme activity (T-SOD), and phosphatidylserine exposure on the cell surface, to investigate the protective effect of APS on early cellular apoptotic responses against oxidative stress. The results showed APS exhibited excellent ability to free radical scavenging by DPPH, ABTS,O2·, H2O2 and ferric reducing antioxidant power. APS also protected pig erythrocytes against AAPH-induced oxidative hemolysis, decreased total superoxide dismutase activity and lipid peroxidation. The oxidant mediated phosphatidylserine exposure was in a concentration and time dependent manner in pig erythrocytes.2.A total of 36 (27 ± 1) d old piglets (Duroc × Landrace × Yorkshire) werer andomly alloeated to 3 treatment groups and were fed with one of three diets, basal dicts (control) or diet ssupplemented with APS 100 mg/kg and 400 mg/kg, for 28 d. The results showed that there was no effect of dietary APS on feed intake(F1), body weight gain (BWG), but APS significantly decreased the feed to gain ratio (F/G)(P<0.05).3.The effects of APS on the biochemical parameters, the levels of hormone and cytokines in blood. The experiment were conducted to examine the effects of APS on cytokines, sensitive enzymes of liver function, the changes of glucose, lipids, proteins and the related hormone in acute phase reaction induced by LPS. Total of 24 piglets (Duroc × Landrace x Yorkshire) were about 27 ± 1d old, weight 7.90 ± 0.43kg, male and female in half. According to single factor experiment design was divided into 4 groups, the control group (injection of saline), the acute phase of stress treatment (intraperitoneal injection of LPS,25 μg/kg·BW), APS dosage (100 and 400 mg/kg)+LPS treatment, for 28d. The results show that:the blood inflammatory cytokines IL-1β, IL-6 and TNF-α in piglet of LPS group were significantly increased (P<0.05), the piglets of APS group significantly decreased in inflammation of blood factor (P<0.05) compared with LPS. The Blood Glu, Leptin, and PGE2 in LPS-induced piglets were significantly increased (P<0.05); the Insulin and IGF-1 in LPS-induced piglets were significantly decreased (P<0.05); APS significantly alleviated the acute phase response caused these hormonal changes in the concentration (P<0.05).The blood glucose, ALB concentration decreased (P>0.05) in LPS-induced piglets; NEFA and TG was significantly higher (P<0.05) in LPS-induced piglets; TP, TC and BUN were no significant changed (P>0.05) in LPS-induced piglets. APS significantly reduced the NEFA concentration induced by LPS (P<0.05); ALT, AST, ALP and LDH were significantly higher (P<0.05) in LPS-induced piglets than in control groups; APS could significantly reduce the ALT, AST, ALP and LDH secretion induced by LPS.4.The effects of APS on inflammatory response in liver of piglet.(1)Inflammatory cytokines. The levels of TLR2 and TLR4 gene expression were significantly higher (P<0.05) in liver of LPS group compared with the saline group, and the IL-1β, IL-6, TNF-α and COX-2 mRNA expression was increased (P<0.05) induced by LPS. APS reduced the level of IL-1β, IL-6, TNF-α and COX-2 mRNA expression in liver of piglet (P<0.05) compared with LPS group. The p-Akt protein was significantly increased stimulated by LPS (P<0.05), APS had a decreasing trend of p-Akt expression, but no significant difference with the LPS group and control group (P>0.05). The NF-κB binding DNA activity in liver was significantly increased induced by LPS (P<0.05). APS in diets significantly reduced the NF-kB binding DNA activity (P<0.05) compared to the LPS group, and there was no significant difference between 400 mg/kg APS group and the control group.(2)Acute phase response proteins. The concentrations of IL-1β, IL-6, and TNF-a were significantly increased (P<0.05) in liver induced by LPS; the concentrations of IL-1β, IL-6, and TNF-α IL-1β, IL-6 in APS group were lower than LPS group (P<0.05). The concentrations of Cort were significantly increased by LPS (P<0.05) and GR mRNA expression level was significantly lower in LPS group (P<0.05); APS had significantly reduced concentrations of Cort and increased the levels hepatic GR mRNA expression, and GR mRNA expression levels in 400 mg/kg APS group were significantly higher than control group (P<0.05). The expression of p-STAT3 protein were significantly increased in liver (P<0.05) after LPS stimulation; the APS in diets significantly reduced the expression of p-STAT3 protein (P<0.05) compared to the LPS group. The levels of CRP and SAA2 mRNA was significantly higher than the control (P<0.05), and 400 mg/kg APS had reduce CRP and SAA2 mRNA expression compared to LPS group (P<0.05). There was no significantly changed of AGP mRNA expression between the control and LPS group (P> 0.05); APS increased AGP mRNA expression compared with LPS group, and 400 mg/kg APS group had significantly increase AGP mRNA expression compared to control and LPS groups (P<0.05). There were no significant changes in LPS and APS group about AAT, CC3 and CP mRNA level (P>0.05).5.The antioxidant functions of APS in liver of piglets induced by LPS. Firstly, the Nrf2 protein expression of antioxidant enzymes, oxidative stress-related gene Ref-1 and its downstream gene mRNA expression levels of major acute phase were determined in liver of piglets; Secondly, the mitochondrial antioxidant enzymes, TCA cycle enzymes, membrane function and ATP content were measured in terms of research on the protective effect of APS in LPS-induced oxidative damage. The results showed that:(1)The protective effect of APS in LPS-induced oxidative stress piglets. The antioxidant enzymes of GSH-Px, T-SOD and CAT were decreased significantly (P<0.05) After LPS stimulation. The APS in 400 mg/kg group significantly increased the T-SOD and GSH-Px activity (P<0.05) compared with the LPS group. The MDA and protein carbonyl content in LPS group increased significantly (P<0.05) compared with the control group; the content of MDA in 100 and 400mg/kg APS group was lower than LPS group about 15.5% (P>0.05)and 31.7%(P<0.05). There was no significant difference (P>0.05) about protein carbonyl content in APS groups, LPS group and the control group. The expression of Ref-1 and p-Nrf2 protein increased significantly after LPS induced (P<0.05),400mg/kg APS group significantly relieved the expression of p-Nrf2 (P<0.05). LPS had significantly increased HO-1, GST, PRX3 and MCP-1 mRNA, and 400 mg/kg APS significantly decreased the PRX3, MCP-1 mRNA (P<0.05) compared with the LPS group.(2)The protective effect of APS on oxidative damage in mitochondria of liver. The mitochondrial antioxidant enzymes of Mn-SOD, GSH-Px and GRe activity were significantly decreased (P<0.05) in LPS group, and APS had significantly alleviated the decrease in antioxidant enzymes (P<0.05). The MDA and GSSG (P<0.05) were increased significantly by LPS stimulated, and the GSH and GSH/GSSG were decreased (P<0.05) by LPS stimulated. The MDA and GSSG content in the400 mg/kg APS group were decreased respectively by 11.67%(P<0.05) and 36.36%(P<0.05). There was no significant (P>0.05) difference between the LPS group and the APS groups about the GSH content. The mtNOS activity and NO content were increased significantly (P<0.05) in the mitochondria after LPS induced. APS significantly alleviated LPS- induced increase in mtNOS activity (P<0.05) and with a decrease trend in NO concentration (P>0.05). The mitochondrial three carboxylic acid cycle enzyme of SDH and MDH activity decreased significantly (P<0.05) after LPS induced compared to control group, and APS alleviated in SDH and MDH activity compared to the LPS group (P<0.05). LPS significantly reduced ATP concentration in liver mitochondrial compared with the saline group, and APS supplementation inhibited the ATP decreasing trend, but there was no significant difference (P>0.05). LPS stimulation caused a decrease in mitochondrial membrane potential (P<0.05), and the 400 mg/kg APS group could significantly (P<0.05) increase the mitochondrial membrane potential, and there was no significant difference with the normal group (P>0.05). The Ca2+-ATP activity of liver mitochondria significantly reduced in piglets (P<0.05) after LPS stimulation, and the addition of APS can inhibit Ca2+-ATP enzyme activity in decreasing, but there was no significant (P>0.05).As stated above, the conclusions are as follows:(DAPS has strong antioxidant activity in vitro. (2)The supplementation of APS reduced Akt expression, then decrease the transcription factor NF-κB binding DNA activity, with decreasing the expression of inflammatory factors, to avoid inflammatory factor/proinflammatory imbalances; on the other hand, APS had inhibited of Cort and increased GR, then the expression of p-STAT3 was reduced, in order to alleviate the acute phase of LPS. (3)APS protected antioxidant enzyme activity, then improving the cellular redox balance. The ROS signal was weaken, to avoid excessive activation of APE/Ref-1 expression and transcription factor was in the active reduced state, thereby reducing the acute phase response from the oxidation pathway. (4)The supplemented with APS could modulate inflammatory factor and euroendocrine function, alleviating animal inflammatory response. (5)APS showed the mitochondrial protection function in the acute response of piglet, reducing the production of ROS from mitochondria, so oxidative stress in mitochondrial was limited, thereby alleviating the acute phase response.