Gastroprotective effects of Kangfuxin against water-immersion and restraint stress-induced gastric ulcer in rats: roles of antioxidation, anti-inflammation, and pro-survival

Abstract

Context: Kangfuxin (KFX) is widely used for the treatment of gastric and duodenal ulcer; however, more research is needed to determine the protective mechanisms of KFX in ameliorating gastric ulcer.Objective: To investigate the efficacy and potential mechanism of Kangfuxin liquid (KFX) in water-immersion and restraint stress (WIRS)-induced gastric ulcer.Materials and methods: Seventy rats were randomly divided into seven groups (n = 10) as follows: the control group (normal saline, i.g.), the model group (normal saline, i.g.), the KFX groups (2.5, 5 and 10 mL/kg, i.g.), the omeprazole group (20 mg/kg, i.p.) and Sanjiuweitai Granules group (1850 mg/kg, i.g.). The WIRS model was applied to induce stress ulcers after 7 days of drug administration. Afterwards, rats were sacrificed at 10 h induced by WIRS.Results: Pre-treatment with KFX (5,10 mL/kg) could effectively reduce the area of gastric ulcers and improve the pathological changes of ulcerated tissue. Moreover, KFX (5,10 mL/kg) increased the prostaglandin E2 (52%) and cyclooxygenase-1 (30%) levels, and improved malondialdehyde (54%), superoxide dismutase (58%), catalase (39%), and nitric oxide (11%) and TNF-α (9%), IL-6 (11%), MMP-9 (54%) and MMP-2 (53%) of ulcer tissue. Furthermore, pre-treatment with KFX dramatically increased IGF-1, PTEN, and Akt protein expression.Conclusions: Our results suggest that KFX has protective effects on WIRS-induced gastric ulcer via inflammatory reactions, oxidative stress inhibition, and pro-survival action, which were the results of activating the IGF-1/PTEN/Akt signalling pathway. Our results provide evidence of KFX for treating gastric ulcer.

Keywords: IGF-1/PTEN/Akt signalling; KFX; Periplaneta americana L.; gastric diseases.

Figure 1.

Effects of KFX on the macroscopic morphology and gastric ulcer area of the gastric mucosa in WIRS-treated rats. The data were expressed as the means ± SD (n = 5). ^##p < 0.01, compared with the control group; *p < 0.05, **p < 0.01 compared with the model group. KFX: Kangfuxin liquid; SJWT: Sanjiuweitai Granules; OME: Omeprazole.

Figure 2.

Histological assessment of the effects of KFX on the acute gastric mucosal injuries in WIRS-challenged rats. (A) The gastric tissue was fixed with 4% paraformaldehyde and sectioned to prepare for HE staining. The gastric tissue sections were routinely dehydrated, and then stained with haematoxylin and eosin followed by observation with a microscope. (B) The statistic results of gastric microscopic damage scores. The data were expressed as the means ± SD (n = 5). KFX: Kangfuxin liquid; SJWT: Sanjiuweitai Granules; OME: Omeprazole.

Figure 3.

The effect of KFX pre-treatment on MDA, SOD, CAT, GSH-Px activities in the gastric tissues of WIRS-induced rat ulcer model. The data were expressed as the means ± SD (n = 5). ^##p < 0.01, compared with the control group; *p < 0.05, **p < 0.01 compared with the model group. KFX: Kangfuxin liquid; SJWT: Sanjiuweitai Granules; OME: Omeprazole.

Figure 4.

KFX modulated inflammatory factors expression in WIRS-induced gastric ulcer rats. (A and B) The gastric tissue homogenate was prepared to determine the levels of TNF-α, MMP-2, PGE2 and NO by ELISA. (C) The gastric samples were prepared with lysis buffer, resolved on 10% SDS-PAGE, and transferred to a PVDF membrane. Western blots were performed to detect the total or MMP-9, IL-6 and using specific primary antibodies. (D) Densitometric analysis was used to quantify the levels of TNF-α, IL-6 and MMP-9. (E) The total RNA in gastric tissues was extracted to detect the COX-1, COX-2, IL-6, TNF-α, MMP-9, and GAPDH mRNAs by QPCR. The data were expressed as the means ± SD (n = 5). ^##p < 0.01, compared with the control group; *p < 0.05, **p < 0.01 compared with the model group. KFX: Kangfuxin liquid; SJWT: Sanjiuweitai Granules; OME: Omeprazole.

Figure 5.

Effects of KFX on COX-1 and COX-2 expression levels in stomach tissue of WIRS-treated rats. (A, B) Photomicrographs of immunohistochemical staining for determination of COX-1 and COX-2 protein expression in gastric mucosa. (C) Quantitative assessment of COX-1 and COX-2 protein expression. The data were expressed as the means ± SD (n = 5). ^##p < 0.01, compared with the control group; *p < 0.05, **p < 0.01 compared with the model group. KFX: Kangfuxin liquid; SJWT: Sanjiuweitai Granules; OME: Omeprazole.

Figure 6.

KFX activated IGF-1/Akt pathway in WIRS-induced ulcer rats. (A) The gastric samples were prepared with lysis buffer, resolved on 10% SDS-PAGE, and transferred to a PVDF membrane. Western blots were performed to detect the total or Akt, p-Akt, FoxO1, PTEN, IGF-1 using specific primary antibodies. (B) Densitometric analysis was used to quantify the levels of p-Akt/Akt, FoxO1, PTEN, IGF-1. The data were expressed as the means ± SD (n = 5). ^##p < 0.01, compared with the control group; *p < 0.05, **p < 0.01 compared with the model group. KFX: Kangfuxin liquid.