Role of LTB4 and nitric oxide in the gastroprotective effect of Prosthechea karwinskii leaves extract in the indomethacin-induced gastric injury in the rat

Abstract Gastric injury is mainly described by inflammation of the gastric epithelium. Recently, our group of work demonstrated that Prosthechea karwinskii leaves extract induces both an in vitro antioxidative action and an in vivo gastroprotective effect in a rat. However, the molecules involved in the gastroprotective action by Prosthechea karwinskii are not known. Thus, the aim of this study is to determine whether Prosthechea karwinskii extract modifies anti-inflammatory and antioxidative biomarkers in an in vivo rat model of indomethacin-induced gastric injury. Rats were orally administered with indomethacin and Prosthechea karwinskii leaf extract. Our results suggest that the gastroprotective effect of Prosthechea karwinskii leaf extract is related to the reduction in leukocyte infiltration and antioxidative action in a model of indomethacin-induced gastric injury. Further studies are warranted to investigate the role of the compounds identified in the gastroprotective action of Prosthechea karwinskii leaves extract. Graphical Abstract


Introduction
The use of NSAIDs is limited by their extensive damage induced in the gastrointestinal (GI) tract. However, NSAIDs are often prescribed for long-term periods to treat chronic musculoskeletal diseases such as arthritis and osteoarthritis (Rannou et al. 2016); thus their chronic use aggravates gastric ulcer condition. New therapeutic alternatives are necessary to better prevent and/or treat NSAID's gastrotoxicity.
Medicinal plants have been a natural source for the treatment of several illness. Herbal remedies are still used by 70% of the population, according to the World Health Organization. In Mexico, some orchids have been used in therapeutic practices since pre-Hispanic times. The mixtecos and zapotecos of Oaxaca used the bulbs of an orchid named Prosthechea michuacana as food, which is eaten raw to eliminate thirst, and it is also used as anti-inflammatory and for the treatment of renal disease and diabetes (Guti errez et al. 2010). Additionally, preclinical studies have shown anti-inflammatory, antioxidative and hepatoprotective effects (Guti errez and Sol ıs 2009). Recently, we demonstrated that Prosthechea karwinskii extract induces both an in vitro antioxidative action and an in vivo gastroprotective effect (Barrag an-Zarate et al. 2020). However, the mechanisms behind the gastroprotective effect of Prosthechea karwinskii are still unknown. Thus, the aim of this study is to determine whether Prosthechea karwinskii extract modifies anti-inflammatory and antioxidative biomarkers in an in vivo model of indomethacin-induced gastric injury.

Results and discussion
Oral pre-treatment with P. karwinskii extract reduced in a dose-dependent manner the gastric haemorrhagic lesions induced by indomethacin. The highest dose evaluated of P. karwinskii extract resulted in a gastroprotective effect similar in magnitude to that induced by omeprazole (Supplementary materials, Figure S1). Histological analysis showed that oral indomethacin induced disruption and neutrophil infiltration into the gastric mucosa (Supplementary materials, Figure S2C) compared with the integrity of the mucosa found in the basal group observed as well-defined pits and foveolas without congestion or cellular infiltration (Supplementary materials, Figure  S2A), normal tissue with preserve architecture of pits and foveolas was shown by control of P. karwinskii (Supplementary materials, Figure S2B). In addition, P. karwinskii extract itself maintained gastric mucosa architecture (Supplementary materials, Figure  S2D), the same as omeprazole (Supplementary materials, Figure S2E). Representative images of the corpus of the stomach are shown in the supplementary material ( Figure S3), and indomethacin administration increases evidently gastric haemorrhagic lesions, while P. karwinskii extract and omeprazole pre-treatment prevent the augment of the lesions.
Pre-treatment with P. karwinskii extract significantly reduced leukotriene B4 levels incremented by indomethacin-induced gastric injury (Supplementary materials, Table  S1). It has been previously reported that NSAIDs such as indomethacin cause leukocyte infiltration and an augmentation on LTB 4 gastric levels, activating inflammatory pathway to attack the pathogen (Pineda-Peña et al. 2018).
In our previous work, we have reported both in vivo anti-inflammatory activity in the carrageenan-induced oedema model and gastroprotective effects by the Prosthechea karwinskii leaves extract. In addition, in this work, we have studied the anti-inflammatory pathway in stomach as a route of Prosthechea karwinskii's gastroprotective effect. LTB 4 lack is an important factor present in the gastroprotective action of several compounds; recently docosahexaenoic acid (Pineda-Peña et al. 2018) have demonstrated their gastroprotective action throw LTB 4 and leukocyte recruitment reduction.
In this study, we found that Prosthechea karwinskii leaves extract significantly prevented the augment of NO gastric levels induced by indomethacin during gastric injury. In addition, indomethacin administration significantly increased nitric oxide levels as compared to the basal group (Supplementary materials, Table S1). Overproduction of nitric oxide (NO) following activation of inducible nitric oxide synthase (iNOS) may be involved in gastrointestinal injury.
Even though there is not clearly participation of SOD, pre-treatment with P. karwinskii extract significantly augmented SOD activity against basal and indomethacininduced gastric injury groups (Supplementary materials, Table S1). Some compounds identified in the leaves ethanolic extract of Prosthechea karwinskii have previously demonstrated antioxidant activity in several models.
The chromatogram obtained from the UPLC-ESI-qTOF-MS/MS of P. karwinskii leaves extract (Supplementary materials, Figure S4 and Table S2) is presented in 0-12 min, and the different colours represent the chromatographic ions of each compound identified. The chromatogram shows that the main bioactive compounds identified were quinic acid, neochlorogenic acid, chlorogenic acid, rutin, kaempferol-3-o-b-rutinoside and embelin. Table S2 shows the chemical structures of the compounds identified in the extract. Neochlorogenic acid inhibits LPS-activated inflammatory response throw (Park et al. 2018). Finally, rutin attenuates paw inflammation in mice (Fikry et al. 2018) and inflammation associated to hepatotoxicity (Caglayan et al. 2019).
As it was published before by our group of work, the ethanolic extract of the leaves of Prosthechea karwinskii contains compounds such phenols, saponins, flavonoids and anthraquinones, and more studies need to be performed to elucidate if they are acting in synergism or alone to induce its gastroprotective effect.

Experimental
See the supplementary material.

Conclusion
In summary, our results suggest that the gastroprotective effect of Prosthechea karwinskii leaves extract is related to the reduction in leukocyte infiltration and antioxidative action in a model of indomethacin-induced gastric injury. The orchid extract of Prosthechea karwinskii might be studied for the pre-treatment with NSAIDs to avoid gastric injury induced by this kind of drugs. More studies need to be performed to investigate the role of the compounds identified in the gastroprotective action. Then, we suggest that Prosthechea karwinskii leaves extract might be used as natural treatment to prevent NSAIDs-induced gastric injury.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Consejo Nacional de Ciencia y Tecnolog ıa;Secretar ıa de Investigaci on y Posgrado, Instituto Polit ecnico Nacional.