PPARG stimulation restored lung mRNA expression of core clock, inflammation- and metabolism-related genes disrupted by reversed feeding in male mice

Abstract

The circadian rhythm system regulates lung function as well as local and systemic inflammations. The alteration of this rhythm might be induced by a change in the eating rhythm. Peroxisome proliferator-activated receptor gamma (PPARG) is a key molecule involved in circadian rhythm regulation, lung functions, and metabolic processes. We described the effect of the PPARG agonist pioglitazone (PZ) on the diurnal mRNA expression profile of core circadian clock genes (Arntl, Clock, Nr1d1, Cry1, Cry2, Per1, and Per2) and metabolism- and inflammation-related genes (Nfe2l2, Pparg, Rela, and Cxcl5) in the male murine lung disrupted by reversed feeding (RF). In mice, RF disrupted the diurnal expression pattern of core clock genes. It decreased Nfe2l2 and Pparg and increased Rela and Cxcl5 expression in lung tissue. There were elevated levels of IL-6, TNF-alpha, total cells, macrophages, and lymphocyte counts in bronchoalveolar lavage (BAL) with a significant increase in vascular congestion and cellular infiltrates in male mouse lung tissue. Administration of PZ regained the diurnal clock gene expression, increased Nfe2l2 and Pparg expression, and reduced Rela, Cxcl5 expression and IL-6, TNF-alpha, and cellularity in BAL. PZ administration at 7 p.m. was more efficient than at 7 a.m.

Keywords: PPARG; core clock genes; inflammation; lung; pioglitazone.

FIGURE 1

Experimental flowchart. Sun ☼ and Moon pictograms indicate the light and dark periods, respectively. Experimental schedule from Day 0 to Day 8: (a) mice entrained to a 12‐h light–dark cycle with ad libitum access to food and water. (b) Mice in RF groups. (c) Mice in NF groups. (d) Mice were sacrificed on Day 8 at noon (HALO 05) and midnight (HALO 17), and lung specimens and BAL samples were collected. Sacrifice for histopathological analysis was performed at HALO 05. White arrows indicate the time of PZ administration. HALO, hour after light onset.

FIGURE 2

Diurnal changes in clock gene mRNA in mouse lung. Expression of mRNA: (a) Per1; (b) Per2; (c) Cry1; (d) Cry2; (e) Clock; (f) Arntl; and (g) Nr1d1. Horizontal lines show significant differences. Y‐axis—relative mRNA levels.

FIGURE 3

Circadian changes in mRNA transcription of inflammation‐/metabolism‐related genes in the lungs of mice. Expression of mRNA: (a) Nfe2l2; (b) Pparg; (c) Rela; and (d) Cxcl5. Significant differences are shown by the horizontal line. Y‐axis—relative mRNA levels.

FIGURE 4

Influence of RF and PZ treatment on inflammatory cell influx in mouse BAL: (a) total cells; (b) macrophages; (c) lymphocytes; and (d) neutrophils.

FIGURE 5

Changes in mouse BAL cytokines levels after RF and PZ treatment: (a) IL‐6; (b) TNF‐alpha; (c) TGF‐beta 1.

FIGURE 6

Lung tissue changes and histopathological scores after RF and PZ treatment (H&E). Scale bars 50 μm. (a) Vascular congestion (asterisks) and cellular infiltrates (white arrows) in RF untreated mice; (b) marked cellular infiltrates (black arrows) in RF PZ 7 a.m. mice; (c) absent of visual changes in vascular congestion in RF PZ 7 p.m. mice; (d) nighttime feeding PZ 7 p.m. and (e) control groups demonstrate similar minimal levels of vascular congestion and cellular infiltrations of lung interstitium; (f) Comparisons of mean scores of vascular congestion and (g) cellular infiltrates: P‐value calculated by Kruskal–Wallis and Dunn's post hoc test; (h) mucus overproduction mice (PAS staining) in the bronchial epithelium of RF untreated; (i) PAS reactivity in bronchial cast after PZ intake at 7 a.m.; (j) mucus overproduction in bronchial epithelium of RF PZ 7 p.m. mice; (k) PAS reactivity mostly absent in goblet cells of control mice; and (l) comparisons of mean mucus scores: P‐value calculated by Kruskal–Wallis and Dunn's post hoc test.

FIGURE 7

Light–dark changes are received by special light‐sensitive receptors in the retina and induced inputs to SCN, which acted as a central pacemaker of the circadian clock. In route SCN generals neurohumoral outputs to the tissue‐specific peripheral clocks (Dibner et al., 2010). A circle shows the core and stabilization loops of the core circadian oscillator. Tissue‐specific circadian transcription is regulated by REV‐ERB and retinoic acid receptor‐related orphan receptors (RORs), which interacted with specific response elements of the clock (including Arntl) and clock‐controlled genes. Peripheral oscillators might have taken away from the SCN control and developed peripheral rhythm due to eating or fasting (Damiola et al., ; Kim et al., 2022). RF induced the diurnal disruption of the core clock gene and inflammation‐/metabolism‐related gene expression pattern. Impaired gene expression increased the activity of the NFκB pathway and decreased NRF2/PPARG. PZ activated PPARG with consequent suppression of the NFκB pathway and decreased concentration of pro‐inflammatory cytokines IL‐6, TNF‐alpha, and cell influx in BAL.