The mixture of Radix isatidis, Forsythiae, and Gypsum alleviates lipopolysaccharide-induced fever in broilers by inhibition of TLR4/NF-κB signaling pathway

Abstract

To determine whether the antipyretic effect of the mixture of Radix isatidis, Forsythiae, and Gypsum (RIFG) on lipopolysaccharide (LPS) induced fever broilers and its related mechanisms. A total of 315 24-day-old yellow-plumed broilers were randomly divided into 7 groups, except for the control group, other groups were injected with LPS. Two hours later, RIFG were given drinking water to relieve fever, and it was evaluated by the expression of genes and proteins of the maximum body temperature rise (∆T), body temperature response index (TRI), serum and hypothalamic pyrogenic heat factor. RIFG could reduce the body temperature of broilers with fever (P < 0.01). It inhibited the expressions of IL-6 and PGE2 (P < 0.01), down-regulated mRNA expression levels of TNF-ɑ and COX-2 (P < 0.01), and promoted the generation of antipyretic factor AVP mRNA (P < 0.01). In addition, the expression level of TLR4 and NF-κB p65 protein can be down-regulated, and LPS + RM group has the best down-regulated effect. RIFG had a good antipyretic effect on reducing LPS-induced fever of broilers by inhibiting the activation of TLR4/NF-κB signaling pathway and thermogenic factors.

INTRODUCTION

With the rapid development of large-scale poultry industry, antipyretic drugs have a greater application prospect in the clinical treatment of mass fever, and the research and development requirements are developing towards the trend of high efficacy and less adverse reactions. As one of the common clinical symptoms, fever is manifested as a body temperature higher than the normal physiological range. It is the immediate response of the immune system to infection, injury or tissue destruction, and has the dual functions of defense and damage (Blomqvist and Engblom, 2018). Its fever mechanism is very complex. At present, pyrogen can be divided into exogenous and endogenous heat sources according to the location of generation. Exogenous heat source is essentially the product of some or the whole microorganism or microorganism (Ogoina, 2011). Established and verified animal models of fever include lipopolysaccharide (LPS; Evans et al., 2015; Pakai et al., 2018; Zhang et al., 2019), yeast (Guo et al., 2014; Ye et al., 2020), 2, 4-dinitrophenol (Okokon et al., 2016), etc.

Previous studies have shown that LPS is an effective pyrogen. After LPS enters the body, our immune system senses LPS through Toll-like receptors TLR4 (Mazgaeen and Gurung, 2020), and it can induce monocytes, macrophages, lymphocytes, etc. to produce pyrogen cytokines by activating the NF-κB signaling pathway, including interleukin-6 (IL-6) (Roth and De Souza, 2001), interleukin-1 (IL-1), tumor necrosis factor (TNF) (Ma et al., 2021), etc. Pyrogenic cytokines can induce the production of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) (Rummel et al., 2006), and cause a febrile response (Roth and De Souza, 2001).

The mixture of Radix isatidis, Forsythia and Gypsum (RIFG) is a kind of antipyretic preparation of traditional Chinese medicine. It is composed of Radix isatidis, Gypsum, Forsythia, Anemarrhena asphodeloides Bunge, and Pogostemon cablin, which can treat chicken fever. Previous studies have shown that Radix isatidis has antiviral, anti-inflammatory and antipyretic effects (Deng et al., 2021). Gypsum is often used for external fever, can clear heat and fire, eliminate boredom and thirst (Yang et al., 2016). Forsythia clearing heat and detoxifying, reducing swelling and dispersing knots, it is often used to treat diseases such as wind heat and cold, it has antiviral and antipyretic effect (Guo et al., 2022). Anemarrhena asphodeloides Bunge has anti-inflammatory, antioxidant, antipyretic and other pharmacological effects (Zhao et al., 2015). Pogostemon cablin strengthens the stomach, relieves fever and relieves pain (Zhou et al., 2014; Kang et al., 2023). The main purpose of this study was to study the antipyretic effect of RIFG on LPS induced fever of broilers, and to explore its antipyretic mechanism, so as to provide more options for clinical antipyretic drugs.

MATERIALS AND METHODS

Plant Material, Chemicals, and Regents

RIFG was prepared from Radix isatidis, Gypsum, Forsythia, Anemarrhena asphodeloides Bunge and Pogostemon cablin in a certain proportion into a traditional Chinese medicine antipyretic mixture, and each 1 mL was equivalent to 1.46 g of raw drugs. Shuanghuanglian Oral Liquid was purchased from Sichuan Tongda Animal Health Technology Co., Ltd (Sichuan, China). Carbaprim Calcium was purchased from Shandong Lukang Shilile Pharmaceutical Co., Ltd (Shandong, China). LPS (O55:B5) was purchased from Sigma-Aldrich Chemical Inc, (MI, USA). Chicken interleukin 6 (IL-6) and chicken prostaglandin E2 (PGE2) ELISA kits were purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd (Shanghai, China). Hiscript Ⅲ RT SuperMix for qPCR (+gDNA wiper) and ChamQ Universal SYBR qPCR Master Mix were purchased from Nanjing Vazyme Biotech Co., Ltd (Nanjing, China). The Enhanced BCA Protein Assay Kit was acquired from Beyotime Biotechnology Co., Ltd (Shanghai, China).

Experimental Animals

A total of 315 healthy 24-day-old yellow-feathered broilers was provided by Enping Kilong Industrial Co., Ltd. (Jiangmen, China). The broiler breed is “Fengshan chicken,” which was introduced from Wenchang chicken in Hainan province, China, and became an independent breed after repeated breeding and improvement. Enping Kilong Industrial Co., Ltd., as a clinical practice cooperation base for experimental animals of South China Agricultural University, meets the basic requirements for Good clinical practice operations.

Before the experiment, the basal body temperature of broilers was measured twice a day at a fixed time. The broilers were reared in Jilong Fengshan Chicken Farm, Jiangmen City, Guangdong Province, with the room temperature of 24∼26°C and relative humidity of 50∼60%. The broilers were fed and drank freely. Feed the basic diet with the formula shown in Table 1.

Table 1.

Main raw materials and additives.

Component	g/kg	Additive	g/kg
Second corn	436.3	Methionine	1.5
Second soybean meal	284	Lysine	1.25
Corn gluten meal	9.7	WM13.5%	7.68
Red Wheat 3090 (Jiangsu,China;Zhejiang,China)	200	438 vitamin premix for poultry	0.36
Fish meal	15.5	Vitamin C	0.1
Oil	6.5	Minerals premix for poultry	1.3
Limestone	10.5	Sodium bicarbonate	1.5
Gypsum powder	5	Fralac34Dry1	0.5
Calcium hydrogen phosphate (osteogenic)	12.5	Salt	2.35
		Choline chloride	1.3
		Bosar Se2	0.05
		Compound enzyme 8411	0.25
		BASF phytase	0.15
		Lipase	0.15
		Xylanase	0.06
		Threonine	0.5

¹Fralac34Dry:Composed of α-glycerol propionate, α-monobutyric acid glyceride, Glyceryl dipropionate butyrate, lactic acid (Bioscwin (Shanghai) Animal Pharmaceutical Co., Ltd.)

²Bosar Se:A selenium-containing feed additive (Yunnan Bosar Biology Group, China).

Experimental Design

A total of 315 broilers with stable body temperature and 2 anal temperature fluctuations less than 0.5°C were selected and randomly divided into 7 groups: Control group (CON), LPS model group (LPS), LPS+Shuanghuanglian oral liquid group (LPS + SHL), LPS + Carbaprim Calcium group (LPS + CBC), LPS + RIFG mixture low dose group (LPS + RL), LPS + RIFG mixture medium dose group (LPS + RM), LPS + RIFG mixture high dose group (LPS + RH).

Control group broilers were intraperitoneally injected 0.9% sodium chloride injection. The other groups were injected with LPS solution composed of 0.9% sodium chloride injection at the weight of 35 μg/100 g to establish fever model. Clinical symptoms were observed after modeling, including changes in diet, drinking water, feces, respiration, mental state, feather limbs, etc. After 2 h of LPS administration, each group of broilers were given water for antipyretic treatment, while broilers in control group and LPS group were given equal volume of distilled water. The specific dosage was shown in Table 2. Body temperature was monitored from 30 min after modeling and continuously monitored for 10 h to calculate ∆T and TRI.

Table 2.

Test grouping and treatment.

Group	LPS induced fever	After injection LPS of 2 h
CON	i.p 0.9% NaCl, 3.5 mL/100 g	Normal drinking water
LPS	i.p LPS, 35μg/100 g	Normal drinking water
LPS + SHL	i.p LPS, 35μg/100 g	Drinking water with 0.75 mL/kg SHL
LPS + CBC	i.p LPS, 35μg/100 g	Drinking water with 120 mg/kg CBC
LPS + RL	i.p LPS, 35μg/100 g	Drinking water with 0.3 mL/kg RIGF
LPS + RM	i.p LPS, 35μg/100 g	Drinking water with 0.6 mL/kg RIGF
LPS + RH	i.p LPS, 35μg/100 g	Drinking water with 1.2 mL/kg RIGF

Sample Collection

After 6 h of LPS injection, cervical venous blood was collected and serum was extracted by centrifugation. After euthanasia, the whole brain was removed quickly on ice. Hypothalamic tissue was frozen in liquid nitrogen and transferred to a -80°C refrigerator for preservation.

ELISA Test for IL-6, PGE2 in Serum

According to the manufacturer's instructions, the content of IL-6 and PGE2 in the serum was detected by ELISA kit.

Hypothalamic RNA Extraction and Fluorescence Quantitative Real-Time PCR Analysis

mRNA levels of IL-6, COX-2 and AVP in hypothalamus were detected. The total RNA was extracted with a total RNA extraction reagent (Vazyme Biotech Co., Ltd, Nanjing, China). The total mRNA was reverse transcribed with HiScript III qRT SuperMix (Vazyme Biotech Co., Ltd, Nanjing, China) and used for qPCR by the use of ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd, Nanjing, China). The qPCR data were acquired with the Roche LightCyler 480 machine (Roche) and the relative quantification was performed with QuantStudio Design and Analysis Software. Transcript levels were relative quantified by the 2^−ΔΔCT method. All molecule expression was normalized against gene expression of specified housekeeping genes, namely β-actin. The primer sequences of target genes and housekeeping gene are shown in Table 3.

Table 3.

Gene-specific primers sequences for quantitative real-time PCR.

Gene	GenBank accession number	Primers sequence (5′–3′)
β-actin-F	NM_205518.2	ATGGCTCCGGTA TGTGCAAG
β-actin-R	NM_205518.2	CAACCATCACACC CTGATGTC
IL-6-F	NM_204628.2	AAATCCCTCCTCG CCAATCT
IL-6-R	NM_204628.2	CCCTCACGGTCTTC TCCATAAA
COX2-F	YP_010037843.1	TGTCCTTTCACTGC TTTCCAT
COX2-R	YP_010037843.1	TTCCATTGCTGTGT TTGAGGT
AVP-F	NM_205185.3	GGCAGTGAGCAGGC AGAAGAG
AVP-R	NM_205185.3	GCATCAGCCGCAGC AGTAGG

Western Blot Analysis for TLR4 and NF-κB

The hypothalamic tissue of broilers was analyzed by standard Western blotting. In short, the protein lysate is added for tissue homogenization and the protein concentration is determined. The protein lysates were separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% nonfat dry milk for 2 h, and incubated overnight at 4°C with the primary antibodies against nuclear factor kappa B p65 (NF-κB p65, 1:2000, abcam, English), TLR4 (1:1000, Hangzhou Jingjie Biotechnology Co., Ltd., Hangzhou, China), GAPDH (1:5000, Proteintech Group., Inc, IL, USA) Subsequently, the membranes were washed 3 times and incubated with the corresponding secondary antibodies (goat anti-rabbit IgG HRP or rabbit anti-mouse IgG HRP, Beyotime Biotechnology Co., Ltd., Shanghai, China) for 1 h. Finally, the bands were examined with luminescent solution. The protein expression level was analyzed by Image J software.

Statistical Analysis

All data obtained in this study were processed statistically and divergence were presented as means ± SD. One-way ANOVA was used to analyze the differences among groups for multi-group data comparison. And the least significant difference method (LSD) was used for multiple comparisons and Duncan's new repolarization difference test. P < 0.05 indicated that difference was statistically significant. Graphpad Prism 9.3 software was used for drawing.

Maximum temperature rise ∆T=fever temperature−basal body temperature. Temperature response curve was plotted with time as the abscess and temperature change ∆T as the ordinate. The area between the body temperature response curve and baseline 1∼10 h after injection of LPS was calculated according to the ordinate coordinate of 0.2°C corresponding to 5 cm and the abscissa coordinate of 1 h corresponding to 1 cm. A total of 9 trapezoid areas were calculated. The lower the value, the better the antipyretic effect.

RESULTS

Establishment of LPS Induced Broiler Fever Model

Body temperature of broilers after LPS injection is shown in Figure 1. The results showed that there was no significant difference in body temperature between the LPS group and the CON group before LPS injection (0 h). After injecting LPS for 2 h, the body temperature of LPS group broilers increased to 42.9°C, which was significantly different from that of healthy broilers (P < 0.01), and could maintain a high temperature for 6 h. At the same time, the broilers in the model group showed decreased appetite, drowsiness, lethargy, crouching, diarrhea, shortness of breath and other symptoms, indicating that the modeling was successful. Body temperature of broilers in LPS group decreased gradually at 6∼7 h after injection, but there was no significant difference between LPS group and CON group (P > 0.05).

Figure 1.

Changes of body temperature in broiler fever models induced by LPS. *P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Effects of Intraperitoneal Injection of LPS on Body Temperature of Broilers

The temperature changes of broilers at different times after LPS injection were shown in Table 4 and Figure 2, and the temperature response index of each group was shown in Table 5. After injecting LPS for 2 h, compared with the CON group, the body temperature of the LPS injection group was significantly increased (P < 0.01) to more than 42.6°C, and the TRI_1-2h index was significantly increased (P < 0.01). Compared with LPS group, the body temperature response index was significantly decreased after administration (P < 0.01). After 30 min of antipyretic treatment, the body temperature of LPS + RL, LPS + RM and LPS + RH groups began to decrease significantly (P < 0.01), and the antipyretic effect of LPS + RL and LPS + RH group was similar to that of LPS + CBC group. After 2 h of administration, the body temperature of LPS + RL and LPS + RM groups decreased to below 42°C, which was lower than that of LPS + SHL group but higher than that of LPS + CBC group (P < 0.05), and the change trend of TRI_3-4h was consistent with that of body temperature. After 4 h of antipyretic treatment, there was no significant difference in body temperature between LPS + RM group and CON group (P > 0.05). The body temperature in LPS + CBC, LPS + RL, and LPS + RH groups were repeated and then increased. These results indicated that RIFG mixture could reduce the body temperature of LPS febrile broilers and had a good antipyretic effect. LPS + RL and LPS + RM groups could exert the cooling effect faster, and the antipyretic effect of LPS + RM group was more stable and better.

Table 4.

Effects of different time on body temperature of broilers in each group.

Time	CON	LPS	LPS + SHL	LPS + CBC	LPS + RL	LPS + RM	LPS + RH
0 h	41.40 ± 0.21	41.39 ± 0.09	41.33 ± 0.15	41.33 ± 0.11	41.34 ± 0.17	41.39 ± 0.12	41.46 ± 0.21
2 h	41.36 ± 0.11	42.91 ± 0.18##	42.87 ± 0.28##	42.82 ± 0.20##	42.72 ± 0.29##	42.80 ± 0.35##	42.67 ± 0.29##
2.5 h	41.30 ± 0.09	42.94 ± 0.17##	42.73 ± 0.26##	42.11 ± 0.35##⁎⁎	42.10 ± 0.12##⁎⁎	42.44 ± 0.34##⁎⁎	42.13 ± 0.33##⁎⁎
3 h	41.42 ± 0.14	42.96 ± 0.13##	42.06 ± 0.31##⁎⁎	41.68 ± 0.25#⁎⁎	42.07 ± 0.16##⁎⁎	42.12 ± 0.27##⁎⁎	42.20 ± 0.14##⁎⁎
4 h	41.49 ± 0.20	42.63 ± 0.28##	42.19 ± 0.28##⁎⁎	41.66 ± 0.25⁎⁎	41.91 ± 0.16##⁎⁎	41.90 ± 0.37##⁎⁎	42.74 ± 0.27##⁎⁎
5 h	41.40 ± 0.32	42.21 ± 0.18##	41.68 ± 0.10⁎⁎	42.07 ± 0.47##	41.68 ± 0.23⁎⁎	41.82 ± 0.44##⁎⁎	41.88 ± 0.22##⁎
6 h	41.52 ± 0.17	42.24 ± 0.13##	41.79 ± 0.38#⁎⁎	41.56 ± 0.24⁎⁎	41.96 ± 0.29##⁎	41.47 ± 0.29⁎⁎	42.11 ± 0.28##
7 h	41.47 ± 0.21	41.44 ± 0.44	41.76 ± 0.29#⁎	41.61 ± 0.29	42.03 ± 0.17##⁎⁎	41.43 ± 0.33	41.82 ± 0.27#⁎⁎

^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, ^⁎⁎P < 0.01 vs. LPS group; Is the time after injection of LPS, 0 h: before injection of LPS, 2 h: after injection of LPS, and so on.

Figure 2.

Temperature response curve of broilers in each group. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Table 5.

Changes of TRI of broilers in different time points.

	CON	LPS	LPS + SHL	LPS + CBC	LPS + RL	LPS + RM	LPS + RH
TRI1-2h	1.86 ± 0.50	6.36 ± 0.52##	6.04 ± 0.60##	6.36 ± 1.67##	6.18 ± 1.50##	6.54 ± 0.47##	5.86 ± 1.79##
TRI2-3h	1.94 ± 0.39	9.67 ± 0.52##	7.31 ± 0.87##⁎⁎	6.39 ± 0.80##⁎⁎	6.97 ± 0.93##⁎⁎	7.31 ± 1.15##⁎⁎	7.36 ± 0.76##⁎⁎
TRI3-4h	2.07 ± 0.51	9.21 ± 0.65##	5.75 ± 0.75##⁎⁎	3.61 ± 0.81##⁎⁎	4.89 ± 0.32##⁎⁎	4.57 ± 0.93##⁎⁎	5.68 ± 0.76##⁎⁎
TRI4-5h	2.22 ± 1.08	7.11 ± 0.95##	4.67 ± 0.87##⁎⁎	4.31 ± 1.74##⁎⁎	3.97 ± 0.78##⁎⁎	4.31 ± 1.41##⁎⁎	4.89 ± 0.77##⁎⁎
TRI5-6h	2.07 ± 0.87	6.25 ± 0.46##	3.71 ± 0.81##⁎⁎	4.14 ± 1.14##⁎⁎	3.71 ± 0.93##⁎⁎	3.32 ± 1.32#⁎⁎	4.82 ± 0.49##⁎⁎
TRI6-7h	2.29 ± 0.64	4.54 ± 0.96##	3.96 ± 1.28##	3.04 ± 0.87⁎⁎	4.61 ± 0.52##	2.29 ± 0.80⁎⁎	4.46 ± 0.60##
TRI7-8h	2.50 ± 0.40	2.65 ± 1.46	2.55 ± 0.67	2.80 ± 0.54	3.85 ± 0.52#⁎	2.30 ± 1.02	3.65 ± 0.42#
TRI8-9h	2.55 ± 0.21	2.30 ± 0.72	2.05 ± 0.87	2.40 ± 0.49	2.80 ± 0.62	2.40 ± 0.91	3.40 ± 0.29#⁎
TRI9-10h	2.00 ± 0.35	2.06 ± 0.80	2.13 ± 0.92	1.94 ± 0.47	3.06 ± 0.24	2.00 ± 1.19	2.88 ± 0.32

^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Effects of RIFG on the Expression Levels of Pyrogen Cytokines in Serum

The results are shown in Figure 3. Compared with CON group, serum IL-6 (P < 0.01) and PGE2 (P < 0.05) contents in LPS group were increased, indicating that LPS induced fever of broilers was closely related to IL-6 and PGE2 contents. After administration, IL-6 content in LPS + RL, LPS + RM, and LPS + RH groups were significantly decreased compared with LPS group (P < 0.01), and no significant difference was found between CON, LPS + SHL, and LPS + CBC groups. PGE2 levels in all administration groups were decreased, LPS + RH group had a significant difference compared with LPS group (P < 0.01), and PGE2 levels in LPS + SHL and LPS + CBC groups were lower than those in LPS + RL, LPS + RM groups compared with LPS group had a decreasing trend. The results showed that RIFG mixture could inhibit the expression of IL-6 and PGE2 in serum induced by LPS.

Figure 3.

Levels of serum IL-6 and PGE2 in each group. ^#P < 0.05, ^##P < 0.01 vs. control group. *P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Effects of RIFG on the Expression Levels of Fever and Antipyretic Genes in Hypothalamus

The results are shown in Figure 4. Compared with CON, mRNA expression levels of TNF-ɑ, COX-2 and AVP in LPS group were significantly increased (P < 0.01), indicating that LPS-induced fever of broilers was closely related to the contents of these 3 factors. Compared with LPS group, mRNA expression levels of TNF-ɑ and COX-2 in all drug administration groups declined, and LPS + RM and LPS + RH groups had significant differences in TNF-ɑ expression compared with LPS group (P < 0.01), and their levels were lower than those of LPA + CBC group (P < 0.05) and LPS+SHL group. The mRNA expression level of COX-2 in LPS + RL group was significantly different from that in LPS + SHL group (P < 0.05). Compared with LPS group, the AVP mRNA expression level of all administration groups was increased, LPS + RL, LPS + RM, LPS + RH group and LPS group were significantly different (P < 0.01), and the AVP mRNA expression level of LPS + RM group was the highest, but the difference was significant (P < 0.01). The results showed that RIFG mixture could inhibit the expression of thermogenic factor TNF-ɑ and COX-2 in hypothalamus of LPS-induced broilers, and promote the generation of AVP mRNA.

Figure 4.

mRNA expression levels of TNF-ɑ, COX-2 and AVP in each group. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, ^⁎⁎P < 0.01 vs. LPS group.

Effects of RIFG on TLR4/NF-κB Gene and Protein Expression Levels in Hypothalamus

As shown in Figure 5A, compared with the CON group, the mRNA expression levels of TLR4 (P < 0.01) and NF-κB (P < 0.05) in LPS group were significantly increased, indicating that LPS-induced fever model of broilers was closely related to the mRNA contents of TLR4 and NF-κB. Compared with LPS group, TLR4 mRNA levels in LPS + RL, LPS + RM, and LPS + RH groups were significantly decreased (P < 0.01), and the inhibition effect of LPS + RM group was better than that of LPS + SHL and LPS + CBC groups (P > 0.05). The mRNA expression of NF-κB in LPS + RL group was significantly decreased (P < 0.05), LPS + RM and LPS + RH groups had a downward trend, and there was no significant difference compared with LPS + CBC (P > 0.05). The results showed that RIFG can inhibit the expression of TLR4 /NF-κB mRNA in chicken hypothalamus induced by LPS. As shown in Figure 5B, LPS stimulation significantly increased the expression of TLR4/NF-κB protein (P < 0.05), LPS + RL, LPS + RM, and LPS + RH groups inhibited the expression of TLR4/NF-κB protein, and LPS + RM group inhibited the expression of TLR4/NF-κB protein (P < 0.01).

Figure 5.

RIFG inhibited the expression level of TLR4/NF-κB gene and protein in hypothalamus. ^#P < 0.05, ^##P < 0.01 vs. control group; *P < 0.05, **P < 0.01 vs. LPS group.(A) mRNA expression levels of TLR4/NF-KB in hypothalamus. (B) Western blot analysis. TLR4/NF-κB protein expression level in hypothalamus, relative protein level normalized to GAPDH.

DISCUSSION

Fever caused by various diseases is a common symptom. If not treated in time, the loss will be serious, especially the impact on chicks, resulting in increased mortality (Hamissou Maman et al., 2019). Animal models of LPS-induced fever are often used to screen antipyretic drugs and explore the mechanism of inflammatory fever (Roth and Blatteis, 2014; Yuan et al., 2017; Mazgaeen and Gurung, 2020). A study has been conducted to evaluate the antipyretic effect of puerarin by intraderitoneal injection of LPS (100 μg/kg) in rats induced by LPS (Yao et al., 2012). In this study, the fever model of broiler was established with LPS. Compared with CON group, the body temperature of broilers increased significantly at 2 h after LPS injection (P < 0.01), which was consistent with previous studies (Li et al., 2021). At the same time, the broilers showed decreased appetite, lethargy, crouching, diarrhea, shortness of breath and other symptoms, suggesting that the LPS-induced broiler fever model was successfully established. In order to observe the antipyretic effect of RIFG mixture on the rapid rise of body temperature, we chose this time point after injection of LPS 2 h to let chickens drink the medicine.

Studies have shown that the degree of fever reaction is closely related to PGE2 (Eskilsson et al., 2017). PGE2 is the main medium that can cause the rise of body temperature setting point, and it is the common pathway of multiple pyrogen induced body temperature rise (Bruno Conti, 2004; Blatteis, 2006). The traditional view of the steps of fever generation is that monocytes, macrophages and lymphocytes stimulated by pathogens produce pyrogen cytokines, which are released and transported to the hypothalamic thermoregulatory center to induce COX-2 to produce PGE2 and produce fever. According to the results of this experiment, the overall difference of PGE2 content in peripheral blood was not significant after 6 h of LPS injection, but the thermal causes of IL-6 and TNF-a were significantly different. We could find that the generation of thermal causes and their transformation into fever mediated PGE2 through pyrogen signal transduction were carried out at a relatively slow rate. It is speculated that IL-6 and TNF-ɑ may be one of the mediators to maintain the febrile stage. In addition, some studies have proposed that PGE2 is the key medium produced in the initial stage of fever. After intravenous injection of LPS, the activation of complement triggers the rapid release of a large amount of PGE2 in KC (Perlik et al., 2005). The sampling time node in this study was in the continuous stage of fever rather than the rapid production stage of PGE2, which may be the reason why the inhibition effect of drugs on PGE2 was not significant.

Currently recognized pyrogen cytokines include interleukin-1, interleukin-6, and tumor necrosis factor alpha (Bruno, 2004; Nilsberth et al., 2009). Studies have shown that IL-6 is a key component of LPS-induced fever and meets many criteria for circulating pyrogen. Rats injected with specific antiserum (IL-6AS) did not produce a febrile response to the peripheral immune response and involved the transcription activator 3 (STAT3) pathway to induce the expression of prostaglandin synthetase COX-2 (Rummel et al., 2006). TNF-α is the first member of an LPS-induced cytokine cascade that activates mPGES-1 activity in vivo to produce PGE2 in response to glutathione (Roth and Blatteis, 2014).

COX-2 is a target gene of NF-κB and STAT3, and activation of NF-κB or STAT3 in brain endothelial cells is associated with COX-2 in these cells (Eskilsson et al., 2014). We found that the contents of IL-6, TNF-α and COX-2 declined after RIFG mixture injection, suggesting that it might inhibit the expression of IL-6 and TNF-α, thereby inhibiting the levels of COX-2 and PGE2, the positive feedback regulatory mediators of body temperature.

Numerous experiments and some clinical studies have proved that arginine vasopressin (AVP), as an important antipyretic substance with negative body temperature feedback, can regulate febrile reaction (Dong et al., 2007). The endogenous antipyretic effect played in the prehypothalamus is mediated by central nervous system effects such as AVP, and endogenous antipyretic agents can counteract the changes in neuronal activity induced by endogenous pyrogen such as cytokines and prostaglandins (Joachim, 2004). Studies have shown that the mechanism of action of Xiangqin Jiere granules on endotoxin-induced fever may be related to promoting the production and release of negative regulator AVP, reducing the level of positive regulator PGE2, and inhibiting the expression of PGE2 receptors EP1 and EP4 (Nie et al., 2019.). In this study, we found that the content of AVP in hypothalamus of broilers increased significantly after LPS injection during fever, indicating that the AVP of central nervous system had begun to play a role during fever. After RIFG mixture was applied for antipyretic treatment, the body temperature of broilers decreased and the content of AVP increased significantly, suggesting that the drug may play an antipyretic effect by promoting the expression of AVP.

Exogenous pyregenic LPS invades the host and triggers a series of immune responses through pathogen-associated molecular patterns (PAMP), recognized by Toll-like receptors-4 (TLR4), which induces transcription of COX-2 into PGE2 via NF-κB, MAPKp38, and extracellular signal-regulated kinases (ERK1/2). Then it causes fever. The pyrogenic cytokines TNF-α, IL-1, and IL-6 act outside the brain by activating cytokine receptors located on the periventricular organs, causing the release of PGE2 to cause fever (Mazgaeen and Gurung, 2020). It has been reported that one of the major signaling pathways of LPS-mediated TNF-α transcription is conducted through the NF-κB induced kinase pathway (Haddad and Land, 2002).Therefore, in order to further study the RIFG mixture antipyretic pathway, we detected the expression of TLR4/NF-κB genes and proteins in the hypothalamus. We found that the expression levels of TLR4 and NF-κB genes and proteins in the hypothalamus increased significantly after injection of LPS, and the expression levels decreased after RIFG mixture, suggesting that the drug may inhibit the transcription of COX-2 into PGE2 through TLR4/NF-κB pathway. It can inhibit the up-regulation of TLR4 mRNA and protein expression, down-regulate the activation of NF-κB and reduce the expression of TNF-α gene.

In conclusion, RIFG mixture has a good antipyretic effect on LPs-induced broilers, and it plays a role by inhibiting pyrogenic cytokines IL-6, TNF-α, PGE2, COX-2 and promoting the release of antipyretic factor AVP, involving TLR4/NF-κB pathway.