Journal of Thermal Biology 84 (2019) 384–397 Contents lists available at ScienceDirect Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio Effect of curcumin supplementation on TLR4 mediated non-specific immune responses in liver of laying hens under high-temperature conditions T Aamir Nawab1, Guanghui Li1, Lilong An*, Jiang wu, Liuwen Chao, Mei Xiao*, Yi Zhao, Muhammad Waseem Birmani, Muhammad Waseem Ghani Department of Animal Science, Agricultural College, Guangdong Ocean University, Haida Road, Mazhang District, Zhanjiang, 524088, Guangdong, China ARTICLE INFO ABSTRACT Keywords: The liver performs a significant role in innate and adaptive immunity. Heat stress causes oxidative stress in liver Anti-inflammatory tissues and reduces the immune responses of laying hens which can cause several diseases affecting poultry- Antioxidant production performance. Hepatic inflammation is a common trigger of liver disease, which is reflected by he- Curcumin patic tissue damage leading to fibrogenesis and hepatocellular carcinoma. Dietary manipulation of curcumin has Heat stress been proposed to ameliorate the immune status of chickens under heat stress. Thus, this study aimed to in- Laying hen vestigate the effect of curcumin supplementation on TLR4 mediated non-specific immune response in liver of Oxidative damage laying hens under high-temperature conditions. Experimental groups contained two controls groups (high temperature and thermo-neutral control (HC and NC) fed basal diet) and three high-temperature curcumin treatments groups (HT100, HT200 and HT300). Laying hens in HC and HT groups exposed 6 h/day heat stress (32 ± 1 °C). The results of present study showed that heat stress curcumin treatment group had reduced in- flammatory responses (IL-6, IL-1β, TNF-α) as compared to HC and NC group. Pathological lesions and DNA damage of immune tissues were decreased in heat stress curcumin supplementation as compared to HC and NC group. Furthermore, PCNA, TLR4 and its downstream gene expression as well as protein expression (TLR4, NF- κB and PCNA) were significantly down regulated in heat stress curcumin supplemented group as compared to HC and NC group. Therefore, it is concluded that heat stressed hens supplemented with dietary curcumin enhance the immunity of laying hens and combat stressful environmental conditions. 1. Introduction circulation. A study has reported that almost 70–80% of the hepatic blood supply is delivered from the gut through the portal vein. This The liver is a key, frontline immune tissue which plays an important blood supply is rich with environmental antigens and harmless dietary function in both innate and adaptive immunity (Kubes and Jenne, toxin as well as molecules from the microbiota of the gut. The liver 2018). It harbors various types of resident immune cells and produces must tolerate this immunogenic load while still providing im- immune mediators as well as regulatory molecules (Robinson et al., munosurveillance for pathogenic infections (Robinson et al., 2016; 2016). It is responsible for the synthesis of cytokines, chemokines, Kubes and Jenne, 2018). complement components and acute phase proteins that involve in the High environmental temperature negatively affects the immune innate immunity (Robinson et al., 2016). The major role of the in- responses in the poultry birds. Thermal stress induces oxidative damage flammatory response is to combat tissue injury and infection. Liver is in liver tissues which, in turn, cause liver injury and tissue damage more sensitive to environmental stress, toxins, food antigens and bac- (Shini and Kaiser, 2009; Shini et al., 2010). A study has revealed that terial components (Gao, 2016). Innate immune cells identify danger broiler constantly exposure to high environmental temperatures had substance or damage cells through surface expressed pattern recogni- reduced the non-specific immunity Fathi et al. (2017). Heat stress dis- tion receptors (PRRs). Subsequently, activated PRRs initiate TLR4 sig- turbs the microecological balance of the intestinal microbiota and naling pathway that trigger the release of inflammatory response causes bacterial translocation which induces the production of in- (Bieghs and Trautwein, 2013). The liver is located at a specific place in testinal endotoxins (LPS) (Gu et al., 2012; Yu et al., 2012). These en- the body which acts as an important barrier between gut and systemic dotoxins reach the liver through circulation and activate the TLR4 * Corresponding authors. E-mail addresses: [email protected] (A. Nawab), [email protected] (L. An), [email protected] (M. Xiao). 1 These two authors have equal contribution in this paper. https://doi.org/10.1016/j.jtherbio.2019.07.003 Received 11 April 2019; Received in revised form 19 June 2019; Accepted 1 July 2019 Available online 17 July 2019 0306-4565/ © 2019 Elsevier Ltd. All rights reserved. A. Nawab, et al. Journal of Thermal Biology 84 (2019) 384–397 mediated response. TLR4 is highly expressed in both parenchymal and permanganate and formalin. Artificial light was provided for 18 h/day non-parenchymal liver cells (Guo and Friedman, 2010). Various authors with the intensity of 10–15 Lx until the end of experiment. Heaters were have reported that most of liver diseases are associated with TLR sig- settled in experimental chickens room to regulate the environmental naling pathway (Takeuchi and Akira, 2010; Qian et al., 2014; Guven- temperature according to the body requirement of chicks. Feed and Maiorov et al., 2015). Activation of TLR4 by LPS induces the initiation water were offered ad-libitum. of NF-kB and MAPK pathways to promote the overproduction of pro- inflammatory cytokines (Kagan and Medzhitov, 2006). Therefore, it is 2.3. Data collection necessary to reduce the inflammatory status by restraining the activa- tion of TLR4-mediated NF- kB pathway. The blood collection was performed from the brachial vein, and the From long time, antibiotics have been considered the first choice for samples were stored in microtubes and then centrifuged under re- liver inflammation. However, several antibiotics have banned due to frigeration (1,500×g, 5 min, 8 °C) to obtain the serum, which was their liver-carcinogenicity (Rahmani et al., 2017). Therefore, there is a stored at −80 °C until analysis. The serum inflammatory cytokines need to find out new therapeutic approaches to treat liver inflamma- concentration was determined using an ELISA kit from Nanjing tion. Recently, dietary curcumin has gained interest as an alternative Jiancheng Bioengineering Institute (Nanjing, China). The liver samples feed additive to treat inflammatory diseases including acute liver and were collected at 3rd, 6th and 9th weeks of experiments for in- lung injury, endometritis and mastitis (Lv et al., 2015; Fathi et al., flammatory cytokines analysis, liver tissues histology, q-RT-PCR genes 2017). Curcumin has potential anti-inflammatory and anti-oxidative analysis and western blotting. activity (Cleary and McFeeters, 2006; Singh et al., 2010; Zhang et al., 2014; Wang et al., 2015; Pulido-Moran et al., 2016; Amalraj et al., 2.4. Serum inflammatory cytokines analysis 2017; Hadisoewignyo et al., 2018). Therefore, the aim of this study was to explore the effects of dietary curcumin on TLR4 mediated non-spe- Blood extracted from the brachial vein was incubated at 37 °C for 30 cific immune responses in liver of laying hens under high temperature mints then centrifuged to obtain the serum. Inflammatory cytokines (IL- conditions for 9 weeks. 1β, TNF-α, and IL-6) were determined using chicken ELISA kits, ac- cording to the manufacturer's instructions (Nanjing Jiancheng 2. Material and methods Bioengineering Institute, Nanjing, China). All the samples and stan- dards along with a blank control were run in duplicate and the readings 2.1. Reagents and antibodies were taken at 450 nm using a microplate reader (Bio-Rad Co., Ltd. Shanghai, China). Curcumin was purchased from the Agricultural Vegetable Limited Company in Xi'an, China. Curcumin was composed of 77% curcumin, 2.5. Histological examination 18% demethoxycurcumin, and 5% bisdemethoxycurcumin (Rahmani et al., 2017). The purity of curcumin used in this study was 95%. Liver samples were immediately collected from the birds for mor- Curcumin was first added to a small amount of basal diet and then phological and molecular studies. Liver tissues were excised and fixed thoroughly mixed with 100 kg feed. ELISA kits of IL-1β, TNF-α, and IL-6 in 4% paraformaldehyde solution in PBS, dehydrated and then em- was obtained from Nanjing Jiancheng Bioengineering Institute bedded in paraffin for morphological analysis. After that, 4-μm tissue (Nanjing, China). Rabbit anti-TLR4 (toll like receptor) polyclonal an- sections were cut using a Leica microtome (Nussloch Gmbh, Germany) tibody (1:400) was purchased from Boster Biological Technology, and mounted on polylysine-coated slides (Boster Corporation, China). Pleasanton CA, USA, and Catalog # PA1484. Mouse anti-PCNA (Pro- The rest of fresh liver samples were frozen in liquid nitrogen and then liferating cell nuclear antigen) monoclonal antibody (1:200) (Catalog # stored at −80 °C for q-RT-PCR and western blotting. Hematoxylin and ab29) and rabbit anti-NF-κB (nuclear factor kappa B cells) polyclonal eosin (HE) staining was performed by routinely used protocol for liver antibody (Catalog # ab16502) was obtained from abcam.com, China. histopathological examination (Kim et al., 2016). Stained tissue sec- Mouse anti-β-actin monoclonal antibody (1:1000) (Catalog