Pure Total Flavonoids From Citrus Protect Against Nonsteroidal Anti-inflammatory Drug-Induced Small Intestine Injury by Promoting Autophagy in vivo and in vitro

Abstract

Small intestine injury is an adverse effect of non-steroidal anti-inflammatory drugs (NSAIDs) that urgently needs to be addressed for their safe application. Although pure total flavonoids from citrus (PTFC) have been marketed for the treatment of digestive diseases, their effects on small intestine injury and the underlying mechanism of action remain unknown. This study aimed to investigate the potential role of autophagy in the mechanism of NSAID (diclofenac)-induced intestinal injury in vivo and in vitro and to demonstrate the protective effects of PTFC against NSAID-induced small intestine disease. The results of qRT-PCR, western blotting, and immunohistochemistry showed that the expression levels of autophagy-related 5 (Atg5), light chain 3 (LC3)-II, and tight junction (TJ) proteins ZO-1, claudin-1, and occludin were decreased in rats with NSAID-induced small intestine injury and diclofenac-treated IEC-6 cells compared with the control groups. In the PTFC group, Atg5 and LC3-II expression, TJ protein expression, and the LC3-II/LC3-I ratio increased. Furthermore, the mechanism by which PTFC promotes autophagy in vivo and in vitro was evaluated by western blotting. Expression levels of p-PI3K and p-Akt increased in the intestine disease-induced rat model group compared with the control, but decreased in the PTFC group. Autophagy of IEC-6 cells was upregulated after treatment with a PI3K inhibitor, and the upregulation was significantly more after PTFC treatment, suggesting PTFC promoted autophagy through the PI3K/Akt signaling pathway. In conclusion, PTFC protected intestinal barrier integrity by promoting autophagy, which demonstrates its potential as a therapeutic candidate for NSAID-induced small intestine injury.

NSAID-Associated Small Intestinal Injury: An Overview From Animal Model Development to Pathogenesis, Treatment, and Prevention

“…Now, diclofenac is one of the most prescribed drugs worldwide, and it is also one of the commonly used modeling drugs for NSAID-related enteropathy. In a diclofenac-induced rat model, intestinal tissue mucosal congestion, edema, erosion, and even ulceration could be observed ( Chen et al, 2021 ). The intestinal injury was evaluated in ulceration, adhesion to the surrounding tissue, and diarrhea using the Reuter BK scoring method.…”

“…NSAID-induced autophagy inhibition impaired the integrity of the mucus layer combined with the inability of intestinal epithelial cells to clear intracellular pathogens, which elicited an uncontrolled inflammatory response eventually ( Chamoun-Emanuelli et al, 2019 ). Pure total flavonoids from citrus could promote autophagy to protect the small intestinal barrier by activating the PI3K/Akt signaling pathway ( Chen et al, 2021 ). It is a flavonoid isolated and purified from citrus peel, and its medicinal value for gastrointestinal protection has been demonstrated.…”

“…In addition, animal models play a significant role in exploring effective therapeutic and preventive measures and analyzing therapeutic modalities’ possible mechanisms of action. Researchers have successfully used a wide variety of NSAIDs to establish disease models in rodents, including indomethacin ( Kawashima et al, 2020 ; Kuzumoto et al, 2021 ; Tawfik et al, 2016 ; Yamada et al, 1993 ), diclofenac ( Beck et al, 1990 ; Chen et al, 2021 ; Xu et al, 2021 ), aspirin ( Bouzenna et al, 2019 ; Brodie et al, 1970a ; Chi et al, 2021 ), flurbiprofen ( Campanella and Jamali, 2009 ), naproxen ( Carvalho et al, 2015 ; Nicolau et al, 2017 ), loxoprofen ( Hayashi et al, 2013 ; Satoh and Urushidani, 2016 ), ibuprofen ( Beck et al, 1990 ; Lu et al, 2018 ), diflunisal ( Niu et al, 2014 ), BFMeT ( Hagiwara et al, 2004 ; Uejima et al, 1996 ), paracetamol ( Chopyk et al, 2019 ; Niu et al, 2020 ), Ketoprofen ( Cheng et al, 2014 ; Saitta et al, 2014 ). Many experimental articles have verified the reproducibility and stability of various modeling modalities.…”

Citrus aurantium ‘Changshan-huyou’—An ethnopharmacological and phytochemical review

“…Reasonable intervention of intestinal microenvironment is of great significance to guide rational drug use in clinic. ( Chen et al, 2014 ; Chen et al, 2021 ) studied the mechanism of small intestinal side reactions induced by non-steroidal antiinflammatory drugs (NSAIDs) in vivo , concluded that the integrity of intestinal barrier of the mice would be protected after treating with PTFC. Authors induced intestinal injury in SD rats via continuous irrigation stomach with diclofenac (7.5 mg/kg).…”

The new exploration of pure total flavonoids extracted from Citrus maxima (Burm.) Merr. as a new therapeutic agent to bring health benefits for people

“…Merr . (PTFC) were found, including naringin, neohesperidin, narirutin, and hesperidin, and the content were 243.78 ± 2.69, 5.96 ± 0.06, 12.11 ± 0.12, and 136.04 ± 4.24 mg/g, respectively ( 9 ). Since PTFC is regarded as the major active ingredient of C. maxima (Burm.)…”