In vivo and in vitro protective effects of omeprazole against neuropathic pain

Abstract

Apart from reducing the acid secretion, omeprazole inhibits activation of the nuclear factor-κB, release of inflammatory cytokines, and chemotaxis of neutrophils. These mechanisms prompted us to evaluate antineuropathic effect of omeprazole in the chronic constriction injury (CCI)-induced rat model of neuropathic pain and LPS mediated ROS-induced U-87 cells. Omeprazole at 50 mg/kg/day/oral for 14 days significantly reduced the intensity of neuropathic pain estimated as paw withdrawal latency, withdrawal pressure threshold and restored the motor nerve conduction velocity in the constricted nerve, when compared with respective groups. The histological findings revealed the protective effect of omeprazole against the CCI-induced damage. Omeprazole significantly decreased the levels of tumor necrosis factor (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) as compared to their respective control groups. It also reduced the oxidative stress by up regulating the SOD, catalase activity and decreasing MDA content. Similarly, in-vitro study, LPS mediated ROS-induced U-87 cells, omeprazole reduced the oxidative stress as well as the release of TNF-α, IL-1β and IL-6. Altogether, these results suggest that, neuroprotective effect of omeprazole is mediated through preventing release of proinflammatory cytokines, augmenting endogenous anti-oxidant defense system, and maintain the structural integrity of sciatic nerve from the CCI-induced structural damage and inflammatory changes.

Figure 1. Omeprazole ameliorated the cold, warm and mechanical stimuli induced allodynia in CCI induced neuropathic pain in rats.

(A) In cold allodynia treatment with omeprazole (50 mg/kg p.o. for 14 days) showed time dependent increase in the paw withdrawal latency in CCI control rats without influencing the measures in sham rats. (B) In warm allodynia treatment with omeprazole (50 mg/kg p.o. for 14 days) showed time dependent increase in the paw withdrawal latency and threshold in CCI-control rats but not in sham rats. (C) In Mechanical allodynia, treatment with omeprazole (50 mg/kg p.o. for 14 days) showed time dependent increase in the paw withdrawal latency or threshold in CCI-control rats but not in sham rats. Data are expressed as the mean ± S.E.M. (n = 8/group). Significance was determined by repeated measures ANOVA followed by the Dennett’s post-hoc test. The statistical significance of difference of CCI-control compared to sham was shown as ^###p < 0.001, whereas the statistical significance of difference in the omeprazole and gabapentin treated groups as compared to CCI-control was designated as ***p < 0.001.

Figure 2. Omeprazole alleviate the MNCV in CCI-induced neuropathic pain in rats.

Administration of omeprazole (50 mg/kg p.o. for 14 days) for CCI rats showed significant improvement in the MNCV. Data are expressed as the mean ± S.E.M. (n = 8/group). Significance was determined by two way ANOVA followed by the Bonferroni’s multiple comparison test. The statistical significance of difference of CCI-control compared to sham was shown as ^###p < 0.001, whereas the statistical significance of difference in the omeprazole and gabapentin treated groups as compared to CCI-control was designated as ***p < 0.001.

Figure 3. Omeprazole reduces the oxidative stress in CCI-induced neuropathic pain in rats.

CCI surgery in rats induces distinct oxidative stress, while treatment of omeprazole (50 mg/kg p.o. for 14 days) alleviates the increase in oxidative stress. (A) Malondialdehyde content (MDA) (B) Reduced glutathione (GSH). (C) Superoxide dismutase (SOD). (D) Catalase. Each value represents the mean of 5–8 experiments ± S.E.M. Data are expressed as the mean ± S.E.M. (n = 8/group). Significance was determined by one-way ANOVA followed by Dennett’s post-hoc test. The statistical significance of difference of CCI-control compared to sham was shown as ^###p < 0.001, whereas the statistical significance of difference in the omeprazole and gabapentin treated groups as compared to CCI-control was designated as ***p < 0.001.

Figure 4. Omeprazole condenses the release of pro-inflammatory cytokines in CCI-induced neuropathic pain in rats.

(A) Tumour necrosis factor-α (TNF-α). (B) Interleukin-6 (IL-6). (C) Interleukin-1β (IL-1β). Data are expressed as the mean ± S.E.M. (n = 8/group). Significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparison test. The statistical significance of difference of CCI-control compared to normal group was shown as ^###p < 0.001, whereas the statistical significance of difference in the omeprazole and gabapentin treated groups as compared to CCI-control was designated as **p < 0.005 and ***p < 0.001.

Figure 5. Omeprazole prevents histological variations in CCI-induced neuropathic pain (Hematoxylin and eosin X100).

The normal rat’s sciatic nerve section showed normal structure, architecture and no inflammation. Sham operated rat’s showed same structure as observed in the normal rats, not any marked changes were observed. CCI-control animals showed swelling of nerve fibers, derangement and showed increase in the inflammatory cell infiltration as well as pronounced inflammation. Omeprazole treated rats showed decrease in the swelling as observed by decrease in inflammation. Gabapentin treatment with rats normalised the derangement observed in the CCI rats. Scale bar 1000 μm.

Figure 6. Effect of omeprazole on LPS mediated ROS, SOD, catalase and cytokines in U-87 cells.

(A) MTT cell viability assay after omeprazole (OPZ) exposure in U-87 cells. The significance of difference in the treatment groups compared to untreated control was designated as ***p < 0.001. (B) Various concentrations of omeprazole were treated in LPS pre-treated (500 ng/ml for 20 min) cells prior to measure the ROS. Graphical representation of ROS positive population analyzed flow cytometrically after DCFH-DA staining. (C) Biochemical detection of SOD, and (D) Catalase after omeprazole treatment in LPS pre-treated U-87 cells. (E), (F) and (G) were the expressions of TNF-α, IL-6 and IL-1β , respectively, after omeprazole treatment in LPS pre-treated cells measured by indirect ELISA. H₂O₂ is used as a positive control for ROS. LPS indicate U-87 cells treated with LPS alone. LPS + OPZ represents indicated concentration of Omeprazole treatment on LPS pre-treated U-87 cells. Data represents mean ± S.E.M. of 8 independent experiments. The significance of difference of H₂O₂ (positive control of ROS in in-vitro experiments) compared to control was designated as ^$$$p < 0.001. The statistical significance of difference of LPS group compared to control group was shown as ^###p < 0.001, whereas the statistical significance of difference in the treatment groups as compared to LPS group was designated as **p < 0.005 and ***p < 0.001.