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.

Keywords: PI3K-AKT pathway; autophagy; non-steroidal anti-inflammatory drugs; pure total flavonoids from citrus; small intestine injury.

FIGURE 1

HPLC chromatographic profile of PTFC and standard flavanones narirutin, naringin, neohesperidin, and hesperidin.

FIGURE 2

Relationship between autophagy and tight junction (TJ) barrier in nonsteroidal anti-inflammatory drug (NSAID)-induced small intestine injury. Levels of mRNA were determined in the NSAID model group and control group using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for (A) autophagy-related 5 (Atg5) and (B) zonula occludens (ZO-1), claudin (CLDN)-1, and occludin (OCLN) (C) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (D,E) Western blotting was used to analyze protein levels in the NSAID model group and control group (D) Protein levels of light chain 3 (LC3) forms LC3-II and LC3-I were determined using ImageJ software and the signal density ratios of LC3-II/LC3-I were calculated (E) Protein levels of ZO-1, CLDN-1, and OCLN. **p < 0.01.

FIGURE 3

Morphological examination of various experimental groups or rats (A) Morphological changes in the intestinal mucous membrane were observed (B) Hematoxylin and eosin (H&E) staining revealed severe injuries in the small intestines of rats in NSAID model group, whereas the PTFC group showed significant improvement in bowel lesions (×200 magnification). Immunohistochemistry (IHC) was used to evaluate expression of (C) light chain 3 (LC3)-II (D) zonula occludens (ZO)-1 (E) claudin (CLDN)-1, and (F) occludin (OCLN) in control, NSAID model, and PTFC groups of rats.

FIGURE 4

PTFC increases TJ-related protein levels and inhibits NSAID-induced small intestine injury progression in rats. Levels of mRNA were evaluated in the NSAID model group, PTFC group, and control group of rats using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for (A) autophagy-related 5 (Atg5) and (B) ZO-1, CLDN1, and OCLN (C) Western blot analysis was used to determine protein expression levels in the groups of rats (D) Levels of light chain 3 (LC3) were determined and the signal intensity ratio of LC3-II/LC3-I was calculated using ImageJ software (**p < 0.01) (E) Protein expression of ZO-1, CLDN1, and OCLN were measured in all groups using western blotting (*p < 0.05 and **p < 0.01).

FIGURE 5

PTFC promotes autophagy and increases TJ-related protein levels in IEC-6 cells. IEC-6 cells were untreated for the 48 h of culture (Control), treated with diclofenac for the 48 h of culture (NSAID), or treated with diclofenac for the 48 h of culture plus treatment with PTFC for the final 24 h or culture (PTFC). The mRNA levels were then detected in all groups using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (A) mRNA levels of autophagy-related 5 (Atg5) (B) mRNA levels of ZO-1, CLDN1, and OCLN (C) Western blot analysis was used to determine protein expression levels in the groups of cells (D) Levels of light chain 3 (LC3) were determined and the signal intensity ratio of LC3-II/LC3-I density ratio was calculated using ImageJ software (**p < 0.01) (E) Protein expression of ZO-1, CLDN1, and OCLN was measured in all groups using western blotting (*p < 0.05 and **p < 0.01).

FIGURE 6

Effect of PTFC on the formation of autophagosome in IEC-6 cells. Immunofluorescence staining of LC3-II (Green) was performed. Nuclei were stained with DAPI. Chloroquine (50 μM) was used as positive control. The results revealed that the LC3-II signal in the diclofenac-treated model group (NSAIDs) was significantly decreased compared with that in the untreated group (Control), but the positive signal in the PTFC treatment group (PTFC) was significantly enhanced.

FIGURE 7

PTFC increases autophagy in rats through PI3K/Akt Signaling (A) Western blot analysis was performed to determine expression levels of phosphoinositide 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), Akt, phosphorylated-Akt (p-Akt), mechanistic target of rapamycin (mTOR), and phosphorylated mTOR (p-mTOR) in the NSAID model group, PTFC group, and Control group of rats (B) Density ratios of phosphorylated to unphosphorylated proteins (p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR) were calculated using ImageJ software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (*p < 0.05 and **p < 0.01).

FIGURE 8

PTFC increases autophagy through PI3K/Akt Signaling in IEC-6 cells (A) Western blot analysis was performed to determine expression levels of levels of light chain 3 (LC3), phosphoinositide 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), Akt, and phosphorylated-Akt (p-Akt) (B–D) Density ratios of LC3-II/LC3-I and phosphorylated/unphosphorylated proteins (p-PI3K/PI3K, p-Akt/Akt) were calculated using ImageJ software. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control (*p < 0.05 and **p < 0.01).