Cyanidin-3-O-Glucoside Improves Colonic Motility During Severe Acute Pancreatitis by Inhibiting the H₂S-Regulated AMPK/mTOR Pathway

Introduction

Acute pancreatitis (AP) is an inflammatory process that occurs in an otherwise healthy pancreas and exhibits wide clinical variation.¹ Although most cases of AP are mild, ~20% of patients with AP develop a severe form of the disease characterized by organ dysfunction,¹ known as severe acute pancreatitis (SAP).² Most patients with SAP experience a diverse range of severe symptoms,³ but current management strategies overlook the key role of intestinal function during SAP development. Previous studies have demonstrated that bacterial infection and intestinal organ sepsis are important factors in SAP development;^4,5 thus, increasing research is being conducted regarding novel therapies to limit colonic injuries.^6–9

Previous studies have reported that H₂S is produced predominantly by cystathionine-γ-lyase (CSE) and other kinases in the transsulfuration pathway. Moreover, H₂S initiates distinct biological responses in the human body,^10–12 including the gastrointestinal (GI) tract, where H₂S is produced by both GI tissues and gut bacterial flora.^13,14 H₂S production was also reported to inhibit GI motility in a fish model.^13,14 Substantial H₂S is produced during SAP attacks, where it inhibits inflammation in the GI system during SAP progression.^15,16 Therefore, H₂S produced during SAP attacks is hypothesized to be associated with impaired intestinal mobility, and modulating H₂S production may be a novel strategy for managing SAP-associated colonic motility loss in clinical settings.

Previous studies investigating the events associated with colonic motility loss have furthered the development of novel therapeutic approaches. For example, dietotherapy is attracting increased attention for its efficacy in improving digestive system functioning with few adverse effects.^17,18 Moreover, anthocyanins, which belong to the flavonoid family, are widely distributed in vegetables and other foods that are part of the human diet¹⁹ and may carry health benefits owing to their antioxidant and anti-inflammatory properties.²⁰ Anthocyanins also affect the intestinal system; thus, studies have focused on their potential to modulate and improve the microflora in the GI tract.²¹ However, no previous studies examining the interaction between anthocyanins and the GI system have assessed the effect of anthocyanins on intestinal motility. As one of the most abundant natural anthocyanins, cyanidin-3-O-glucoside (C3G) contributes to modulating numerous biological processes, particularly those involved in immunoregulation.^22–24 C3G exerts its functions in the GI tract via multiple mechanisms; therefore, the present study was conducted to investigate the protective effect of C3G against SAP-induced colonic motility loss by focusing on the effect of C3G on H₂S and its downstream pathways.

To evaluate the study’s hypothesis, rats were injected with sodium taurocholate to induce SAP, then C3G was administered. The effects of C3G on colonic motility, H₂S production, and inflammatory cytokine levels were detected to identify the exact effect of C3G on SAP-induced colonic motility loss. Moreover, the changing pattern of H₂S-mediated AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling²⁵ was detected to elucidate the mechanism by which C3G restores colonic mobility. To validate the in vivo assay results, colonic muscle cells (CMCs) were isolated and subjected to plasma isolated from SAP-model rats, then C3G and the AMPK inhibitor, MK-3903, were used to elucidate the interaction between C3G and the H₂S-mediated AMPK/mTOR pathway.

Materials and Methods

Chemicals and Antibodies

C3G (purity >98%; cat. no. HY-N0640) was purchased from MedChemExpress. Antibodies against CSE (cat. no. 12,217-1-AP) were purchased from ProteinTech Group, Inc. Antibodies against total (t)-AMPK (cat. no. ab32047), phosphorylated (p)-AMPK (cat. no. ab23875), total (t)-mTOR (cat. no. ab2732), p-mTOR (cat. no. ab109268) and GAPDH (cat. no. ab181602) were purchased from Abcam. Secondary antibodies (cat. no. A0277, goat anti-rabbit; cat. no. A0208, goat anti-rabbit; cat. no. A0216, goat anti-murine) were provided by Beyotime Institute of Biotechnology. MK-3903 (an AMPK activator) was purchased from Sigma-Aldrich (Merck KGaA). The AMPK activator, MK-3903 (cat. no. HY-107,988), was purchased from MedChemExpress.

SAP-Model Rats and C3G Administration

Adult male Wistar rats weighing 200–250 g were housed per routine protocols and grouped into the sham, SAP or SAP + C3G groups, with n=10 rats per group. The sham group was induced with SAP without injecting the corresponding agents. The SAP group rats were anesthetized with 50 mg/kg body weight pentobarbital sodium, subjected to a laparotomy, then injected with 5% sodium taurocholate into the pancreatic and bile ducts using a microinjection pump (1 mL/kg, 0.1 mL/min) for 10 min. The incision was then sutured (the overall survival rate of the SAP-model rats was ~60% over a 24-hour period; Figure S1). The SAP + C3G group rats were gavaged with 100 mg/kg body weight C3G and injected with sodium taurocholate. Before the subsequent assays, all rats were injected with 10 mL normal saline and fasted for 24 h. All animal experiments were conducted in accordance with the Institutional Animal Ethics Committee and Animal Care Guidelines for the Care and Use of Laboratory Animals of Southwest Hospital of Army Medical University (Ref no. A-20,170,505) and the Guide for the Care and Use of Laboratory Animals (1985), NIH, Bethesda, MD, USA, or the European Guidelines on Laboratory Animal Care.

Colonic Motility Measurements

Colonic motility was assessed by measuring fecal pellet output numbers before and after SAP induction²⁶ and detected 1 h prior to model induction and 4, 8, 12, 16, 20, and 24 h after model induction. Upon completing the measurements, the rats were euthanized with an overdose of pentobarbital sodium (200 mg/kg).

Measurement of Serum H₂S Levels

Serum H₂S levels were measured according to a previous study.²⁷ Aliquots (75 μL) of sera were mixed with 100 μL distilled water and 300 μL 10% trichloroacetic acid. The reaction was stopped with 150 μL of 1% zinc acetate. N,N-dimethyl-p-phenylenediamine sulfate (20 μM) in 7.2 M HCl and FeCl₃ (30 μM; 133 μL) in 1.2 M HCl were then added to the mixture and incubated for 15 min. The absorbance at 670 nm was measured, and the H₂S concentration was calculated.

Detecting the Inflammatory Response

After the colonic motility measurements, the rats were euthanized with an overdose of pentobarbital sodium (200 mg/kg body weight), and plasma and colonic muscle tissue samples were collected. Tumor necrosis factor (TNF)-α (cat. no. H052) and interleukin (IL)-6 (cat. no. H007) production in the samples was detected using enzyme-linked immunosorbent assay (ELISA) kits (Nanjing Jiancheng Bio-engineering Institute Co., Ltd.) per the manufacturer’s instructions.

Western Blotting

Extracted protein was subjected to routine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then primary antibodies against CSE (1:500), t-AMPK (1:2000), p-AMPK (1:1000), t-mTOR (1:2000), p-mTOR (1:1000), and GAPDH (1:1000) were incubated on polyvinylidene difluoride membranes at 4°C overnight. After incubation with secondary horseradish peroxidase-conjugated IgG antibodies (1:5000), the relative protein expression levels were calculated using Gel-Pro-Analyzer (Media Cybernetics, Inc.).

Cell Preparation and Administration

CMCs were separated from the mucous membrane of each rat’s proximal colon and cultured in solution containing 0.15% collagenase II, 0.1% trypsin inhibitor, and 0.25% fetal bovine serum at 37°C. CMCs were identified via immunofluorescence detection of calponin and α-SMA (Figure S2), then treated with different combinations of 90 mM H₂S solution,²⁸ 10 μg/mL C3G and 5 μmol/l MK-3903 for 24 h. Inflammatory responses in the CMCs were detected as described above.

Ca²⁺ Assay

CMCs (1×10⁶) were subjected to repeated freezing and thawing to release the intracellular components. The suspension was then centrifuged at 3000 rpm for 20 min to collect the supernatant. The Ca²⁺ concentration was detected using an ELISA kit (Shanghai Keshun Biotechnology Co., Ltd.) per the manufacturer’s instructions.

Statistical Analysis

Data are presented as the mean ± standard deviation. One-way analysis of variance followed by Duncan’s post hoc multiple comparisons test were conducted. Statistical analyses were conducted, and graphs were created using GraphPad Prism 6 (GraphPad Prism Software, Inc.). P<0.05 was considered statistically significant.

Results

Administering C3G Improved Colonic Motility in SAP-Model Rats

The effect of C3G on colonic motility in the rats was assessed by measuring the fecal pellet output among the rats. Before inducing SAP, fecal pellet output numbers did not differ between the groups (sham vs SAP: P=0.993; sham vs SAP + C3G: P=0.991; SAP vs SAP + C3G: P=0.968; Figure 1). After inducing SAP, symptoms were induced via sodium taurocholate, and significant differences were detected between the sham and SAP groups after 4 h (P=0.04). C3G administration significantly alleviated colonic motility loss after 14 h (14 h: P=0.000; 24 h: P=0.000; Figure 1), indicating that C3G administration improved the colonic motility during SAP progression.

Figure 1 C3G administration improved colonic motility in SAP-model rats. *P<0.05 vs sham group; #P<0.05 vs SAP group. Abbreviations: C3G, cyanidin-3-O-glucoside; SAP, severe acute pancreatitis.

C3G Suppressed the Inflammatory Response and H₂S Levels in SAP-Model Rats

Inducing SAP increased the levels of H₂S (P=0.001; Figure 2A), TNF-α (P=0.000) and IL-6 (P=0.000; Figure 2B and C). H₂S suppressed the inflammatory response; however, its anti-inflammatory effect was offset by its negative effect on colonic motility. Thus, C3G administration inhibited both H₂S and inflammatory cytokine production (H₂S: P=0.048; TNF-α: P=0.006; IL-6: P=0.004; Figure 2).

Figure 2 C3G administration suppressed H2S production and cytokine levels in SAP-model rats. Serum (A) H2S, (B) TNF-α and (C) IL-6 levels. *P<0.05 vs sham group; #P<0.05 vs SAP group. Abbreviations: C3G, cyanidin-3-O-glucoside; SAP, severe acute pancreatitis; TNF, tumor necrosis factor; IL, interleukin.

Administering C3G Inhibited CSE Levels and Deactivated the AMPK/mTOR Signaling Pathway in SAP-Model Rats

To identify the mechanism by which SAP improved colonic motility, AMPK/mTOR pathway activity was detected. SAP increased the CSE expression (P=0.000) and activated the AMPK/mTOR pathway by increasing p-AMPK levels (P=0.000) and the p-AMPK/t-AMPK ratio (P=0.000) and suppressing p-mTOR levels (P=0.000) and the p-mTOR/t-mTOR ratio (P=0.000; Figure 3). Moreover, C3G reversed the expression patterns of these indicators (CSE: P=0.001; p-AMPK: P=0.014; p-mTOR: P=0.035; p-AMPK/t-AMPK ratio: P=0.003; p-mTOR/t-mTOR ratio: P=0.018); thus, the effect of C3G on colonic motility was associated with inhibiting the H₂S/AMPK/mTOR pathway.

Figure 3 C3G administration suppressed CSE expression and deactivated the AMPK/mTOR pathway. *P<0.05 vs sham group; #P<0.05 vs SAP group. Abbreviations: C3G, cyanidin-3-O-glucoside; SAP, severe acute pancreatitis; CSE, cystathionine-γ-lyase.

Activation of AMPK/mTOR Blocked the Protective Effect of C3G Against SAP Plasma-Induced CMC Impairments

To further assess the pathway underlying the effects of C3G, CMCs were isolated and subjected to H₂S solution, C3G, and an AMPK activator in different combinations. H₂S administration increased CSE expression (P=0.000) and activated AMPK/mTOR signaling in CMCs (p-AMPK: P=0.000; p-mTOR: P=0.002; p-AMPK/t-AMPK: P=0.000; p-mTOR/t-mTOR: P=0.011; Figure 4A), which also induced cytokine production (TNF-α: P=0.000; IL-6: P=0.000; Figure 4B and C) and suppressed Ca²⁺ accumulation (P=0.002; Figure 4D) in CMCs. C3G administration suppressed CSE expression (P=0.000) and AMPK/mTOR pathway activity (p-AMPK: P=0.000; p-mTOR: P=0.041; p-AMPK/t-AMPK: P=0.002; p-mTOR/t-mTOR: P=0.016), which contributed to inhibiting the inflammatory response (TNF-α: P=0.008; IL-6: P=0.000) and restored Ca²⁺ accumulation (P=0.011). However, the AMPK activator impaired the protective effect of C3G against SAP plasma, which reactivated the AMPK/mTOR pathway by increasing p-AMPK (P=0.001) and p-AMPK/t-AMPK (P=0.004) levels but suppressing p-mTOR (P=0.043) and p-mTOR/t-mTOR (P=0.024) levels, which also contributed to increasing the CSE level (P=0.029; Figure 4A). These changes in CSE and AMPK/mTOR signaling increased TNF-α (P=0.041) and IL-6 (P=0.023; Figure 4B and C) production and decreased Ca²⁺ production (P=0.025; Figure 4D). Thus, the protective effect of C3G against SAP-induced colonic motility loss depended on H₂S-mediated AMPK/mTOR pathway inhibition.

Figure 4 AMPK activation offset the effect of C3G on H2S-treated CMCs. (A) CSE levels and AMPK activity, (B) TNF-α levels, (C) serum IL-6 levels, (D) Ca2+ accumulation. &P<0.05 vs CMC + H2S + C3G group.

Discussion

H₂S is the third member of gasotransmitter family synthesized endogenously via the transsulfuration pathway, which is an important mechanism for providing cells with cysteine.^29,30 Being increasingly recognized as a functionally relevant mediator of a number of physiological functions, deficiencies in the H₂S production can cause a chronic inflammatory response by inducing pro-inflammatory molecule production, thus resulting in development of various diseases.²⁹ Regarding the protective effects on GI system, H₂Scan decrease production of TNF-α and leukocytes.¹⁵ However, the Being increasingly recognized as a functionally relevant mediator of a number of physiological functions. Tamizhselvi et al³¹ revealed that H₂S induced inflammation in AP rats. Therefore, the functions and related mechanisms of H₂S in GI diseases should be assessed.

Consistent with previous studies,^16,32,33 SAP symptoms initiated CSE synthesis and increased H₂S levels. The enhanced release of H₂S should be associated with a weakened inflammatory response, but it seemed that the anti-inflammation effects of H2S were blocked by its and suppressive effects on ed colonic motility during SAP progression. However, C3G administration suppressed the plasma cytokine levels and improved suppressed colonic motility in SAP rats. Therefore, an interaction was hypothesized to have occurred among C3G, H₂S, and inflammation: H₂S exerted an anti-inflammatory effect during SAP progression, but its positive effect was offset by its negative effect on colonic motility in SAP rats. C3G administration improved colonic motility by suppressing H₂S production. In the meanwhile, the anti-inflammatory effects of C3G compensated for the lack in anti-inflammatory factors induced by the deficient production of H₂S. Thus, applying C3G as a treatment agent for impairments associated with SAP not only improved the colonic motility loss but also contributed to the control of inflammatory response.

H₂S regulates multiple pathways. In the present study, activity of the AMPK/mTOR pathway was detected to examine the signaling pathway mediating the effect of C3G. The results indicated that SAP and H₂S solution induced AMPK/mTOR pathway activity both in vivo and in vitro, whereas C3G inhibited this activity. In addition, the CMCs were also treated with AMPK activator MK-3903. The set of the MK-3903 group was employed to validate that the effects of C3G were dependent on the inhibition of AMPK/mTOR pathway. Activation of the AMPK/mTOR pathway in CMCs impaired the protective effect of C3G against H₂S, increased the cytokine production, and inhibited Ca²⁺ accumulation in CMCs, indicating suppressed motility potential in the cells. The results clearly demonstrated the inhibition of AMPK/mTOR pathway was indispensable for the protective effects of C3G on SAP-induced colonic motility loss. Thus, the changes in AMPK/mTOR pathway partially explained the hypothesis we proposed above: the interaction between C3G and H₂S influenced the activation of AMPK signaling, which finally led to the improved colonic motility and suppressed inflammatory responses associated with SAP initiation.

In conclusion, the in vivo and in vitro assay results demonstrated that administering C3G increased colonic motility in rats by suppressing H₂S production. Moreover, the effect of C3G depended on the H₂S-mediated AMPK/mTOR pathway. H₂S administration and AMPK activation impaired the motility potential of CMCs, even after C3G administration. However, the present study examined only the downstream pathways involved in the protective effect of C3G against SAP on the GI system. Therefore, further studies are required to improve our understanding of the mechanisms underlying C3G functions.