Clopidogrel-Induced Gastric Injury in Rats is Attenuated by Stable Gastric Pentadecapeptide BPC 157

Abstract

Aim: Although Clopidogrel is safe in healthy volunteers, it can induce recurrence of gastric ulcers in high-risk patients. Here, we investigated the protective effect of the natural product, stable gastric pentadecapeptide 157 (BPC 157) on Clopidogrel-induced gastric injury.

Methods: We used acetic acid to induce gastric ulcer in Sprague Dawley rats. Clopidogrel alone or in combination with BPC 157 or L-NAME (nitric oxide system blockade) were administered after healing of acetic acid-induced ulcer. One percent methylcellulose solution was used as control. Ulcer recurrence rate and the ulcer index were compared between these groups. Gastric mucosal apoptosis rate, microscopic inflammation activity and angiogenesis markers vascular endothelial growth factor A (VEGF-A) and CD34 were examined by TUNEL, histological evaluations (HE) and immunohistochemistry (IHC). Pathways involved, expressions of endoplasmic reticulum (ER) stress apoptosis marker CHOP, angiogenic markers VEGF-A and its receptor VEGFR1, and endothelial NO synthase (eNOS) were all analyzed by Western blot.

Results: This study indicated that Clopidogrel significantly induced the gastric ulcers recurrence, severe inflammation and ER stress related apoptosis of the gastric mucosa, suppressed the synthesis of angiogenic markers and eNOS. Furthermore, Clopidrogel intervention resulted in the activation of protein kinase B (AKT) and p38 mitogen-activated protein kinase (p38/MAPK). BPC 157 attenuated the gastric mucosal damage caused by Clopidogrel and reversed these molecular effects. However, NO blockade L-NAME weakened the protective effect and thus the molecular effects of BPC 157 on gastric mucosa.

Conclusion: In conclusion, these results suggest that BPC 157 inhibited Clopidogrel-induced gastric mucosa injury partially by inhibition of gastric mucosa cell ER stress-mediated apoptosis and inflammation, and promoting gastric mucosa angiogenesis via VEGF-A/VEGFR1 mediated-AKT/p38/MAPK signaling pathways.

Keywords: AKT; BPC 157; angiogenesis; clopidogrel; p38/MAPK.

Figure 1

Representative gross morphologies of stomach at the time of necropsy. (A) Control group (1% methylcellulose solution) without visible lesions. (B) The Clopidogrel (10 mg/kg) group with severe lesions and deep ulcers. (C) Clopidogrel (10 mg/kg) plus BPC 157 (10 ng/kg) treatment group with very few lesions and superficial ulcers. (D) L-NAME (5 mg/kg) group with more lesions and ulcers compared with the Clopidogrel (10 mg/kg) plus BPC 157 (10 ng/kg) treatment group. Arrows: lesions and ulcers.

Figure 2

(A) Histopathological features and lesion score of stomach. a, Control group showed intact mucosal lining of flattened epithelial cells. Mucosal crypts appear arranged normally; b, The Clopidogrel group showed denudation of lining epithelium, areas of hyperemia, disturbed glandular structure, damaged crypts, irregular arrangement and inflammatory cells infiltration; c, Clopidogrel plus BPC 157 group with minimal changes compared to Clopidogrel group; d, L-NAME exposed mucosal tissue showed similar pathological changes as in Clopidogrel group. Scale bar = 200 μm. The lesion score is shown in (B). ***P < 0.001 vs Control; ^###P < 0.001 vs Control + Clopidogrel; ⁺⁺P < 0.01 vs Control + Clopidogrel + BPC 157.

Figure 3

TUNEL staining. Epithelial apoptosis in gastric mucosa was evaluated by TUNEL staining. (A) Representative images of TUNEL staining of stomach from rats in four groups. a, Control group; b, Clopidogrel group; c, Clopidogrel plus BPC 157 group; d, Clopidogrel plus BPC 157 and L-NAME group. Scale bar = 200 μm. (B) Ratio of TUNEL-positive (brown) cells in each group was shown. ***P < 0.001 vs Control; ^###P < 0.001 vs Clopidogrel; ⁺⁺P < 0.01 vs Clopidogrel + BPC 157.

Figure 4

Immunohistochemical detection for the angiogenesis markers VEGF-A and CD34. IHC staining for VEGF-A and CD34 in rats’ gastric mucosal tissue. The levels of VEGF-A and CD34 were highly expressed in Control group. In Clopidogrel group, the levels of VEGF-A and CD34 were both decreased compared with control group. And in the BPC 157 co-treatment group, the levels of these two angiogenesis markers were all up-regulated compared with Clopidogrel group. But the NO synthase blocker L-NAME inhibited up-regulation of angiogenesis markers VEGF-A and CD34 induced by BPC 157. Dark brown represents positive result. Scale bar = 50 μm.

Figure 5

Role of VEGF-A-mediated-AKT or p38/MAPK signaling pathway in the inhibition of BPC 157 on Clopidogrel-induced gastric mucosal injury. (A) Clopidogrel group down-regulated the VEGF-A and VEGFR1 compared with Clopidogrel group. But after administration of BPC 157, the VEGF-A and VEGFR1 were up-regulated. However, the L-NAME reversed this effect of BPC 157. *P < 0.05 vs Control; ^#P < 0.05 vs Clopidogrel; ⁺P < 0.05 vs Clopidogrel + BPC 157. (B) As the VEGF-A was downregulated, Clopidogrel dephosphorylated the AKT signaling pathway, phosphorylated p38/MAPK and ERK/MAPK signaling pathways; but the effect of BPC 157 on these three signal pathways was completely opposite to that of Clopidogrel. Notably, L-NAME reversed the effect of AKT and p38/MAPK induced by BPC 157, but not ERK/MAPK. ***P < 0.001 vs Control; ^#P < 0.05, ^###P < 0.001 vs Clopidogrel; ⁺⁺⁺P < 0.001 vs Clopidogrel + BPC 157. (C) Clopidogrel increased the expression of CHOP and decreased the p-eNOS. These were reversed by BPC 157 treatment. But L-NAME partly abolished the effect of BPC 157. *P < 0.05, ***P < 0.001 vs Control; ^#P < 0.05, ^##P < 0.01 vs Clopidogrel; ⁺P < 0.05, ⁺⁺⁺P < 0.001 vs Clopidogrel + BPC 157.