Andrographolide derivative CX-10 alleviates ulcerative colitis by modulating autophagy and cellular stress

Abstract

Ulcerative colitis (UC) is a debilitating inflammatory bowel disease that poses significant challenges in clinical management. Despite existing therapies, many patients fail to achieve adequate symptom relief, underscoring the need to address the underlying mechanisms contributing to the pathogenesis of UC. Andrographis paniculata has been extensively studied in traditional Chinese medicine for its anti-inflammatory properties. This study aimed to evaluate the effects of CX-10, a derivative of andrographolide, on autophagy, oxidative stress, and inflammation in UC. Using dextran sulfate sodium (DSS)-induced mouse model of UC, our findings demonstrated that CX-10 treatment resulted in significant reductions in body weight loss, Disease Activity Index (DAI), and histopathological injury scores, characterized by decreased inflammatory cell infiltration and mucosal damage compared to DSS-treated controls. Quantitative real-time PCR (qRT-PCR) revealed a marked restoration of autophagy-related genes Becn1 and Atg5 in CX-10-treated colonic tissues. Western blot analysis further confirmed enhanced autophagic flux, evidenced by significant increases in the LC3-II/I ratio. CX-10 treatment also led to reduced endoplasmic reticulum (ER) stress, indicated by decreases in the transcript and protein levels of GRP78 and CHOP. Consistent with the in vivo findings, in vitro studies demonstrated that CX-10 effectively enhanced autophagy and reduced oxidative stress in lipopolysaccharides (LPS)-treated HT-29 colonic epithelial cells and RAW 264.7 macrophages. This was accompanied by a marked decrease in reactive oxygen species (ROS) levels, as determined by DCFDA assays. In conclusion, CX-10 exerts protective effects against DSS-induced UC through modulation of autophagy and oxidative stress pathways, suggesting its potential as a novel therapeutic agent for managing UC.

Introduction

Ulcerative colitis (UC) is a chronic inflammatory bowel disease (IBD) characterized by inflammation and ulceration of the colonic mucosa, which significantly affects the quality of life and often requiring long-term treatment.⁽¹⁾ Despite the availability of various therapeutic approaches, including aminosalicylates, corticosteroids, and biologics, many patients fail to achieve sustained remission, highlighting the need for novel therapeutic strategies.⁽²⁾ The pathogenesis of UC is multifactorial, involving genetic factors, dysregulation of the immune response, increased intestinal permeability, and altered microbial composition.^(3–5) Recent studies have emphasized the critical role of cellular stress responses, particularly autophagy and oxidative stress, in the development and progression of UC.^(6,7)

Autophagy is an evolutionarily conserved process in eukaryotes, responsible for degrading damaged organelles and proteins within lysosomes, thereby maintaining cellular homeostasis and regulating immune responses.⁽⁸⁾ Dysregulation of autophagy has been implicated in the pathophysiology of IBD, contributing to exacerbated inflammation and tissue damage.⁽⁹⁾ For instances, during bacterial infections, cytoplasmic vesicles engulf pathogens to form autophagic vesicles, limiting the pathogens’ nutrient access.⁽¹⁰⁾ This process preserves cell survival by protecting intestinal epithelial cells and macrophages from bacterial toxins.⁽¹¹⁾ In summary, impaired autophagy leads to intestinal cell dysfunction, microbial imbalance (dysbiosis), and an unchecked immune response, ultimately resulting in intestinal inflammation.^(12,13)

Oxidative stress, caused by excessive production of reactive oxygen species (ROS), plays a key role in the progression of IBD by damaging cellular structures, activating pro-inflammatory pathways, and compromising the integrity of the intestinal epithelial barrier. Increased ROS levels during inflammation activate macrophages, which further intensify the inflammatory response. This, in turn, triggers the release of pro-inflammatory cytokines and further ROS production. This vicious cycle of ROS and inflammation creates self-perpetuating loop that drives the pathogenesis of IBD.⁽¹⁴⁾ Although prior research has explored the impact of pharmacological agents on autophagy and oxidative stress, there remains a significant gap in understanding how specific natural compounds can modulate these pathways effectively.

Andrographis paniculata (AP), a medicinal plant widely used in traditional medicine, has gained attention for its potent anti-inflammatory and immunomodulatory properties.⁽¹⁵⁾ The primary bioactive compound of AP, andrographolide, has been extensively studied for its therapeutic potential in various inflammatory diseases, including IBD. Andrographolide exerts its effects by modulating key signaling pathways involved in inflammation, such as nuclear factor-kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases (MAPKs).^(16–18) A derivative of andrographolide, 3,14,19-triacetyl andrographolide (CX-10), has hydroxyl groups at C-3, C-14, and C-19 that can be acylated simultaneously or selectively. CX-10, a monomeric compound, has been found to have more potent anti-inflammatory, anti-tumourigenic, and immunosuppressive properties than andrographolide.⁽¹⁹⁾ It has emerged as a promising therapeutic agent due to its anti-inflammatory, antioxidant, and neuroprotective effects.^(17,20) However, research on CX-10 remains limited.

Therefore, this study aimed to investigate the effects of CX-10 on autophagy and oxidative stress in both in vivo murine models and in vitro human colonic epithelial cells and murine macrophages. We hypothesized that CX-10 would enhance autophagic activity and reduce oxidative stress, thereby ameliorating the severity of dextran sulphate sodium (DSS)-induced colitis or LPS-induced inflammation. Our findings indicate that CX-10 not only alleviates colitis symptoms but also modulates key cellular pathways involved in UC pathogenesis, suggesting its potential as a therapeutic agent for managing this debilitating condition.

Materials and Methods

UC mouse model

Male C57BL/6 mice (6–8 weeks old) were purchased from Shanghai Slac Laboratory Animal Co., Ltd, (Shanghai, China). Mice were randomly assigned to four groups: (1) Control, (2) DSS alone, (3) DSS + low-dose CX-10, and (4) DSS + high-dose CX-10. UC was induced by administering 3% DSS (molecular weight 36,000–50,000; MP Biomedicals, Santa Ana, CA) in drinking water for 7 days, followed by regular water for 5 days. Mice in the CX-10 treatment groups received either low (50 ‍mg/kg) or high (200 ‍mg/kg) doses of CX-10 (Sigma-Aldrich, St. Louis, MO) via oral gavage once daily at a dose of 0.2 ‍ml/10 ‍g for 1–8 days. DSS and CX-10 were co-administered. Control mice received regular drinking water without DSS.

Samples collection

Mice were euthanized and sacrificed at the end of experiment, and the full-length colon was carefully removed and rinsed with normal saline. Colon length and weight were measured as indirect markers of inflammation, with images captured for further analysis. These parameters were compared across all experimental groups. Portions of the colon tissue were fixed with 10% formaldehyde and embedded in paraffin for histological analysis using H&E staining. The remaining tissue samples were stored at −80°C for RNA extraction and protein analysis using Western blotting.

Assessment of colitis severity

Body weight, stool consistency, and gross bleeding were recorded daily to monitor colitis progression. The disease activity index (DAI) was assessed using an established scoring system, as described in Table 1.^(17,21) For histopathological examination, colon tissues were fixed in 10% formalin for 48 ‍h, dehydrated using ethanol, cleared in xylene, and embedded in paraffin. Thin sections (4 ‍μm) were prepared using a microtome (Kedi), deparaffinized, rehydrated through alcohol gradients, and stained with hematoxylin for 5 ‍min and eosin for 10 ‍min. After dehydration, images were captured using an Olympus microscope. Histopathological changes were scored by a blinded pathologist using the criteria outline in Table 2.^(17,21)

Table 1.

Disease activity index (DAI) scoring system

Weight loss (%)	Stool consistency	Occult/gross bleeding	DAI score
0	Normal	Normal	0
1–5	Soft		1
6–10	Loose	Hemoccult positive	2
11–15			3
>15	Diarrhea	Gross bleeding	4

Table 2.

Histopathological scoring system

Histological parameters	Score
Inflammation severity
None	0
Slight	1
Moderate	2
Severe	3
Inflammation extent
None	0
Mucosa	1
Mucosa and submucosa	2
Transmural	3
Crypt damage
None	0
Basal 1/3 damaged	1
Basal 2/3 damaged	2
Only surface epithelium intact	3
Entire crypt and epithelium lost	4

Cell culture and LPS treatment

HT-29 human colonic epithelial cells and RAW 264.7 murine macrophage cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HT-29 cells were cultured in McCoy’s 5A Medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, while RAW 264.7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS and 1% penicillin-streptomycin. Both cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO₂. To induce inflammation, HT-29 cells and RAW 264.7 cells were treated with 1 ‍μg/ml and 100 ‍ng/ml of lipopolysaccharide (LPS; Sigma-Aldrich), respectively, for 24 ‍h.

CX-10 treatment

CX-10 (C₂₆H₃₆O8, MW: 476), a hemi-synthetic compound derived from andrographolide, was provided by Shandong Target Drug Research Co. Ltd. (Shandong, China). Mice received either a low dose (50 ‍mg/kg) or high dose (200 ‍mg/kg) of CX-10 via oral gavage at a volume of 0.2 ‍ml/10 ‍g body weight once daily for 1–8 days, co-administered with DSS. HT-29 and RAW 264.7 cells were treated with 10 ‍μM or 50 ‍μM of CX-10 for 24 ‍h to assess its effect in vitro.

RNA extraction and qRT-PCR

Mouse colon tissues were homogenised using the FastPrep-24TM Tissue Homogeniser (MP Biomedicals). Total RNA was extracted from the colon tissues, HT-29, and RAW 264.7 cells using RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was then reverse transcribed into cDNA using the PrimeScript^TM RT reagent kit (Takara Bio, Kusatsu, Japan). qRT-PCR was performed using TaqMan Fast Advanced Master Mix (Life Technologies, Carlsbad, CA) on an ABI StepOnePlus^TM Real-Time PCR System (Applied Biosystems, Walfham, MA). The mRNA levels of autophagy-related genes (Becn1 and Atg5) and stress response genes [Hspa5 (also known as Grp78) and Ddit3 (also known as Chop)] were measured. B2m was used as an internal control for normalization. The relative expression levels were calculated using the 2^−ΔΔCt method. Primers (Life Technologies) used in this study are as follows: B2m (Mm00437762_m1), human B2M (Hs00984230_m1), Becn1 (Mm01265461_m1), BECN1 (Hs01007018_m1), Atg5 (Mm01187303_m1), ATG5 (Hs00355494_m1), Hspa5 (Grp78; Mm01187303_m1), HSPA5 (Hs99999174_m1), Ddit3 (Chop; Mm01135937_g1), and DDIT3 (Hs00358796_g1).

Western blotting

Proteins were extracted from colon tissues and cultured cells using RIPA buffer (Thermo Fisher Scientific, Waltham, MA) supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were then separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA). Membranes were blocked with 5% non-fat milk in TBST (Tris-buffered saline containing 0.1% Tween-20) for 1 ‍h at room temperature, followed by overnight incubation at 4°C with primary antibodies. The following primary antibodies were used: anti-LC3 and anti-Beclin-1 (Abcam, Cambridge, UK), and GRP78, CHOP, and β-actin from (Cell Signaling Technology, Danvers, MA). After washing, the membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 1 ‍h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Bio-Rad Laboratories, Hercules, CA). The LC3-II/I ratio was calculated, and protein expression levels were normalized to β-actin.

ROS measurement

ROS levels in HT-29 cells were assessed using the DCFDA/H₂DCFDA Cellular ROS Assay Kit (Abcam) according to the manufacturer’s protocols. Briefly, cells were incubated with 10 ‍μM DCFDA at 37°C for 30 ‍min in the dark. Following incubation, cells were washed and imaged using a fluorescence microscope (Leica Microsystems, Wetzlar, Germany) to capture fluorescence images of DCFDA-stained cells. Fluorescence intensity, reflecting ROS levels, was quantified using ImageJ software. Results were expressed as relative fluorescence units (RFU) and compared to untreated controls.

Statistical analysis

All statistical tests and graphing were performed using GraphPad Prism 10. Data are expressed as mean ± SEM, and all experiments were conducted in triplicate. Group comparisons were analyzed using one-way ANOVA followed by Tukey’s post hoc test. A p value of less than 0.05 was considered statistically significant.

Results

CX-10 ameliorates DSS-induced UC in mice

The therapeutic potential of CX-10, a derivative of andrographolide, was evaluated using DSS-induced mouse model of UC. To assess the progression of colitis and the protective effects of CX-10, various parameters were measured, including body weight loss, DAI, colon length, and colon weight. CX-10 treatment was administered at both low (50 ‍mg/kg) and high doses (200 ‍mg/kg) to determine its dose-dependent effects.

In the control group, mice exhibited consistent weight gain over the 7-day experimental period, reaching a 7% gain by Day 7 (107% of their initial weight). In contrast, DSS-treated mice displayed a decline in body weight, starting from Day 2, with a total decrease of 25% by Day 7 (75% of initial weight), indicative of severe colitis (Fig. 1A). Treatment with CX-10 significantly mitigated this weight loss in a dose-dependent manner. By Day 7, the low-dose CX-10 group experienced a 14% weight reduction (86% of initial weight), while the high-dose group showed only a 10% reduction (90% of initial weight) (p<0.05 compared to DSS group) (Fig. 1A), highlighting the protective effect of CX-10, especially at the higher dose.

Fig. 1.

CX-10 attenuates DSS-induced ulcerative colitis in mice. (A) Percentage of initial body weight measured over 7 days in control, DSS-treated, and CX-10-treated mice at low (50 ‍mg/kg) and high (200 ‍mg/kg) doses. (B) Disease Activity Index (DAI) scores recorded daily over 7 days for control, DSS-treated, and CX-10-treated mice (low and high doses). (C) Representative images of colons harvested from control, DSS-treated, and CX-10-treated mice on Day 7, illustrating differences in colon length and appearance. (D) Quantification of colon length on Day 7 across all groups. (E) Colon weight normalized to length (mg/cm) on Day 7. Data are presented as mean ± SEM (n = 6 mice per group); One-way ANOVA followed by Tukey’s post-hoc test, *p<0.05 compared to the DSS group; **p<0.01 compared to the DSS group; ***p<0.001 compared to the DSS group; ^#p<0.05 compared to the control group; ^##p<0.01 compared to the control group; ^###p<0.001 compared to the control group.

In parallel, the DAI, a composite measure of weight loss, stool consistency, and rectal bleeding, was monitored daily. Control mice maintained low DAI scores between 0.25 to 0.34 throughout the study (Fig. 1B). DSS-treated mice, on the other hand, showed a progressive increase in DAI, beginning at 0.35 on Day 1 and peaking at 3.78 by Day 7, confirming the severity of colitis. CX-10 treatment significantly reduced DAI in a dose-dependent manner. While mice in the low-dose group exhibited a final DAI of 3.26 by Day 7, which was not significantly different from the DSS group, interestingly, those in the high-dose group had a much lower DAI of 1.25 by Day 7 (p<0.001 compared to the DSS group) (Fig. 1B), demonstrating the efficacy of high dose of CX-10 in reducing colitis severity.

Colon length, a well-established marker of colonic inflammation and disease severity in DSS-induced colitis, was also assessed. DSS-treated mice showed pronounced colonic shortening, with an average colon length of 5.6 ‍cm, compared to 7.6 ‍cm in control mice (Fig. 1C and D). CX-10 treatment ameliorated this colonic shortening in a dose-dependent manner. Mice in the low-dose group had an average colon length of 6.0 ‍cm, while those in the high-dose group displayed near-complete recovery, with an average colon length of 7.1 ‍cm (p<0.05 compared to DSS group), and the colon length of the high-dose group was not statistically different from that of control mice (Fig. 1D), indicating substantial protection against colonic damage.

Further assessment of colonic inflammation and edema was performed by measuring colon weight normalized to length (mg/cm). DSS-treated mice exhibited significantly increased colon weight, averaging 32 ‍mg/cm, indicative of severe inflammation and edema, compared to 24 ‍mg/cm in control mice (p<0.001) (Fig. 1E). CX-10 treatment led to a dose-dependent reduction in colon weight, with the low-dose group averaging 30 ‍mg/cm and the high-dose group 28 ‍mg/cm (p<0.05 compared to the DSS group) (Fig. 1E). These data suggest the ability of CX-10 to alleviate inflammation-induced colonic swelling at a high dose.

Taken together, these findings demonstrate that andrographolide derivative CX-10, provides significant protection against DSS-induced UC in mice. CX-10 treatment mitigates body weight loss, reduces disease activity, and restores colon length and weight towards normal levels. Notably, the high dose of CX-10 produced the most pronounced therapeutic benefit, highlighting a dose-dependent response in mitigating the severity of colitis.

CX-10 reduces histopathological injury score in UC mouse model

Histological analysis of colon tissues on Day 7 demonstrated significant differences in mucosal injury and inflammation among the various treatment groups, highlighting the protective effects of CX-10 in UC mouse models.

In the control group, colon sections (Fig. 2A) displayed normal mucosal architecture, with no signs of inflammation or epithelial damage. The intact epithelial layer and well-preserved mucosal structure resulted in a histological score of 0. In contrast, the DSS-treated group (Fig. 2B) exhibited severe inflammation and extensive mucosal damage. This group showed significant epithelial injury, loss of goblet cells, and marked infiltration of inflammatory cells, leading to significantly elevated histological scores (8.5 ± 0.22, p<0.001 compared to the control group; Fig. 2E). The histological damage was much more pronounced, reflecting the detrimental effects of DSS in inducing colitis.

Fig. 2.

CX-10 reduces histopathological damage in a UC mouse model. (A, D) Representative images of haematoxylin and eosin (H&E)-stained colonic sections from the control group (A), DSS-treated group (B), DSS + low-dose CX-10 group (50 ‍mg/kg) (C), and DSS + high-dose CX-10 group (200 ‍mg/kg) (D). (E) Quantification of histopathological injury scores in colonic tissues across the experimental groups. Data are presented as mean ± SEM (n = 6 mice per group); One-way ANOVA followed by Tukey’s post-hoc test, **p<0.01 compared to the DSS group; ^##p<0.01 compared to the control group; ^###p<0.001 compared to the control group.

Treatment with a low dose of CX-10 (Fig. 2C and E) led to moderate improvements in mucosal integrity. The histological sections (Fig. 2C) indicated reduced epithelial damage and decreased inflammatory cell infiltration compared to the DSS group. Despite some remaining mucosal damage and not significantly different from the DSS group, the histological scores were reduced (5.0 ± 0.26, p<0.01 compared to the control group). The high dose DSS + CX-10 treatment (Fig. 2D and E) resulted in the most significant recovery. Colon sections (Fig. 2D) exhibited near-complete restoration of the epithelial layer, with minimal inflammatory cell infiltration. This group achieved an approximately 71% reduction in histological scores compared to the DSS group (2.5 ± 0.22, p<0.01), indicating significant improvement in mucosal health and a more robust protective effect against DSS-induced colitis.

Overall, these results indicate that CX-10 significantly reduces histopathological injury in a dose-dependent manner, with the high-dose treatment showing the most pronounced protective effects in mitigating UC-induced mucosal injury.

CX-10 modulates autophagy and cellular stress in colonic tissues of a UC mouse model

Given the critical role of autophagy and cellular stress responses in the pathogenesis of UC, we investigated the effects of CX-10 on these pathways in colonic tissues harvested from the UC mice. DSS treatment significantly downregulated the expression of key autophagy-related genes, Becn1 (Beclin-1) and Atg5 (autophagy protein 5), reducing their levels to 0.5- and 0.6-fold, respectively, compared to the control (p<0.001) (Fig. 3A and B). CX-10 treatment, particularly at higher dose, significantly restored the expression of these genes. Specifically, high-dose CX-10 increased Becn1 expression by 1.5-fold and Atg5 by 1.6-fold compared to the control (p<0.001 vs DSS for both). Even at a low dose, CX-10 significantly elevated Becn1 and Atg5 transcript levels compared to the DSS group (p<0.001).

Fig. 3.

CX-10 regulates autophagy and alleviates cellular stress in colonic tissues of a UC mouse model. (A, B) Histograms showing the transcript levels of autophagy-related genes Becn1 and Atg5 in control, DSS-treated, DSS + low-dose (50 ‍mg/kg) CX-10, and DSS + high-dose (200 ‍mg/kg) CX-10 mice. (C) Representative Western blot images showing the protein levels of LC3-I and LC3-II across the control, DSS-treated, DSS + low-dose CX-10, and DSS + high-dose CX-10-treated groups. Protein levels were normalized to β-actin. (D) Quantification of the LC3-II/I ratio in these groups. (E, F) Histograms displaying the transcript levels of ER stress-related genes Hspa5 (GRP78) and Ddit3 (CHOP) in the control, DSS-treated, DSS + low-dose CX-10, and DSS + high-dose CX-10 groups. (G) Western blot images showing the levels of ER stress markers GRP78 and CHOP in the same groups. (H, I) Quantification of GRP78 and CHOP protein levels, normalized to β-actin, across all experimental groups. Data are presented as mean ± SEM (n = 6 mice per group); One-way ANOVA followed by Tukey’s post-hoc test, **p<0.01 compared to the DSS group; ***p<0.001 compared to the DSS group; ^#p<0.05 compared to the control group; ^##p<0.01 compared to the control group; ^###p<0.001 compared to the control group.

At the protein level, Western blot analysis supported these findings, revealing a significant reduction in the LC3-II/I ratio in DSS-treated mice, down to 0.26-fold compared to controls (p<0.001) (Fig. 3C and D), indicating impaired autophagic flux. CX-10 treatment dose-dependently restored autophagy, with low-dose CX-10 increasing the LC3-II/I ratio by 1.2-fold (p<0.001 vs DSS), and high-dose CX-10 increasing it by 1.5-fold (p<0.001 vs DSS) (Fig. 3C and D).

Beyond autophagy, we also investigated the effect of CX-10 on markers of endoplasmic reticulum (ER) stress, specifically Hspa5 (GRP78) and Ddit3 (CHOP). DSS treatment significantly elevated the mRNA level of Hspa5 and Ddit3 (p<0.001 vs control) (Fig. 3E and F), indicating increased ER stress. CX-10 treatment, particularly at high doses, effectively reduced the transcription levels of both markers in a dose-dependent manner compared to DSS-treated mice (p<0.001 vs DSS).

Western blot analysis further corroborated these results, showing a substantial increase in GRP78 and CHOP protein levels in DSS-treated mice, with 3.3-fold and 2.4-fold increases, respectively, compared to controls (p<0.001) (Fig. 3G–I). High-dose CX-10 significantly decreased the protein levels of GRP78 and CHOP to 1.8-fold and 1.2-fold, respectively, levels that were not statistically different from the controls, but significantly lower compared to the DSS group (p<0.01). However, low-dose CX-10 had no significant effect on GRP78 and CHOP protein levels, suggesting that higher doses of CX-10 are required to effectively modulate ER stress.

Overall, these findings demonstrate that CX-10 enhances autophagy and alleviates ER stress in UC mice in a dose-dependent manner, with high-dose CX-10 providing more pronounced protective effects.

Dose-dependent effects of CX-10 on autophagy and stress responses in human colonic epithelial cells and murine macrophages

To further elucidate the molecular mechanisms underlying the protective effects of CX-10, we conducted in vitro experiments using HT-29 human colonic epithelial cells and RAW 264.7 murine macrophages, both exposed to lipopolysaccharides (LPS) to mimic the oxidative stress and inflammation characteristic of UC.

In HT-29 cells, LPS significantly downregulated BECN1 and ATG5 mRNA expression to 0.5-fold and 0.6-fold of control levels, respectively (p<0.001) (Fig. 4A and B). Treatment with CX-10, at both low and high doses, significantly restored the expression of these genes compared to LPS-treated cells (p<0.001) (Fig. 4A). This restoration was confirmed at the protein level by Western blot analysis. LPS exposure caused a notable decrease in the LC3-II/I ratio, reducing it to 0.4-fold compared to controls (p<0.001), indicating disrupted autophagic flux. Both low- and high-dose CX-10 significantly restored the LC3-II/I ratio in LPS-treated cells (p<0.001) (Fig. 4B and C), suggesting that CX-10 enhances autophagy under inflammatory conditions.

Fig. 4.

CX-10 modulates autophagy and stress responses in human colonic epithelial HT-29 cells and RAW 264.7 murine macrophages in a dose-dependent manner. (A) Histograms showing the transcript levels of autophagy-related genes BECN1 and ATG5, alongside ER stress marker genes HSPA5 (GRP78) and DDIT3 (CHOP), in the untreated control, LPS-treated, LPS + low-dose (10 ‍μM) CX-10, and LPS + high-dose (50 ‍μM) CX-10-treated HT-29 cells. (B) Representative Western blot images showing the protein levels of LC3-I, LC3-II, GRP78, and CHOP across untreated control, LPS-treated, LPS + low-dose CX-10, and LPS + high-dose CX-10-treated HT-29 cells. Protein levels were normalized to β-actin. (C) Quantification of the LC3-II/I ratio in these groups. (D, E) Quantification of GRP78 and CHOP protein levels, normalised to β-actin, across all experimental groups. (F) The transcript levels of autophagy-related genes Becn1 and Atg5, alongside ER stress marker genes, Hspa5 and Ddit3, in untreated control, LPS-treated, LPS + low-dose (10 ‍μM) CX-10, and LPS + high-dose (50 ‍μM) CX-10-treated RAW 264.7 macrophages. (G) Representative Western blot images showing the protein levels of LC3-I, LC3-II, GRP78, and CHOP across untreated control, LPS-treated, LPS + low-dose CX-10, and LPS + high-dose CX-10-treated RAW 264.7 macrophages. Protein levels were normalized to β-actin. (H) Quantification of the LC3-II/I ratio in these groups. (I, J) Quantification of GRP78 and CHOP protein levels, normalized to β-actin, across all experimental groups. Data are presented as mean ± SEM (n = 3 independent experiments); One-way ANOVA followed by Tukey’s post-hoc test, *p<0.05 compared to the LPS group **p<0.01 compared to the LPS group; ***p<0.001 compared to the LPS group; ^#p<0.05 compared to the control group; ^##p<0.01 compared to the control group; ^###p<0.001 compared to the control group.

Similar effects were observed in RAW 264.7 macrophages. LPS significantly suppressed Becn1 and Atg5 transcript levels compared to controls (p<0.01), but CX-10 treatment restored their expression (Fig. 4F). Western blotting also confirmed a reduced LC3-II/I ratio in LPS-treated macrophages, which was significantly improved by both low and high doses of CX-10 (p<0.001) (Fig. 4G and H).

In addition to its effects on autophagy, CX-10 modulated ER stress markers in both cell types. LPS treatment significantly increased the mRNA and protein expression of Hspa5 (GRP78) and Ddit3 (CHOP) (Fig. 4A, B, D–G, I, and J). High-dose CX-10 markedly reduced Hspa5 and Ddit3 mRNA levels in HT-29 cells and RAW 264.7 macrophages (p<0.001 vs LPS) (Fig. 4A and F). At the protein level, Western blotting revealed significantly elevated GRP78 and CHOP in LPS-treated HT-29 cells and macrophages, but high-dose CX-10 significantly lowered their expression (p<0.05 vs LPS) (Fig. 4B, D, E, G, I, and J). Although low-dose CX-10 also decreased GRP78 and CHOP protein levels, these reductions were not statistically significant.

Collectively, these findings suggest that CX-10 effectively counteracts LPS-induced autophagy suppression and alleviates ER stress in both human colonic epithelial cells and murine macrophages. High-dose CX-10 showed the most significant improvements in autophagic activity and reductions in ER stress markers, highlighting its potential therapeutic role in controlling cellular stress responses in inflammatory conditions.

CX-10 reduces ROS production in HT-29 human colonic epithelial cells

To further explore the effects of LPS and CX-10 on oxidative stress, we measured intracellular ROS levels in HT-29 cells using the DCFDA assay. LPS treatment resulted in a significant increase in ROS production, with RFU rising to 343 compared to 100 RFU in the untreated control group (p<0.001) (Fig. 5A and B). CX-10 treatment effectively reduced ROS levels in a dose-dependent manner. Specifically, low-dose CX-10 lowered the RFU to 241 (p<0.01 vs LPS), while high-dose CX-10 almost normalized ROS levels, reducing RFU to 117 (p<0.001 vs LPS) (Fig. 5A and B).

Fig. 5.

CX-10 reduces ROS production in human colonic epithelial cells. (A) Representative images of DCFDA-stained HT-29 cells in the untreated control, LPS-treated, LPS + low-dose (10 ‍μM) CX-10, and LPS + high-dose CX-10 groups. Fluorescent signals were captured using a Leica fluorescence microscope. (B) Quantification of relative fluorescence units (RFU), expressed as percentage relative to the untreated controls. Data are presented as mean ± SEM (n = 3 independent experiments); One-way ANOVA followed by Tukey’s post-hoc test, **p<0.01 compared to the LPS group; ***p<0.001 compared to the LPS group; ^###p<0.001 compared to the control group.

These findings indicate that CX-10 not only modulates autophagy and stress response pathways but also significantly reduces oxidative stress, contributing to its overall anti-inflammatory effects in colonic epithelial cells.

Discussion

This study investigates the therapeutic potential of CX-10, a derivative of andrographolide, in addressing key cellular processes implicated in UC, particularly autophagy, oxidative stress, and ER stress. Our findings demonstrate that CX-10 exerts protective effects against colitis both in vivo and in vitro, using DSS-induced UC mouse model, human colonic epithelial cells (HT-29), and murine macrophages. These results highlight CX-10 as a promising candidate for UC therapy, providing new mechanistic insights into the modulation of pathways central to disease pathology.

UC is a chronic inflammatory condition of the colon, characterized by mucosal inflammation that leads to symptoms such as abdominal pain, diarrhea, and rectal bleeding.⁽²²⁾ Although various treatments are available, a significant proportion of patients fail to achieve long-term remission, and current therapies often have adverse effects, including immune suppression and increased infection risk.^(2,23) This underscores the urgent need for more effective therapies with better safety profiles. Emerging evidence identifies autophagy, oxidative stress, and ER stress as key contributors to UC pathogenesis.^(9,14) Autophagy, a process involved in cellular homeostasis and inflammation regulation, is often impaired in UC, leading to exacerbated inflammation and tissue damage.⁽²⁴⁾ Additionally, oxidative stress, driven by excessive ROS, further worsens the inflammatory environment, while ER stress promotes the unfolded protein response (UPR), contributing to ongoing inflammation.⁽¹⁴⁾ Given CX-10’s known anti-inflammatory and antioxidant properties,^(17,19,20) we hypothesized that it could modulate these pathways and offer a novel therapeutic option for UC.

In our study, CX-10 significantly ameliorated colonic inflammation in a DSS-induced UC mouse model, as demonstrated by improvements in body weight, DAI, colon length, and weight. Notably, high-dose CX-10 (200 ‍mg/kg) was more effective than the low dose, indicating a dose-dependent efficacy. These findings align with previous studies on natural compounds that modulate inflammation and oxidative stress.⁽²⁵⁾ At the molecular level, CX-10 restored autophagy in DSS-treated mice. DSS-treated mice displayed downregulation of autophagy-related genes (Becn1 and Atg5) and a reduced LC3-II/I ratio, indicative of impaired autophagic flux. CX-10 treatment, particularly at higher doses, reversed these alterations, restoring both autophagy-related gene expression and LC3-II/I ratios. This supports previous findings that autophagy plays a protective role in IBD and that restoring autophagic activity may help resolve inflammation.^(7,24)

In addition to its effects on autophagy, CX-10 alleviated ER stress in both in vivo and in vitro models. DSS treatment significantly upregulated ER stress markers GRP78 and CHOP, which was reversed by CX-10 treatment. The higher dose of CX-10 was particularly effective in reducing these markers, further supporting a dose-dependent response. This is consistent with studies showing that reducing ER stress can mitigate inflammation and protect colonic tissues in UC.^(26–28) Furthermore, CX-10 effectively reduced oxidative stress in HT-29 cells and murine macrophages exposed to LPS. LPS-induced ROS production was significantly reduced by CX-10 in a dose-dependent manner, further highlighting its antioxidant potential.

The ability of CX-10 to target multiple pathways involved in UC pathogenesis suggests its potential as a broad-spectrum therapeutic agent with anti-inflammatory effects. By restoring autophagic flux, CX-10 may help reduce intestinal inflammation and promote tissue repair. Its antioxidant and ER stress-modulating properties further enhance its anti-inflammatory action, positioning CX-10 as a multifunctional therapeutic candidate for chronic inflammatory diseases such as UC. Importantly, the dose-dependent effects observed in this study suggest that optimizing CX-10 dosing could maximize its therapeutic benefits while minimizing potential side effects.

However, several limitations of this study should be noted. While this study provides compelling evidence that CX-10 alleviates UC by modulating autophagy, oxidative stress, and ER stress, the underlying molecular mechanisms require further exploration. Future studies should investigate specific signaling pathways involved, such as the mTOR pathway in autophagy and the UPR pathways in ER stress. The interrelationship between autophagy, oxidative stress, and ER stress in the context of CX-10 treatment was not directly examined in this study. However, our findings suggest potential connections. Specifically, the observed restoration of autophagic flux by CX-10 may contribute to the reduction of oxidative and ER stress, as autophagy is known to mitigate cellular stress by degrading damaged organelles and proteins. Future investigations should aim to delineate the precise molecular crosstalk between these pathways to better understand the therapeutic mechanisms underlying CX-10’s efficacy in UC.

Furthermore, although our findings demonstrate the effects of CX-10 on colonic epithelial cells and macrophages, we did not investigate its impact on lymphocytes, which are also critical contributors to UC pathogenesis. Future studies should aim to explore how CX-10 influences lymphocyte subsets, including T and B cells, and their associated cytokine profiles to provide a more comprehensive understanding of its immunomodulatory potential.

In this study, we demonstrated CX-10’s efficacy in a murine model and in vitro cell lines, clinical studies in human UC patients are necessary to confirm its therapeutic potential. The long-term effects of CX-10 treatment, particularly on the gut microbiota, which plays a significant role in UC pathogenesis, also warrant further investigation. Finally, exploring the potential synergistic effects of CX-10 with existing UC therapies could provide valuable insights into its use in combination therapies.

In conclusion, CX-10 demonstrates significant therapeutic potential for UC by modulating key processes such as autophagy, oxidative stress, and ER stress in a dose-dependent manner. These findings suggest that CX-10 could offer a novel, multifunctional approach for treating UC and other chronic inflammatory diseases. Future studies focused on clinical trials and mechanistic pathways will be crucial in determining the full therapeutic potential of CX-10 in human patients.

Author Contributions

ZC and XT designed and conducted the study. ZC, YHe, YHong, FY, and TG supervised the data collection. ZC, YHe, YHong, FY, and TG analyzed the data. ZC, YHe, YHong, FY, and TG interpreted the data. ZC and XT prepared and reviewed the manuscript. All authors have read and approved the manuscript.

Funding

This work was supported by National Natural Science Foundation of China (Grant No. 8167151491).

Ethics Approval

Ethical approval was obtained from the Ethics Committee of Tongde Hospital of Zhejiang Province.

Data Availability

The authors declare that all data supporting the findings of this study are available within the paper and any raw data can be obtained from the corresponding author upon request.

Conflict of Interest

No potential conflicts of interest were disclosed.