Brusatol-Enriched Brucea javanica Oil Ameliorated Dextran Sulfate Sodium-Induced Colitis in Mice: Involvement of NF-κB and RhoA/ROCK Signaling Pathways

Brucea javanica oil (BJO) is beneficial for the treatment of ulcerative colitis (UC), and that quassinoids in particular brusatol are bioactive components. However, it is still uncertain whether or not other components in BJO, such as oleic acid and fatty acids, have an anti-UC effect. The present study is aimed at comparing the anti-UC effects between brusatol-enriched BJO (BE-BJO) and brusatol-free BJO (BF-BJO) and at exploring the effects and mechanisms of BE-BJO on colon inflammation and intestinal epithelial barrier function. Balb/C mice received 3% (wt/vol) DSS for one week to establish the UC model. Different doses of BE-BJO, BF-BJO, or BJO were treated. The result illustrated that BE-BJO alleviated DSS-induced loss of body weight, an increase of disease activity index (DAI), and a shortening of colon, whereas BF-BJO did not have these protective effects. BE-BJO treatment improved the morphology of colon tissue, inhibited the production and release of TNF-α, IFN-γ, IL-6, and IL-1β in the colon tissue, and reversed the decreased expressions of ZO-1, occludin, claudin-1, and E-cadherin induced by DSS but augmented claudin-2 expression. Mechanistically, BE-BJO repressed phosphorylation of NF-κB subunit p65, suppressed RhoA activation, downregulated ROCK, and prevented phosphorylation of myosin light chain (MLC) in DSS-treated mice, indicating that the protective effect of BE-BJO is attributed to suppression of NF-κB and RhoA/ROCK signaling pathways. These findings confirm that brusatol is an active component from BJO in the treatment of UC.


Introduction
Ulcerative colitis (UC) is a chronic bowel disorder characterized by diffuse inflammation of the mucosa of the colon and rectum [1,2]. The hallmark clinical symptoms of UC include abdominal pain, rectal bleeding, and diarrhea [3]. Persistent UC leads to the development of colorectal cancer [4]. Currently, the etiopathogenesis of UC has not been completely understood. Although several categories of therapeutic drugs, such as anti-inflammatory drugs and immunosuppressants, have improved the life expectancy of patients, available effective therapies are still limited and the resurgence of diseases often occurs [5][6][7]. Therefore, development of effective therapeutic drugs for the management of UC would have important clinical implications.
Brucea javanica (L.) Merr. (Ya-dan-zi in Chinese, Simaroubaceae) is a crucial Chinese folk medicine widely used for the treatment of dysentery, which was also known as inflammatory bowel disease (IBD) [8,9]. Brucea javanica oil (BJO), an extract of the desiccative ripe fruit of the Brucea javanica (L.) Merr., exhibits various bioactivities, including anti-inflammatory, antimalarial, and antitumor activities [10]. Our previous studies have shown that BJO protects against IBD in both 2,4,6-trinitrobenzenesulfonic acid-(TNBS-) induced Crohn's disease model and dextran sulfate sodium-(DSS-) induced UC model, via suppression of NF-κB activation [11,12]. Chemically, BJO is composed of flavonoids, alkaloids, volatile oils, sterols, fatty acids, and polyphenolic acids. Among all these components, quassinoids are vital bioactive compounds that display various types of biological activity [13,14]. Indeed, we have reported that quassinoids, in particular, brusatol and bruceine D, had potential therapeutic effects against UC [15,16]. However, it is still uncertain whether or not other components such as oleic acid and fatty acids have an anti-UC effect. Thus, we prepared BJO enriched with brusatol and brusatol-free BJO which mainly consists of oleic acid and fatty acids [17]. The present study is aimed at comparing the anti-UC effects between brusatol-enriched BJO (BE-BJO) and brusatol-free BJO (BF-BJO).
Moreover, inflammation and epithelial barrier defects in the alimentary system can damage the normal function of colon and promote the pathogenesis of UC. Since the antiinflammatory effects of BJO and brusatol have been proven, the present study focused on investigating the potential effect of BJO on improving intestinal epithelial barrier function and repairing mucosal epithelium. This study will also try to elucidate the possible mechanisms of BJO, by investigating the involvement of the RhoA/Rho-associated serinethreonine protein kinase (ROCK) signaling pathway, which is pivotal in the process of intestinal inflammation and epithelial barrier dysfunction [18].

Animals.
Healthy male Balb/C mice, weighing 22-25 g, were purchased from the Laboratory Animal Center of Guangzhou University of Chinese Medicine (Guangzhou, China). The animals were housed under standard environmental conditions where the temperature (23 ± 1°C), lighting condition (12 h light/dark cycles), and humidity (40-60%) are tightly controlled. The animals had free access to standard diet and water ad libitum. Animal procedures used in this study were in line with institutional guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).
Body weight was measured daily by using a digital weight scale. After 7 days, all mice were sacrificed after being fasted for 12 h and anesthetized by carbon dioxide (CO 2 ) inhalation; then, the blood was collected immediately. Colon tissues were quickly removed, and its length was measured. Then, the colon tissues were collected for morphological observation, Western Blot, ELISA, and quantitative real-time polymerase chain reaction (qRT-PCR).
2.5. Evaluation of Disease Activity Index (DAI). The index of disease activity was evaluated according to a previously reported procedure [12]. Briefly, DAI was scored as follows: DAI = ðscore of body weight loss + score of stool properties + score of hematocheziaÞ/3. The criteria for evaluation are listed in Table 1. 2.6. Cytokine Analysis by ELISA. The levels of TNF-α, IL-6, IL-1β, IFN-γ, and IL-10 in colon tissues were measured quantitatively by using ELISA kits in accordance with the manufacturer's protocol. The levels of cytokine were quantified by standard curves.

Histological
Analysis. The colon tissues were immediately fixed with 10% buffered formalin following harvest, then  [19] and is shown in Table 2.
2.8. Western Blotting Analysis. The colon segments from each group were homogenized and lysed using cold Pro-prep protein lysis buffer (Intron Biotechnology, Seoul, Republic of Korea) with addition of protease inhibitor cocktail and phosphatase inhibitors, for 30 min on ice. The tissue lysates were centrifuged, and the supernatant was collected. The BCA assay kit (Thermo Scientific Pierce, IL, USA) was used to determine the protein concentrations of the supernatant. Equal amounts of protein extract for each sample were separated in 8% or 10% of sodium dodecyl sulfate-(SDS-) polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membranes were blocked with 5% skimmed milk for 1 h at room temperature.
After that, the membranes were incubated overnight at 4°C with primary antibodies (dilution, 1 : 1000) against ZO-1, MLC, PMLC, claudin-1, claudin-2, p65, p-p65, RhoA, p-  RhoA, E-cadherin, ROCK-1, and β-actin. The membranes were washed with TBS-T, followed by incubation with appropriate horseradish peroxidase-(HPR-) conjugated secondary antibody (dilution, 1 : 5000) for 1 h at room temperature. Next, the membranes were rinsed 3 times with TBS-T buffer and the signals of protein level were visualized using a Western imaging system. The intensity of the protein bands was quantified using Image J software. β-Actin was used as an internal control for total proteins.

Quantitative Real-Time Polymerase Chain Reaction
(qRT-PCR). qRT-PCR was implemented as described previously [20]. Briefly, the colon samples were homogenized with Trizol reagent. And then, total RNA was extracted according to the manufacturer's instructions. Quantitated RNA was reverse-transcribed into cDNA, and quantitative real-time PCR amplification was performed using the SYBR Green reagent to examine the mRNA expressions of ZO-1, occludin, and claudin-1. The primer sequences are listed in Table 3.
2.10. Statistical Analysis. The results were analyzed by using GraphPad Prism software 8.0 (GraphPad Software, La Jolla, CA, USA) and presented as mean ± standard error of mean (SEM). The numbers of mice in each group were shown as "n" number. Statistical significance was identified by using the one-way analysis of variance (ANOVA). P < 0:05 was considered to be statistically significantly different.

BE-BJO Alleviated DSS-Induced Colitis in Mice.
Firstly, the existence of brusatol was examined in BE-BJO and BF-BJO. As demonstrated in Figure 1, brusatol was detected in BE-BJO, but not BF-BJO. To investigate the potential therapeutic effect of BE-BJO and BF-BJO on UC, the mice were treated with 3% DSS for 7 days to induce colitis. The body weights of the mice were tested; DAI including body weight loss, stool properties, and hematochezia were evaluated; and the length of the colon was measured. As shown in Figure 2, the DSS model group demonstrated a significant decrease in body weight, an increase in DAI scores, and a shortening of colon length. Treatment with BE-BJO alleviated DSS-induced colitis in a dose-dependent manner, as implied by the rebound of body weight and colon length, and the improvement of DAI ( Figure 2). The effects of BE-BJO were similar as that of the positive control drug 5-ASA and BJO (Figure 2). By contrast, treatment with BF-BJO did not display protective effects against DSS-induced colitis ( Figure 2). Thus, these observations suggest that BE-BJO, but not BF-BJO, is the active constituent from BJO that alleviates DSS-induced colitis in mice.  BioMed Research International scores were evaluated. DSS induced a destruction of the colon architecture, which was exhibited by obvious necrosis, epithelial damage or shedding, and submucosal infiltration of inflammatory cells, accompanied by crypt hyperplasia (Figure 3(a)). Treatment of BJO, 5-ASA, and BE-BJO (305, 610 mg/kg/day), but not BF-BJO, significantly improved the collapse of the colon structure and infiltration of inflammatory cells (Figure 3(a)). As shown in Figure 3(b), the histopathological score of the DSS group was significantly higher than that of the normal group; treatment with BJO and 5-ASA significantly reduced the histopathological score as compared to the DSS group; treatment of BE-BJO (305 and 610 mg/kg/day) significantly reversed DSS-induced increase of histopathology score, with a similar trend as BJO and 5-ASA. These results confirm that BE-BJO prevents colon histopathological change induced by DSS.

BE-BJO Attenuated the Levels of Proinflammatory
Cytokines in the Colon of DSS-Treated Mice. Colon inflammation plays a key role in the development of UC [21][22][23][24]. The effects of BE-BJO on the production of proinflammatory cytokines, including TNF-α, IL-1β, IFN-γ, and IL-6, were examined in the colon tissue of DSS-treated mice. As displayed in Figure 4, the levels of these proinflammatory cytokines were significantly upregulated in colorectums of the DSS group; treatment of BE-BJO dose-dependently reversed DSS-induced production of proinflammatory cytokines. In addition, the anti-inflammatory effect of BE-BJO at a high dose (610 mg/kg/day) was equivalent to that of BJO and 5-ASA ( Figure 4). Taken together, these data indicate that BE-BJO could ameliorate colon inflammation in the DSS model.

BE-BJO Restored the Impairment of Intestinal Epithelial
Barrier Function in DSS-Induced Colitis. Since UC is characterized by damage of intestinal mucosal barrier function, therapeutics that could enhance intestinal mucosal healing is a major goal of UC therapy [20,25]. The intestinal epithelial barrier function depends on the integrity of the mucus layer, which is determined by the expression and assembly of tight junction proteins [26,27]. The tight junction complex is constituted by transmembrane proteins, like occludin and the claudin family, and by linker proteins, like zonula occludens-1 (ZO-1), that affiliate with the actin cytoskeleton [28]. To examine the effect of BE-BJO on intestinal mucosal healing, the expressions of tight junction proteins were   Figure 5, treatment of DSS for 7 days resulted in a remarkable decrease in the protein expressions of ZO-1, occludin, claudin-1, and E-cadherin but augmented the expression of claudin-2 as compared to the control group. Treatment of BE-BJO reversed the effect of DSS on expressions of these tight junction proteins in a dose-dependent manner. A high dose of BE-BJO shares a parallel effect with BJO and the positive drug 5-ASA. These observations imply that BE-BJO could enhance intestinal epithelial barrier function by altering the expression levels of tight junction proteins.

BE-BJO Suppressed NF-κB Activation in DSS-Induced
Colitis. The phosphorylation of p65 (p-p65) is a hallmark of NF-κB activation and leads to colon inflammation during the pathogenesis of UC [29,30]. Thus, the levels of p-p65 and total p65 in the colon tissues of mice were determined by Western Blot. The results demonstrated that DSS treatment significantly upregulated the expression of p-p65, without altering the expression of total p65 ( Figure 6). Treatment with 5-ASA, BJO, and BE-BJO (152.5, 305, and 610 mg/kg/day) significantly reduced the level of p-p65 and the ratio of p-p65/p65 ( Figure 6). Thus, the results indicate that the BE-BJO could repress NF-κB activation through inhibiting the phosphorylation of p65.
3.6. BE-BJO Inhibited the Activation of the RhoA/ROCK Signaling Pathway in DSS-Induced Colitis. RhoA/ROCK is closely associated with the regulation of adhesion, migration, and proliferation of intestinal crypt cells, thus playing a pivotal role in the development of UC [7]. We therefore  (Figures 7(a) and 7(d)). Therefore, these data confirm that BE-BJO could suppress the RhoA/ROCK signaling pathway.

Discussion
BJO is well-accepted to have antidiarrheal properties and has been reported to have an anti-inflammatory effect in UC disease models, thus suggesting therapeutic potential in the treatment of UC [12]. Besides quassinoids including brusatol and bruceine D, BJO also contains oleic acid, linoleic acid, stearic acid, palmitic acid, arachidonic acid, and other unsaturated fatty acids [31]. In order to elucidate whether or not these components contribute to the anti-UC properties of BJO, the present study compared the effects of BE-BJO and BF-BJO in the DSS-induced UC model. According to our observations, BE-BJO alleviated DSS-induced loss of body weight, an increase of DAI, and a shortening of colon, whereas BF-BJO did not have these protective effects. The effect of BE-BJO is equivalent to that of BJO. It thus suggests that quassinoids in particular brusatol, but not oleic acid or other unsaturated fatty acids, serve as the active components for the anti-UC properties of BJO. UC is a progressive alimentary system disorder that is characterized by dysregulated immune response, chronic inflammation, unbalanced gut microbiota (dysbiosis), and defective mucosal barrier function [32,33]. The present study further explored the effects and mechanisms of BE-BJO on colon inflammation and intestinal epithelial barrier function.
In line with our previous observations [11,12], the present results showed that treatment of BE-BJO suppressed the production and release of proinflammatory cytokines including TNF-α, IFN-γ, IL-6, and IL-1β in the colon tissue. TNF-α is a proinflammatory molecule that mediates multiple physiological and pathological processes of UC. Excess production of TNF-α leads to the secretion of chemokines by colonic epithelial cells and damage of epithelial barrier [34,35]. IL-1β emerges as a requisite role in the development of inflammatory reaction, and the increase of IL-1β level can cause autoimmune process and then impairs colon tissue [36,37]. IL-6 is an immunomodulatory cytokine that participates in the progression of UC [38,39]. IL-6 and IL-1β are team players in the development of UC. INF-γ is an activator of phagocytes and neutrophil with immunomodulatory activity [40]. Thus, inhibiting the release of these NF-κB is pivotal in the inflammatory cascades during the pathogenesis of colitis [41]. The p65 subunit is the major functional subunit in NF-κB. In response to stimulation, p65 undergoes phosphorylation and is transferred to the nucleus, where it binds to promoters of target genes to enhance the inflammatory response [6,42]. Our results demonstrated that BE-BJO dose-dependently reversed DSSinduced p65 phosphorylation, thus suggesting that BE-BJO could repress NF-κB activation.
Moreover, the present study indicates that the anti-UC effect of BE-BJO is attributed to improvement of intestinal stability and structural integrity by enhancing tight junctions. The primary structure of intestinal barrier is the apical junctional complex, which consists of the tight junctions and the subjacent adherent junctions that contribute to apical-basal cell polarity maintenance and to cell signaling events [43].
The tight junction complex is constituted by transmembrane proteins such as occludin and the claudin family and by linker proteins like ZO-1 [28]. These proteins regulate the permeability and integrity of intestinal mucosal barrier [44,45]. In this study, BE-BJO rebounded the decreased expressions of ZO-1, occludin, claudin-1, and E-cadherin induced by DSS but augmented claudin-2 expression, thus indicating that BE-BJO could regulate the expressions of colonic epithelial barrier-associated proteins, thereby repairing the damaged colonic mucosa during UC. Taken into considerations that therapeutics targeting enhancing intestinal mucosal healing is a major goal of UC therapy [25,46], the present findings provide important evidence for the therapeutic potential of BJO in the treatment of UC.
To elucidate the mechanisms underlying the effect of BJO on intestinal epithelial barrier function, we tested the possible involvement of RhoA/ROCK signaling. ROCK and myosin light chain kinases (MLCK) are involved in the , and E-cadherin (f) Western Blotting bands. The mRNA expression of occludin (g), ZO-1 (h), and claudin-1 (i) and claudin-2 (j) in colon tissue. All values are presented as the mean ± SEM. ## P < 0:01 and # P < 0:05 versus normal group; * P < 0:05, * * P < 0:01, and * * * P < 0:001 versus DSS group. 9 BioMed Research International regulation of tight junctions during the intestinal inflammatory response [47,48]. Activated RhoA/ROCK signals deactivate myosin phosphatase (MLCP), leading to failure of dephosphorylation of MLC, resulting in increased levels of intracellular phosphorylation and increased actin-myosin cross-linking, finally promoting the polymerization of the actin microfilaments [49,50]. In addition, RhoA/ROCK elevates MLC phosphorylation levels. The present study   All values are presented as the mean ± SEM. ## P < 0:01 versus normal group; * P < 0:05, * * P < 0:01, and * * * P < 0:001 versus DSS group. 10 BioMed Research International indicates that BE-BJO inhibits the RhoA/ROCK signaling pathway. This conclusion is supported by the following observations: (1) BE-BJO repressed DSS-induced activation of RhoA; (2) BE-BJO treatment downregulated ROCK expression; and (3) BE-BJO prevented phosphorylation of MCL in DSS-treated mice. Since the RhoA/ROCK signaling pathway is associated with colon inflammation and regulation of intestinal epithelial barrier function, it is probably that BE-BJO ameliorated UC by repressing RhoA/ROCK. Intriguingly, there is a crosstalk between NF-κB and RhoA/ROCK which work together to accelerate the progression of UC [33,51], the inhibitory effect of BE-BJO on both signaling pathways might help to explain the remarkable anti-UC effect of BE-BJO.

Conclusion
In conclusion, the present study has shown that BJO enriched with brusatol could ameliorate DSS-induced UC by preventing colon inflammation and enhancing intestinal epithelial barrier function. Mechanistically, these anti-UC effects of BE-BJO are probably associated with repression of NF-κB and RhoA/ROCK signaling pathways. These findings confirm that brusatol is the active compound from BJO and suggest the therapeutic potential of brusatol and BE-BJO in the treatment of UC. Disease activity index DSS: Dextran sulfate sodium Elisa: Enzyme-linked immunosorbent assay HE: Hematoxylin-eosin HPLC: High-performance liquid chromatography IBD: Inflammatory bowel disease IFN: Interferon IL-1β: Interleukin-1β IL-6: Interleukin-6 MLC: Myosin light chain MLCK: Myosin light chain kinase NF-κB: Nuclear factor kappa-B RT-PCR: Reverse transcription-polymerase chain reaction ROCK: Rho-associated kinase TGF-β: Transforming growth factor-β TNF-α: Tumor necrosis factor-α TJ: Tight junction UC: Ulcerative colitis ZO: Zonula occluden.

Data Availability
The data that support the findings of this study are available from the corresponding author upon request.