Capsaicin Inhibits Inflammation and Gastric Damage during H pylori Infection by Targeting NF-kB-miRNA Axis

Abstract

Helicobacter pylori (H. pylori) infection is considered as one of the strongest risk factors for gastric disorders. Infection triggers several host pathways to elicit inflammation, which further proceeds towards gastric complications. The NF-kB pathway plays a central role in the upregulation of the pro-inflammatory cytokines during infection. It also regulates the transcriptional network of several inflammatory cytokine genes. Hence, targeting NF-kB could be an important strategy to reduce pathogenesis. Moreover, treatment of H. pylori needs attention as current therapeutics lack efficacy due to antibiotic resistance, highlighting the need for alternative therapeutic approaches. In this study, we investigated the effects of capsaicin, a known NF-kB inhibitor in reducing inflammation and gastric complications during H. pylori infection. We observed that capsaicin reduced NF-kB activation and upregulation of cytokine genes in an in vivo mice model. Moreover, it affected NF-kB-miRNA interplay to repress inflammation and gastric damages. Capsaicin reduced the expression level of mir21 and mir223 along with the pro-inflammatory cytokines. The repression of miRNA further affected downstream targets such as e-cadherin and Akt. Our data represent the first evidence that treatment with capsaicin inhibits inflammation and induces antimicrobial activity during H. pylori infection. This alternative approach might open a new avenue in treating H. pylori infection, thus reducing gastric problems.

Keywords: H. pylori; NF-kB; capsaicin; cytokines; inflammation; miRNA.

Figure 1

Capsaicin ameliorates H. pylori induced inflammation in both in vitro and in vivo conditions. (A) ELISA was performed with the media collected from Control (CON), H. pylori infected (HP) and H. pylori infected + Capsaicin treated (HP + Caps) and Capsaicin (Caps) treated samples to check the levels of cytokines IL-6 and TNFα. AGS gastric cells were pretreated with capsaicin (100 μM) for 2 h and then infected with H. pylori (MOI 100) for 4 h. (B) Serum samples collected from control (CON), infected (HP) and infected mice treated with capsaicin (HP + Caps) were subjected to ELISA, showing the effect of capsaicin treatment in the levels of cytokines IL-6 and TNFα in H. pylori infected mice. Data are represented as mean ± SEM. Values as tested by ANOVA followed by Tukey’s post hoc test. ‘*’, ‘**’, and ‘***’ represent significant differences between groups at p < 0.05, p < 0.01, and p < 0.001, respectively. Experiments were performed in triplicates. (C) Mice were treated with antibiotic cocktail every day for 7 days. After an interval of 1 week, mice were infected with H. pylori by gastric intra-gavage every alternate day for a week. After 14 days of quarantine period, mice in the H. pylori inoculated group were treated with capsaicin (5 mg/kg body weight) for 40 days as the H. pylori + capsaicin-treated group. At the end of the treatment regime, all mice were sacrificed, and gastric tissues and serum were collected.

Figure 2

Treatment with capsaicin affects the expression of NF-kB regulated genes. (A–D) Capsaicin treatment decreased the mRNA expression levels of IL6, TNFα, IL-1β and NLRP3 as compared to infected tissues. Values were normalized to GAPDH as housekeeping gene for indicated genes. Control (CON), H. pylori infected (HP), H. pylori infected + Capsaicin treated (HP + Caps). (E) Immunoblot showing the expression of phospho NF-κB and NF-κB after capsaicin treatment in H. pylori infected mice. Experiments were performed in triplicates. α Tubulin was used as internal control. Densitometry of representative blot was performed. Data are represented as mean ± SEM. Values were tested by ANOVA followed by Tukey’s post hoc test. ‘*’and ‘**’ represent significant differences between groups at p < 0.05, p < 0.01, respectively.

Figure 3

Capsaicin reduces NF-kB-regulated miRNA expression and H. pylori infection. (A,B) Real-time PCR showing the expression levels of miR21 and mir223 in capsaicin-treated mice samples as compared to H. pylori infected samples. In case of miRNAs, U6 was used as control to normalize miRNAs. Control (CON), H. pylori infected (HP), H. pylori infected + Capsaicin treated (HP + Caps). (C) Real-time PCR was performed to detect H. pylori specific 16s ribosomal DNA. (D) Control (CON), H. pylori infected (HP), H. pylori infected + Capsaicin treated (HP + Caps) and only Capsaicin (CAPS) treated AGS cells were subjected to NF-kB promoter assay. Promoter assay was performed to show the activation of NF-kB binding to the promoter sequence. (E) Western blot showing H. pylori infection increased expression of virulence factor CagA, while capsaicin treatment (100 μM) decreased it significantly in AGS cell line. β actin was used as loading control. Densitometry of representative blot was performed, and values are tested by ANOVA followed by Tukey’s post hoc test. ‘*’, ‘**’ and ‘***’ represent significant differences between groups at p < 0.05, p < 0.01, p < 0.001, respectively.

Figure 4

Capsaicin exposure affects e-cadherin and pAkt expression. E-cadherin, phospho Akt and Akt levels were determined by Western blot in control (CON), H. pylori infected (HP) and capsaicin treated gastric tissue samples (HP + Caps). Densitometric analysis was performed keeping total Akt and α tubulin as the internal control. Data are represented as mean ± SEM. Values as tested by ANOVA followed by Tukey’s post hoc test. ‘*’, ‘**’, and ‘***’; represent a significant difference between groups at < 0.05, p < 0.01, p < 0.001, respectively.

Figure 5

Capsaicin reduced gastric tissue damage. (A) H. pylori infection causes overall inflammation along with distortion of gastric tissue. Inflammatory changes are represented by black arrows. Capsaicin treatment ameliorates the effects of H. pylori infection significantly. Control gastric tissue at 10× and 20× (left panel-CON). H. pylori treated gastric tissue at 10× and 20× (middle panel-HP) and capsaicin-treated infected gastric tissue (Right panel-HP + Caps) after H. pylori infection in mice. (B) Capsaicin binds strongly to NF-κB promoter binding domain. 3D representation of docking complexes of NF-κB with capsaicin as visualized in BIOVIA discovery studio after docking. Docking was performed using Patchdock and Firedock web servers. (C) 2D representation of docked complexes to show hydrogen bonds and amino acid interactions.