Inhibition of Oxidative Stress and Inflammation by Fisetin Ameliorates Heat Stress-Induced Intestinal Injury in Rats

Intestinal injury and dysfunctions play an important role in the pathophysiology of heat stress. The objective of this study was to determine whether fisetin could ameliorate heat stress-induced intestinal oxidative stress and inflammation, and explore the possible mechanisms at transcriptional levels. Twenty- four male Sprague-Dawley rats aged 8 weeks were randomized to 3 groups, namely, control, heat stress, and heat stress + fisetin (HS-FIS). The experiment lasted for 3 days with daily 1.5 h of heat treatment (40°C) for the heat stress and HS-FIS groups. Rats of the HS-FIS group were orally given 100 mg fisetin /kg body weight/day before the heat treatment. The results showed that fisetin restored the heat stress- induced jejunum morphological damage and increased intestinal permeability, which may be attributed to the improved redox status, the decreased myeloperoxidase activity, the suppressed toll-like receptor 4 signaling pathway mediated expression of pro-inflammatory cytokine tumor necrosis factor alpha at translational and transcriptional level, and the increased gene expression of interleukin 10 in the jejunum. In conclusion, fisetin alleviated the intestinal injury in rats caused by heat stress through inhibiting of oxidative stress and inflammation. This may offer a useful nutritional strategy for improving the health status of individuals exposed to heat stress.


INTRODUCTION
W ith climate change and global warming, the adverse effects of heat stress (HS) on human and animal health are becoming serious. The HS causes damage to multiple organs and high rate of mortality. Individuals exposed to HS are vulnerable to intestinal injury and dysfunction, as indicated by morphological alteration (He et al., 2015), reduced absorption and digestion of nutrients Wu et al., 2021), enhanced oxidative stress , over activation of inflammation and increased paracellular permeability . Importantly, intestinal injury and dysfunctions play a pivotal role in the pathophysiology of HS, which were observed in clinical studies (Snipe, 2019) and animal models (Ye et al., 2019). Therefore, the search for preventive or/and therapeutic strategies that could alleviate the adverse effects of HS on intestine is a main concern.

F i r s t A r t i c l e
oral FIS administration elevated glutathione (GSH) level, suppressed the infiltration of inflammatory cells, production of pro-inflammatory cytokines (e.g., tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1β), reactive oxygen species (ROS) and reactive nitrogen species in the colon tissues of colitis mice exposed to dextran sulphate sodium. However, data related to FIS modulation of intestinal health are limited. We hypothesized that FIS could attenuate the HS-induced intestinal damage in rats due to its excellent antioxidant and anti-inflammatory properties. In the present study, the beneficial effects of FIS on intestinal morphology, oxidative and immune status in heat-stressed rats, as well as the possible mechanisms at transcriptional levels were explored.

Animals and treatments
Male Sprague-Dawley rats, aged 8 weeks, weighing 200±20 g, were acclimated to the environment (temperature, 20-24 °C; humidity, 40-60%; 12 h light/ dark cycle) for 1 week. During the entire experimental period including acclimation, rats were provided with tap water and standard chow diet ad libitum under the normal condition. And then, rats were allocated into 3 groups (n=8): (1) the control (CON) group: rats were fed with 0.5% carboxymethylcellulose sodium (CMC-Na, diluted in 0.86% normal saline; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) by oral gavage administration for 3 days; (2) the HS group: rats were fed with 0.5% CMC-Na by oral gavage administration for 3 days under HS environment (1.5 h per day at 40 °C from 11:30 am to 1:00 pm for 3 consecutive days); and (3) the HS-FIS group: rats were fed with 100 mg FIS/kg body weight/day (purity 98%; diluted in 0.5% CMC-Na; Yuanye Biotechnology Co. Ltd, Shanghai, China) by oral gavage administration for 3 days under HS environment. The CMC-Na or FIS were provided for 3 consecutive days at 2 h before HS treatment. The FIS dose in the present study was selected according Lee et al. (2015).

Sample collection
After heat treatment for third day, all rats were anesthetized and sacrificed quickly. Blood was collected through eyeball of each rat and centrifuged at 2000 g (15 min, 4 °C) to harvest serum. The serum was stored at -80 °C until subsequent analysis. The procedure of jejunum sample collection was performed according to the method of Lu et al. (2011). Part of the jejunum was fixed in 4% buffered paraformaldehyde for histological analysis, and another part was immediately snap-frozen in liquid nitrogen for further analysis.

Diamine oxidase (DAO) activity
The activity of DAO (catalog No. A088-1) in the serum was determined using a commercial kit purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Histology analysis
The fixed jejunum sample was dehydrated and embedded in paraffin. Five-µm sections were cut and then stained with hematoxylin and eosin (HE). Ten welloriented, intact villi and their associated crypts per rat were selected, and images were recorded using an optical binocular microscope (Olympus BX5; Olympus Optical Co. Ltd, Tokyo, Japan) equipped with a digital camera (Nikon H550L; Nikon, Tokyo, Japan). Measurements of the villus length, crypt depth, and villus width of the jejunum were detected using the Image-Pro Plus software (version 6.0, Media Cybernetics, Inc., Rockville, MD, USA). According to the method described in the previous study (Dong et al., 2014), the villus: crypt ratio and villus surface area were calculated.

Oxidative status assay
As described in the previous study , the jejunal malondialdehyde (MDA, catalog No. A003-1) concentration, total superoxide dismutase (T-SOD, catalog No. A001-1) and glutathione peroxidase (GPX, catalog No. A005) activities, as well as total antioxidant capacity (T-AOC, catalog No. A015-1) and GSH (catalog No. A006-2) levels were determined using assay kits according to the guidelines of manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All results were normalized to total protein concentration in each sample for inter-sample comparison. The jejunal total protein concentration was determined according to the guidelines of the manufacturer (catalog No. A045-3, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Cytokine assays by ELISA
A commercial ELISA kit (catalog No. EK382/3-96) purchased from Multisciences Biotech Co., Ltd (Hangzhou, China) was employed to analyze the jejunal TNFαconcentration following the manufacturer's instructions. The detection limit was 0.43 pg/mL; the inter-and intraassay coefficients of variation were less than 7% and 9%, respectively. All results were normalized to total protein concentration in each sample for inter-sample comparison. The jejunal total protein concentration was determined according to the guidelines of the manufacturer (catalog No. A045-3, Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Myeloperoxidase (MPO) activity assay
The MPO activity (catalog No. A044) and total protein concentration (catalog No. A045-3) in the jejunum were analyzed using a commercial kit following the instructions of the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). All results were normalized to total protein concentration in each sample for inter-sample comparison. CON, rats were orally fed with 0.5% carboxymethylcellulose sodium; HS, rats were orally fed with 0.5% carboxymethylcellulose sodium and then subjected to heat treatment; HS-FIS, rats were orally fed with 100 mg FIS /kg body weight/day and then subjected to heat treatment. Results are expressed as mean and standard error (n = 6). * P<0.05 was compared with the CON group; # P<0.05 was compared with the HS group.  (n=5); T-SOD, total superoxide dismutase (n=8); T-AOC, total antioxidant capacity (n=8); CON, rats were orally fed with 0.5% carboxymethylcellulose sodium; HS, rats were orally fed with 0.5% carboxymethylcellulose sodium and then subjected to heat treatment; HS-FIS, rats were orally fed with 100 mg FIS /kg body weight/day t and hen subjected to heat treatment. Results are expressed as mean and standard error. * P<0.05 was compared with the CON group; # P<0.05 was compared with the HS group. Fig. 2. Effects of fisetin on the jejunal diamine oxidase (DAO) activity in heat-stressed rats. CON, rats were orally fed with 0.5% carboxymethylcellulose sodium; HS, rats were orally fed with 0.5% carboxymethylcellulose sodium and then subjected to heat treatment; HS-FIS, rats were orally fed with 100 mg FIS /kg body weight/day and then subjected to heat treatment. Results are expressed as mean and standard error (n=8). * P<0.05 was compared with the CON group; # P<0.05 was compared with the HS group.

Quantitative RT-PCR analysis
Total RNA from liver samples were extracted using TRIzol Reagent (TaKaRa, Dalian, China) according to the guidelines of the manufacturer. The integrity, concentration, and purity of RNA, reverse transcription, as well as qRT-PCR were performed according to the previous studies (Cheng et al., 2016. The primer sequences of genes used in this study are presented in Table I. The target genes expression levels were normalized by the housekeeping gene β-actin, and then were calculated via the 2 -ΔΔCt method (Livak and Schmittgen, 2001). The values of CON group were used as a calibrator.

Statistical analysis
Results are expressed as mean and standard error and analyzed by SPSS 17.0. The individual rat was used as the experimental unit. Statistical differences between different groups were determined via one-way analysis of variance (ANOVA) and Tukey's post hoc test for multiple comparisons. Significant difference was accepted at P < 0.05.

FIS reduces the severity of intestinal injury in rats subjected to HS
In Figure 2, the DAO activity in the serum was O n l i n e

F i r s t A r t i c l e
Inhibition of Oxidative Stress and Inflammation 5 significantly higher in rats exposed to HS compared with the CON group (P<0.05). Also, the jejunal matrix metalloproteinase 3 (MMP3, P=0.079) and heat shock protein 70 (HSP70, P<0.05) genes expression were increased in the HS group compared with the CON group (Fig. 3A). Rats in the HS group exhibited jejunal villus atrophy and shedding (Fig. 4). The jejunal villus height and villus height: crypt depth ratio were significantly lower (Table II, P<0.05) in rats exposed to HS compared with the CON group. Administration of FIS to rats exposed to HS significantly decreased (P<0.05) the serum DAO activity and the jejunal HSP70 gene expression, improved jejunum morphology, increased (P<0.05) jejunal villus height, villus surface area, villus width and villus height: Crypt depth ratio in the HS-FIS group compared with the HS group (P<0.05). However, crypt depth, the IFABP and villin mRNA expression in the jejunum of rats were not affected among the 3 groups (P>0.05).

FIS attenuates the jejunal oxidative stress in rats subjected to HS
The higher MDA content, T-SOD activity, GPX activity in the jejunum of heat-stressed rats were reduced by FIS administration (Table III, P<0.05). However, the T-AOC and GSH levels in the jejunum were comparable among the 3 groups (P>0.05). At transcriptional level, administration of FIS to rats exposed to HS alleviated the increased nuclear factor, erythroid 2-like 2 (Nrf2) and GPX1 expression compared with the HS group (Fig. 3B,  P<0.05). The genes expression of SOD1 and Kelch-like ECH-associated protein 1 (Keap1) in the jejunum were not influenced by HS and FIS treatment (P>0.05).

FIS relieves the jejunal inflammation in rats subjected to HS
The TNF-α concentration was higher in the jejunum of the HS group compared with the CON group (Fig. 5A,  P<0.05). However, FIS treatment to heat-stressed rats caused decreases in jejunal TNF-α concentration and MPO activity (Fig. 5B) compared with the HS group (P<0.05). At transcriptional level, rats in the HS group exhibited higher jejunal TNF-α, IL10 and toll-like receptor 4 (TLR4) mRNA expression compared with the CON group (Fig.  3C, P<0.05). Expectedly, the increased IL10, decreased TNF-α and TLR4 genes expression were observed in the jejunum of the HS-FIS group compared with the HS group (P < 0.05). The jejunal IL6 and interferon γ (IFN-γ) genes expression in rats were not affected (P > 0.05) among the 3 groups. Fig. 3. Effects of fisetin on the genes expression related to jejunal injury markers (A), redox status (B) and inlammation (C) in heat-stressed rats. HSP70, heat shock protein 70; I-FABP, intestinal fatty acid-binding protein; MMP3, matrix metalloproteinase 3; GPX1, glutathione peroxidase 1; Nrf2, nuclear factor, erythroid 2-like 2; SOD1, superoxide dismutase 1; Keap1, Kelch-like ECHassociated protein 1; IL6, interleukin 6; IL10, interleukin 10; TLR4, toll-like receptor 4; TNF-α, tumor necrosis factor alpha; IFN-γ, interferon γ; CON, rats were orally fed with 0.5% carboxymethylcellulose sodium; HS, rats were orally fed with 0.5% carboxymethylcellulose sodium and then subjected to heat treatment; HS-FIS, rats were orally fed with 100 mg FIS /kg body weight/day and then subjected to heat treatment. Results are expressed as mean and standard error (n=8). * P<0.05 was compared with the CON group; # P<0.05 was compared with the HS group.

O n l i n e F i r s t A r t i c l e
K. Cheng et al. Fig. 4. The jejunal histological appearance (hematoxylin and eosin). (A) CON, rats were orally fed with 0.5% carboxymethylcellulose sodium; (B) HS, rats were orally fed with 0.5% carboxymethylcellulose sodium and then subjected to heat treatment; (C) HS-FIS, rats were orally fed with 100 mg FIS /kg body weight/day and then subjected to heat treatment. Original magnification 100 ×, Scale bars = 100 μm. Fig. 5. Effects of fisetin on the jejunal tumor necrosis factor alpha (TNF-α) level (A) and myeloperoxidase (MPO) activity (B) in heat-stressed rats. CON, rats were orally fed with 0.5% carboxymethylcellulose sodium; HS, rats were orally fed with 0.5% carboxymethylcellulose sodium and then subjected to heat treatment; HS-FIS, rats were orally fed with 100 mg FIS /kg body weight/day and then subjected to heat treatment. Results are expressed as mean and standard error (n=6). * P<0.05 was compared with the CON group; # P<0.05 was compared with the HS group.

DISCUSSION
In the present study, the effects of oral FIS administration on the jejunum of rats subjected to HS were investigated for the first time. As expected, FIS relieved the heat stress-induced intestinal damage demonstrated by the decreased serum DAO activity, improved histologic structure, inhibited oxidative stress and inflammation. In this study, the beneficial effects of FIS on intestine of heat-stressed rats may be attributed to the decreased gene expression of TLR4, reduced TNF-α expression, increased IL10 gene expression, and suppressed MPO activity.
The intestine is one of the first and more susceptible organs negatively affected by hyperthermia challenges due to the fact that animals redistribute blood to the periphery to maximize radiant heat dissipation (Pearce et al., 2014). When exposed to HS, the synthesis of most proteins is delayed, but HSP is rapidly synthesized (Al-Aqil and Zulkifli, 2009). Among the HSP, HSP70 is the most conserved and most common family, which is abundant in various tissues in most organism. There is ample evidence that the transcription of HSP70 is rapidly induced by high temperature (Tedeschi et al., 2015;Song et al., 2017;Cheng et al., 2019). Therefore, the expression of HSP70 in intestine is a reliable biomarker for measuring a thermotolerance response. Similarly, in this study, heat exposure led to an increase in the jejunual HSP70 mRNA expression. As expected, FIS restored the heat stressinduced upregulated jejunal HSP70 gene expression, suggesting that FIS reduced the heat responses of rats exposed to high temperature. Similar results observed in scrotal hyperthermia model showed that administration of FIS decreased the gene expression of HSP72 (Pirani et al., 2021).
Heat stress results in the increased intestinal permeability in animals. The serum DAO activity is O n l i n e

F i r s t A r t i c l e
Inhibition of Oxidative Stress and Inflammation recognized as a sensitive marker for monitoring the alteration of intestinal barrier permeability Cheng et al., 2019). In the present study, the serum DAO activity was increased during HS, suggesting that the intestinal barrier function was compromised. In addition, MMP3 expression was analyzed as an indicator of intestinal damage, as has been reported by other authors Yi et al., 2018), results in this study showed that this parameter was affected by HS, which further confirmed heat stress-induced intestinal injury. As expected, FIS treatment could attenuate the intestinal morphologic damage of heat-stressed rats as indicated by the increased villus height, villus width, the ratio of villus height to crypt depth and villous surface area. Meantime, FIS significantly decreased circulating DAO activity in response to HS exposure in this study. Thus, our results showed that FIS could be used as a potential regulator in improving intestinal morphologic damage and permeability of rats under HS. Emerging evidence revealed that an increase in the generation of ROS such as superoxide anions, hydrogen peroxide and hydroxyl radicals was observed in individuals exposed to HS, which eventually led to intestinal damage . As it is known, the enzymatic antioxidant defense against excessive ROS has an important function on maintaining redox homeostasis; SOD catalyzes superoxide radicals to molecular oxygen and hydrogen peroxide, which is decomposed by CAT and GPX to harmless compounds such as water and oxygen. However, the overwhelming ROS will harm DNA, proteins and lipids, even leading to cellular injury and death. The results in our study presented that HS induced increases in the activities of GPX and T-SOD; nevertheless, these increases were shown to be inadequate to counteract the oxidative damage in the intestine of rats as indicated by the increased MDA concentration, which supported the findings of Cheng et al. (2019). In addition, Nrf2 and its target antioxidant enzyme GPX genes expression were upregulated in the intestine of heat-stressed rats, which may explain the increased GPX activity. Accumulating studies have confirmed that HS can result in the increased adaption of Nrf2 and its target antioxidants genes (Zhang et al., 2002;Bhusari et al., 2008). However, the decreased T-SOD activity in the jejunum of heat-stressed rats was not parallel with its gene expression, which need to be further investigated. In this study, FIS administration alleviated the increased GPX and T-SOD activities, MDA content, Nrf2 and GPX mRNA expression, suggesting that FIS can attenuate intestinal oxidative damage induced by HS. Our data in rats are consistent with the experiment in HS-induced oxidative stress broilers, in which FIS supplementation improved the circulating redox status (Ogbuagu et al., 2018). The beneficial effects of FIS on redox status of heat-stressed rats in the present study were attributed to its hydroxyl groups and anti-inflammation property rather than enhancing antioxidant defense systems.
Previous studies have shown that the overproduction of pro-inflammatory cytokines such as TNF-α induced by HS contributed significantly to the intestine tissues necrosis and dysfunction (Cheng et al., 2019). Similarly, results observed in our study also demonstrated that the protein and mRNA levels of jejunal TNF-α were increased in heatstressed rats, which may be attributed to the up-regulation of TLR4 gene expression. The TLR4, a well-known pattern recognition receptor, simulation of which triggers the biosynthesis and release of inflammatory cytokines, including IL6 and TNF-α (Shi et al., 2006). Our results were in accordance with the previous studies in different animal models such as broilers , mice (Mohyuddin et al., 2021) and rats (Cheng et al., 2019) in which HS increased TLR4 mRNA abundance and its targeted inflammatory cytokines production in intestine. In addition, in the present study, the upregulated expression of jejunal IL10 mRNA during heat exposure may be due to the fact that heat stress-induced inflammation and the cells undergoing inflammation will respond by producing anti-inflammatory cytokines. These results in this study further supported the notion that heat stress-induced jejunal inflammation could be due to the increased TLR4 mRNA expression. Expectedly, FIS counteracted the increased TNF-α concentration, and the upregulated genes expression of TLR4 and TNF-α in the jejunum of heatstressed rats, suggesting that FIS could play a positive role in inhibiting jejunal inflammation. Likewise, FIS has been reported to reduce the colonic the protein expression of pro-inflammatory cytokines, TNF-α, IL6, and IL-1β, in colitis mice subjected to dextran sulphate sodium (Sahu et al., 2016). On the other hand, in this work, FIS treatment upregulated the transcription of IL10 gene in the jejunum of rats exposed to HS. The IL10 is considered a more potent inhibitor of many pro-inflammatory cytokines produced by monocytes and dampens many inflammatory responses (Patel and Davidson, 2014). Moreover, in the current study, FIS administration also inhibited the MPO activity in the jejunum of heat-stressed rats. The MPO system of neutrophil plays a critical role in intestinal mucosal inflammation. Additionally, MPO has been implicated as a participant in intestinal damage under many inflammation conditions, which catalyzes the production of cytotoxic oxidant hypochlorous acid (Hampton et al., 1998;Nicholls and Hazen, 2005). Hypochlorous acid can react avidly with cellular bio-macromolecules such as proteins, lipids and DNA, which consequently contributes to oxidative O n l i n e F i r s t A r t i c l e K. Cheng et al. stress and inflammation in tissues including small intestine (Smith, 1994). Thus, the antioxidant property of FIS is partly attributable to the decreased MPO activity. Taken together, the FIS anti-inflammation property probably results from the downregulated TLR4 mRNA expression, upregulated IL10 mRNA abundance and inhibited MPO system of neutrophil.

CONCLUSION
The data obtained from the present study indicates that FIS administration confers protection against heat stress-induced intestinal damage partly by mitigating oxidative stress and inflammation via the downregulated TLR4 mRNA expression, upregulated IL10 mRNA abundance and inhibited MPO system of neutrophil. This study offers a useful nutritional strategy for improving the health status of individuals exposed to HS.