Honokiol Ameliorates DSS-Induced Mouse Colitis by Inhibiting Inflammation and Oxidative Stress and Improving the Intestinal Barrier

Ulcerative colitis (UC) is a multifactor intestinal disease with increased morbidity. Recently, pleiotropic drugs with exact biosafety have been urgently needed. Honokiol (HKL) is the major bioactive component of traditional Chinese medicine “Houpu,” with almost no toxic effects and approved anti-inflammation, antioxidant, antispasmodic, etc. effects. This study examined the therapeutic effect of HKL in dextran sulfate sodium- (DSS-) induced experimental colitis. In vivo, C57BL/6 mice received 3% DSS for seven days to generate UC, and HKL was pretreated for five days and given during the whole DSS-induced period. In vitro, RAW264.7 macrophages were stimulated with lipopolysaccharide (LPS) to induce inflammation, and mouse colon epithelial cells (MCEC) were treated with HKL or pretreated with HKL and then stimulated with LPS-induced macrophage supernate to investigate the barrier enhancement roles. HKL significantly ameliorated disease activity index (DAI), colon length, and histopathological scores in DSS-induced colitis. The inflammatory mediators of interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX2) were decreased, and the tight conjunction proteins were increased in the HKL-treated group both in vivo and in vitro. Above all, HKL can relieve experimental UC through anti-inflammation, antioxidant, and epithelial barrier enhancement roles. These effects were associated with peroxisome proliferator-activated receptor γ (PPARγ)/nuclear factor-κB (NF-κB) p65, sirtuin3 (SIRT3)/adenosine 5′-monophosphate- (AMP-) activated protein kinase (AMPK), and nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase 1 (HO1) signaling pathways. In conclusion, after further clinical studies, HKL may be a promising drug for UC.


Introduction
UC is a chronic relapsing inflammatory disease limited to the mucosal layer of the colon [1]. The exact pathogenesis of UC is still unknown; the most widely accepted view is that it is multifactorial, including dysregulated immune responses, altered gut microbiota, genetic susceptibility, and environmental factors [1][2][3]. The global burden of UC continues to increase, for instance, in the US market, from $5.0 billion in 2020 to predicated $11-12 billion by 2026 [4]. The new drugs for UC treatment have been impressive in recent years. However, none is effective in most patients, let alone the biologic-experienced patients [5]. In a nationwide study in Israel, colectomy and steroid dependency rates have remained unchanged in patients with UC during the biological era [6]. The large proportion of primary nonresponders to each molecularly targeted agent may be due to different dominant cytokine levels depending on disease stage and patient characteristics [7]. The pathology of UC is very complex. Up to now, there is no treatment and unlikely to have a therapy suitable for all. So, there is an urgent need for low cost, few side effects, and pleiotropic drugs to enrich current treatment options.
HKL is a simple biphenyl neolignane found in Magnolia species [8]. Its structure is shown in Scheme 1. It is brown to white fine powder, aromatic, spicy taste, and slightly bitter. It is soluble in general organic solvents; easily in benzene, ether, chloroform, ethanol, etc.; and insoluble in water [9]. It is a potent bioactive compound of traditional Chinese medicine "Houpu." According to the Chinese medicine meridian theory, "Houpu" returns to the large intestine meridian and is widely used to treat gastrointestinal diseases. Modern investigations have confirmed that HKL has anti-inflammation [10], antimicrobial [11,12], antioxidant [13][14][15], antispasmodic [16], anticancer [8,[17][18][19], antithrombotic [20], antidepressant, anxiolytic [21], and other actions [22,23]. As known to all, synthetic drugs have many side effects, including damage to the endocrine system, immune system, and gastrointestinal tract. Regarding the numerous pharmacologic benefits without noticeable side effects of HKL, we hypothesize that HKL might have therapeutic effects on UC.
In the present study, we investigated the anti-inflammation, antioxidant, and intestinal barrier enhancement roles of HKL on UC treatment in vitro and in vivo. The mechanisms responsible for its actions have also been explored.

Animal Model and Treatments.
Six-to eight-week-old female C57BL/6 mice weighed 15-20 g and were purchased from the Laboratory animal center of Shanxi Provincial People's Hospital (Shanxi, China, animal license number: SYXK (Jin) 2019-0003) and kept in the specified pathogen-free (SPF) condition at 22-23°C with a standard 12 h light/dark cycle. All mice have free access to food and water. Forty mice were randomly divided into four groups (n = 10 per group). The control group was given a normal diet; the HKL group received HKL (40 mg/kg wt. purity≥98%, Yuanye, China) dissolved in olive oil (Yuanye, China) via oral gavage (10ul/g) from D1 to D12; the DSS group received DSS (3% w/v, g/ml, 36-50 kDa, Yeasen, China) via drinking water D6-D12; the DSS+HKL group pretreated with HKL for five days and then received 3% DSS for seven days with HKL gavage once a day. The control and DSS groups received olive oil (10ul/g) once daily by gavage during the experimental period. The Animal Ethics Committee approved all animal procedures of the Second Hospital of Shanxi Medical University (Approval No. DW2022051).

Macroscopic Grading and Histological Analysis of Colitis.
The macroscopic grading of DSS-induced colitis is based on a standard scoring system called the disease activity index (DAI) [24]. The details are shown in Table 1. The body weight, stool consistency, and rectal bleeding of each mouse were recorded in fact daily. All mice were anesthetized and sacrificed by cervical dislocation 24 h after the last drug treatment. Their colons were excised, and the length was measured promptly. The distal colonic tissues were fixed in 4% paraformaldehyde solution (Servicebio, China), embedded in paraffin (Solarbio, China), sectioned, and then stained with hematoxylin and eosin (HE) (Solarbio, China) for optical microscopic (CKX53, Olympus, Japan) observation. Histopathological scores were determined according to the previous criteria [25] shown in Table 2. 2.3. Immunohistochemistry. After dewaxing the paraffinembedded colon sections, citric acid (Solarbio, China) was used for antigen reparation. Then, the slices were rinsed with 3% H 2 O 2 (Solarbio, China) for 25 min at room temperature to block endogenous peroxidase activity and incubated with 3% bovine serum albumin (Solarbio, China) for 30 min to block nonspecific binding. Followed by setting with the first antibodies (Proteintech, USA) in a wet box at 4°C overnight, the sections were incubated with the corresponding secondary antibody: anti-rabbit IgG antibody conjugated to horseradish peroxidase (1 : 200) (Proteintech, USA). Then, the freshly prepared diaminobenzidine (DAB) (Solarbio, China) chromogenic solution was dripped on the slices for coloration. The coloration time was controlled under a microscope (CKX53, Olympus, Japan), rinsing with tap water to stop. Counterstaining was performed using HE (Solarbio, China), sealing the sections with synthetic resin (Servicebio, China). Finally, we conducted the microscope examination and the positive results were shown in brown.   Figure 1(e). After that, the resistance value of each group was measured as mentioned above. The TEER calculation formula is as follows: TEER = ðR − RBÞ × surface area (R is the resistance value of the testing well, RB is the resistance value of the blank well, and surface area = 1:12 cm 2 ).      Oxidative Medicine and Cellular Longevity (Proteintech, USA), Claudin1 (Proteintech, USA), and GAPDH (Proteintech, USA) (dilution 1 : 1000)) overnight at 4°C and washed with TBST thrice, followed by incubation with an HRP-conjugated anti-mouse or anti-rabbit second-ary antibody (ABclonal, USA) (dilution 1 : 2000) at room temperature for one hour and rinsed thrice with TBST buffer. Finally, protein bands were reacted to an enhanced chemiluminescence solution (Boster, China) and detected     Oxidative Medicine and Cellular Longevity using the Bio-Rad Image Lab system (ChemiDoc XRS+, Bio-Rad, USA). The intensity of the bands was quantified using the ImageJ software, and the results of GAPDH normalized the relative protein expression levels.

Cell
2.9. Real-Time Quantitative Polymerase Chain Reaction (qRT-PCR). Total RNA was extracted from the colon tissue homogenates, and RAW264.7 cells or MCEC using RNAiso Plus (Takara, Japan) and their concentrations were determined. The primer sequences of interleukin IL-1β, IL-6, TNF-α, ZO1, occludin, and Claudin1 are shown in Table 3. 1 μg RNA was reverse-transcribed into complementary DNA using a commercial RT-PCR kit (Takara, Japan). RT-PCR assays were performed using the 2x M5 HiPer SYBR  2.10. Statistical Analysis. All data are shown as mean ± SEM. The statistical analyses were performed in the GraphPad Prism software version 8.0. The significance of the differences between groups was analyzed by one-way ANOVA followed by Tukey's multiple range tests. P < 0:05 was regarded as statistically significant. For every result in this study, equal or greater than three biological repetitions were conducted.

HKL Ameliorated DAI and Colon Length in DSS-
Induced Colitis in Mice. DSS-induced experimental colitis is a classical ulcerative colitis animal model for its similar symptoms and histopathological characteristics. In this study, the HKL treatment group had lower disease activity index, which consists of weight loss, stool character, and fecal occult blood, compared with the DSS group (Figure 2(c)). Among these indexes, the body weight change is a quantifiable visual indicator of the state of the mice. As shown, the body weight of the model group mice decreased remarkably from the fifth day when they received special water containing 3% DSS. In contrast, the HKL pretreated group showed a mitigated decrease in body weight (Figure 2(b)). The shortening of colon length revealed the severity of DSS-induced colitis. The HKL+DSS group showed an attenuated degree of this trend compared with the DSS group (Figure 2(a)).

HKL Treatment Decreases Inflammatory Mediators in
DSS-Induced Mice. The qRT-PCR results showed that the levels of IL-1β (Figure 3(a)), IL-6 ( Figure 3(b)), TNF-α (Figure 3(c)), iNOS (Figure 3(d)), and COX2 (Figure 3(e)) significantly increased in colon tissue homogenates of the DSS group compared with the control group. In contrast, the levels of the above inflammatory mediators were marked decreased in the HKL treatment group compared with the DSS group. These data suggested that HKL played an antiinflammatory role in vivo.

HKL Treatment Improved the Histopathological Features of the DSS-Induced
Mice. The histopathological features of the HE-stained sections showed marked infiltration of inflammatory cells, loss of crypts, formation of crypt abscesses, destruction of the mucosal layer, and thickening of the muscular layer in the DSS group. In contrast, the DSS+HKL group showed alleviated pathological injuries of the above features (Figure 4(a)). This is also measured by the histological score to show more intuitively (Figure 4(b)). HKL could mitigate the histopathological injuries of experimental colitis from this study, which is also the golden standard of clinical cure.

HKL Improved the Barrier Function of the DSS-Induced
Mice. The gene-expressed levels ( Figure 5(a)) and the protein-expressed levels ( Figure 5(b)) of ZO1, occludin, and Claudin1 increased dramatically in the DSS+HKL group compared with the DSS group. Additionally, the colon tissue immunohistochemistry showed the expression of MUC2, MUC3, and trefoil factor-3 (TFF3) decreased in DSSinduced mice; however, HKL treatment ameliorated the levels of the above proteins ( Figure 5(c)). These results indicated that HKL improved barrier function in DSS-induced colitis partially by restoring the tight junction proteins, mucins, and TFF3 levels.

HKL Suppressed Proinflammatory Cytokine Expression in LPS-Stimulated Macrophages.
Cell viability assay showed that for RAW264.7 cells, HKL did not affect cell viability up to 10 μM; for MCEC, the concentration was up to 40 μM for no significant difference (Figure 6(a)); combined with previous studies [22], 10 μM and 40 μM concentrations were chosen for RAW264.7 cells and MCEC, respectively, for subsequent experiments. The qRT-PCR analyses indicated that the proinflammatory cytokines, including IL-1, IL-6, and TNF-α, increased markedly in 6h-LPS-induced RAW264.7 cells (Figure 6(b)), and the western blot analyses showed that the expression of iNOS and COX2 also significantly increases in 12h-LPS-induced RAW264.7 cells

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Oxidative Medicine and Cellular Longevity ( Figure 6(c)). In contrast, the LPS+HKL group showed reduced gene and protein expression levels of the above inflammatory mediators. Collectively, these data demonstrated that HKL had an inhibiting effect on inflammatory response in vitro.

HKL Maintained a Normal Epithelium Barrier in MCEC.
To investigate the protective role of HKL on the intestinal epithelium barrier in vitro, the MCEC were stimulated with the supernatant of 12h-LPS-induced RAW264.7 cells. The qRT-PCR data indicated that compared with the LPSstimulated supernatant group, the HKL pretreated group had an average level of the ZO1 and occludin mRNA expression (Figure 1(a)). Immunofluorescence was also used to show more intuitively the ZO1, occludin, and Claudin1 protein expression levels of the MCEC with the same cell stimulation condition (Figure 1(b)). In addition, the western blot analysis showed that the ZO1, occludin, and Claudin1 protein expressions were significantly increased with the HKL treatment compared with the control group. However, the optimum effects occurred at the distinct time point for different proteins (Figure 1(c)). A Transwell plate was also used to observe the TEER change. The data showed that the supernatant of 12h-LPSinduced RAW264.7 cells significantly decreased the resistance value of the MCEC monolayer, whereas pretreating with HKL observably inhibited this reducing effect (Figure 1(d)).
Above all, these data demonstrated that HKL exerts a strengthening and repairing role in the intestinal epithelium barrier of MCEC.
3.7. HKL Regulated DSS-Induced Activation of PPARγ/NF-κB Signaling in Mice. It is well known that NF-κB is a crucial nuclear transcription factor that regulates inflammatory response. So, the effect of HKL on phosphorylation levels of p65 in colonic tissue homogenates was analyzed using a western blot. Compared to the control group, the DSS treatment group caused elevated phosphorylation levels of p65. The DSS+HKL group showed markedly reduced phosphorylation of p65 protein. Regarding the PPARγ signaling pathway that is essential for NF-κB activation [26], HKL has been proven to be a PPARγ agonist [27]. The expression of PPARγ was further investigated. As shown in this study, PPARγ significantly reduced in the colonic tissue of the DSS group, while the HKL treatment could recover this trend. Besides, HKL alone could stimulate the expression of PPARγ (Figure 7).
3.8. The Effects of HKL on PPARγ/NF-κB p65, SIRT3/AMPK, and NRF2/HO1 Signaling Pathways In Vitro. Similar to the findings in vivo, HKL treatment markedly reduced the elevated phosphorylation level of p65 and normalized the reduced level of PPARγ (Figure 8(a)). To further explore the underlying mechanism of the epithelial barrier reinforced the role of HKL, the SIRT3/AMPK and NRF2/HO1 signaling pathways were investigated. As shown in the present study, the phosphorylation level of AMPK at Thr172, SIRT3, NRF2, and HO1 expressions was significantly increased at the different time points in MCEC, which were treated by HKL (Figure 8(b)).

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Oxidative Medicine and Cellular Longevity LPS blocked the nuclear translocation of NRF2, while HKL compensated for this effect (Figure 8(c)). In contrast, LPS significantly stimulated the nuclear translocation of NF-κB p65, while HKL inhibited this process (Figure 8(d)).

Discussion
Nowadays, UC has been considered more than a superficial disease confined to the mucosa, which includes the risk of proximal extension, reduced rectal compliance, increased risk of neoplasia, reduced likelihood of responding to medical therapies, fibrosis in the submucosa, altered motility, overlapping functional symptom effects on the myenteric plexus, and even increased risks of cardiovascular morbidity [28]. With all this in-depth knowledge of UC, new individualized treatment is urgently needed. At present, histo-endoscopic remission might be a complete definition of mucosal healing, which is a desirable therapeutic goal in UC [29]. HKL showed a remarkable effect on clinical presentations and histopathological recovery of DSS-induced UC in the present study. Furthermore, chemically induced models mimic some key immunological and histopathological features of IBD in humans. The strengths of the acute models are the similar alterations of barrier function, innate immune effects, and flares [24]. So, the current results provided potential therapeutic strategies for UC in a preclinical setting. However, further investigations about HKL on the chronic models of UC should be carried on. Previously, HKL has been proven to affect stem cell viability to inhibit colon tumorigenesis by suppressing oncogenic YAP1 function [30]. In that study, an AOM/DSSinduced colitis-associated cancer model was used.
Regardless of the unclear etiology of UC, the ultimate symptom is barrier disruption, so restoration of intestinal barrier function is the primary therapeutic aim, which can be achieved by promoting inflammation resolution and accelerating mucosa reparation [2,31]. Consequently, both the anti-inflammation and the intestinal mucosa reparation roles of HKL had been observed in this study. On the antiinflammation aspect, inflammatory mediators, such as iNOS and COX2, and inflammatory genes, such as IL-1β, IL-6, and TNF-α, were increased in the LPS-treated RAW264.7 macrophages, and the LPS+HKL group showed decreased levels. Similar effects were also observed in the DSS and DSS+HKL in vivo groups. The anti-inflammatory effects may work through PPARγ/NF-κB signaling pathway. The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor highly expressed in the colon. It has been proposed as a critical inhibitor of colitis by attenuating nuclear factor-κB (NF-κB) activity [26,32]. Many studies about natural agents, such as PPARγ agonists, could protect mice from DSS-induced UC through its anti-inflammatory role [33][34][35][36][37]. The activation of the PPARγ response elements (PPRE) in the nuclear is based on forming a heterodimer between PPARγ and retinoid X receptor (RXR). So, agents with synergistic effects on PPARγ/RXR heterodimer may be of better efficacy than pure PPARγ agonists. Interestingly, HKL, apart from its activation of PPARγ, also works as an RXR agonist [38], coincidently meeting this condition.
Besides inflammation, the overwhelming ROS generation and defective antioxidant defense contribute to IBD's progression. Oxidative stress is now regarded as a pathogenic and critical factor in the initiation, progression, and severity of IBD rather than a consequence of chronic inflammation in the intestinal mucosa [39]. NRF2 is a stress-responsive transcription factor associated with cellular homeostasis and has been speculated to have a protective effect on UC by regulating the oxidative stress response and repressing inflammation [40][41][42]. When the homeostatic balance is interrupted, NRF2 translocates into the nucleus and activates the expression of various genes, one of which is the antioxidant enzyme: HO-1 [43]. Our results showed the activation of NRF2/HO-1 expression in MCEC and the translocation of NRF2 from the cytoplasm to nuclear in RAW264.7 macrophages when treated by HKL, indicating the antioxidant effects of HKL. As previously described, there is a cross-talk between the AMPK and NRF2 axes, and that AMPK works upstream of NRF2 [44][45][46]. So the activation of AMPK may have both energy homeostatic maintenance and oxidative clearance roles.  AMPK is a crucial cellular energy sensor [47]. AMPK can promote autophagy, which is a process that maintains intestinal barrier function by regulating tight junction proteins [48]. In addition, AMPK has been demonstrated to enhance intestinal barrier function and epithelial differentiation via promoting caudal type homeobox 2 (CDX2) expression [49]. The previous study shows that mitochondrial deacetylase SIRT3 activates AMPK to enhance macroautophagy and chaperonmediated autophagy to protect hepatocytes against lipotoxicity [50]; since HKL has an apparent activation on SIRT3, the potential mechanism of the interaction between SIRT3 and AMPK in intestinal epithelial barrier enhancement can be further investigated. Apart from its roles in intestinal barrier maintenance, AMPK activators can reduce macrophage inflammation through various signaling pathways, such as AMPK/MAPK [51], AMPK/SIRT1/NF-κB [52], and AMPK/ NRF2/HO-1 [44], which indicates the further investigation of HKL as an anti-inflammation agent through AMPK activation. Above all, the upregulated levels of tight junction proteins in the MCEC monolayer when treated by HKL and in the colons of DSS+HKL-treated mice compared to the DSSinduced subjects may be acted through AMPK/NRF2/HO-1 antioxidant pathway and SIRT3/AMPK energy regulation pathway. The cross-talk between these pathways should be further investigated.
Besides new drugs, advanced colon-targeting delivery systems, which exploit multiple characteristics of the colonic environment and exert their actions in parallel to achieve more robust and reliable site-specific drug delivery, can also be utilized to achieve better therapeutic effects [53]. This system can be used to realize the targeted delivery of HKL to the colon focal zone for a better therapeutic action of UC in the future.

Conclusion
In conclusion, the present study demonstrated that HKL could alleviate DSS-induced colitis by reducing inflammation, inhibiting oxidative stress, and strengthening intestinal barrier integrity. The underlying signaling pathways likely involve PPARγ/NF-κB, NRF2/HO-1, and SIRT3/AMPK (Figure 9). The noticeable alleviating effects suggest that HKL is a promising treatment strategy for the prevention of UC.