Treatment of chronic obstructive pulmonary disease by traditional Chinese medicine Morin monomer regulated by autophagy

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is a frequently occurring disorder. The aim of this study is to explore the mechanism of traditional Chinese medicine Morin monomer in the treatment of COPD via regulating autophagy based on the long non-coding RNA (lncRNA) H19/microRNA (miR)-194-5p/Sirtuin (SIRT)1 signal axis.

Methods: The COPD rat model was constructed, and the lung tissues were collected. The pathological analysis was performed using hematoxylin-eosin (HE), Masson, and periodic acid-Schiff (PAS) staining. Autophagosomes were observed using transmission electron microscope. LncRNA H19, miR-194-5p, SIRT1 genes in the rat lung tissues were detected using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The autophagy-related proteins including SIRT1, mammalian/mechanistic target of rapamycin (mTOR), phosphorylated (p)-mTOR, microtubule-associated protein light chain 3 (LC3), Beclin-1, autophagy-related (ATG)7, and p62 in each group were detected using Western blot.

Results: The rats in the control group had normal lung structure. Alveolar enlargement and destruction could be found in the rat lung tissues in the model group, accompanied with obvious infiltration of inflammatory cells, thickened bronchial walls, enlarged alveolar septum, collagen fibers deposition, and goblet cells proliferation. In comparison with the model group, Morin treatment relieved the lung injuries, which was optimized in the moderate- and high-dose groups. The number of autophagosomes in the lung tissues of the model rats was dramatically increased compared with the normal rats. However, the number of autophagosomes in each Morin treatment group was obviously less than that in the model group. LncRNA H19 and SIRT1 expression was significantly increased in the model group, and miR-194-5p was significantly decreased (P<0.05). Morin and 3-methyladenine (3-MA) could obviously reduce the lncRNA H19 and SIRT1 expression, and increase the miR-194-5p expression (P<0.05). Relative to control rats, ATG7, Beclin-1, LC3II/I and SIRT1 levels in the model group increased obviously, while the expression of p62, and p-mTOR/mTOR decreased (P<0.05). Morin treatment reduced the expression of ATG7, Beclin-1, SIRT1, LC3II/I significantly, and increased the p-mTOR/mTOR and p62 expression (P<0.05).

Conclusions: Morin decreased lncRNA H19 expression, resulting in upregulation of miR-194-5p expression, downregulation of SIRT1 expression, and increased of p-mTOR/mTOR expression. Furthermore, cell autophagy was inhibited, contributing to the COPD treatment.

Keywords: Chronic obstructive pulmonary disease (COPD); autophagy; long non-coding RNA H19 (lncRNA H19); microRNA-194-5p/Sirtuin 1 (miR-194-5p/SIRT1).

Figure 1

Histological examination results detected using HE, Masson and PAS staining on the lung tissues of rats in each group (×200) (n=6/group). The lung structure of the rats in the control group is normal. In the model group, the pulmonary alveoli of rats were not completely expanded (black arrow), the inflammatory cells were significantly infiltrated (red arrow), the bronchial tube wall was significantly thickened (green arrow), the alveolar septum was thickened and a large number of collagen fibers were deposited (yellow arrows), and the goblet cells were significantly proliferated (purple arrows). Compared with the model group, different degrees of relief were observed under light microscopy in each treatment group, with the medium- and high-dose Morin groups being the most significant. HE, hematoxylin-eosin; PAS, periodic acid-Schiff.

Figure 2

Autophagy in the lung tissues of rats in each group observed using TEM (n=6/group). The autophagosome is indicated by the arrows. Scale bar =200 nm. 3-MA, 3-methyladenine; TEM, transmission electron microscope.

Figure 3

The lncRNA H19, SIRT1 and miR-194-5p levels within the lung tissues of rats in each group detected using RT-qPCR (n=6/group). The error bars indicate the mean ± SD. *, P<0.05 and ^#, P<0.05 relative to blank control and model groups, respectively; ^@, P<0.05, ^, P<0.05, and ^&, P<0.05 relative to low-, medium-, and high-dose Morin groups, respectively. LncRNA, long non-coding RNA; miR, microRNA; SIRT, Sirtuin; SD, standard deviation; RT-qPCR, reverse transcription-quantitative real-time polymerase chain reaction; 3-MA, 3-methyladenine.

Figure 4

The expression of SIRT1, mTOR, p-mTOR, LC3, Beclin-1, ATG7 and p62 proteins in the lung tissues of rats in each group detected using WB (n=6/group). *, P<0.05 and ^#, P<0.05 relative to blank control and model groups, respectively; ^@, P<0.05, ^, P<0.05, and ^&, P<0.05 relative to low-, medium-, and high-dose Morin groups, respectively. mTOR, mammalian/mechanistic target of rapamycin; p, phosphorylated; LC3, microtubule-associated protein light chain 3; ATG, autophagy-related; WB, Western blot; SIRT, Sirtuin; 3-MA, 3-methyladenine.