[β-Carotene alleviates PM_(2.5)-induced pulmonary fibrosis by regulating the function of mitochondria-associated membranes].

OBJECTIVE: To investigate the protective effect of β-carotene on PM_(2.5)-induced pulmonary fibrosis in mice.

METHODS: Seventy 6-week-old male C57BL/6 mice(SPF grade) were randomly divided into four groups based on body weight: control group(n=25), β-carotene group(n=10), PM_(2.5) group(n=25), and β-carotene+PM_(2.5) group(n=10). The control group received oral gavage of corn oil and intratracheal instillation of sterile saline. The β-carotene group was administered β-carotene orally and saline intratracheally. The PM_(2.5) group received corn oil orally and PM_(2.5) intratracheally. The β-carotene+PM_(2.5) group was treated with β-carotene orally and PM_(2.5) intratracheally. Oral doses of corn oil and β-carotene were administered daily at 50 mg/kg. PM_(2.5) was instilled intratracheally at 5 mg/kg every 3 days. After 25 cycles, 3 mice from the control and PM_(2.5) groups were randomly euthanized for lung tissue collection and Masson's trichrome staining to validate fibrosis model establishment. After the successful establishment of the model, the mice were euthanized. Lung sections were prepared for hematoxylin-eosin(HE) and Masson's staining under light microscopy, collagen volume fraction(CVF) in Masson-stained sections was analyzed using ImageJ. Serum, bronchoalveolar lavage fluid(BALF), and lung tissues were collected to measure serum levels of hydroxyproline(HYP), glutathione(GSH), superoxide dismutase(SOD), malondialdehyde(MDA), and BALF total protein using commercial kits. Western blotting was performed to assess protein expression of collagen type I(COL-I), epithelial cadherin(E-cadherin), inositol 1, 4, 5-trisphosphate receptor(IP_3R), voltage-dependent anion channel(VDAC), and mitochondrial calcium uniporter(MCU)in lung tissues, normalized to β-actin.

RESULTS: After 30 cycles, Masson staining showed that the model was successfully established. Compared to the control group, PM_(2.5)-exposed mice exhibited significantly reduced weight and increased lung coefficient(1.54±1.97)g and 1.13%±0.26%, P<0.01). No significant abnormalities were observed in the lung tissue of control and β-carotene group mice, CVF showed no statistically significant difference. Compared to the control group, PM_(2.5)-exposed mice pathological analysis revealed substantial inflammatory cell infiltration and erythrocyte exudation in alveolar spaces and pulmonary interstitium, along with interstitial thickening, alveolar structural deformation(including alveolar rupture or fusion), and compromised alveolar integrity. Pulmonary tissue exhibited significant blue collagen deposition, The CVF in the PM_(2.5) group(18.34%±1.96%) was significantly higher than that in controls(P<0.01). Serum levels of GSH((40.56±5.77)μmol/L, P<0.01) and SOD((9.24±1.46)U/mL, P<0.01) were markedly reduced, while MDA increased((16.33±1.40)nmol/mL, P<0.01). Total protein content in BALF was significantly elevated((1.35±0.09)mg/mL, P<0.01), accompanied by increased counts of leukocytes and lymphocytes in peripheral blood((2.17±0.15) and(1.77±0.12)10~9/L, P<0.05). Additionally, serum HYP levels were significantly higher((20.99±1.61)μg/mL, P<0.01), alongside downregulated E-cadherin(0.49±0.01) and upregulated COL-I(1.63±0.09), IP_3R(1.38±0.07), VDAC(0.92±0.03), and MCU(1.32±0.03) protein expression in lung tissues(P<0.01). Compared to the control group, β-carotene-treated mice showed no significant differences in all indicators. In contrast, compared with the PM_(2.5) group, the β-carotene+PM_(2.5 )group showed improved body weight gain((2.84±1.04)g, P<0.01) and a significantly reduced lung coefficient(0.86%±0.24%, P<0.01). Histological evaluation revealed relatively preserved alveolar architecture, attenuated interstitial thickening, and diminished inflammatory cell infiltration and erythrocyte exudation. Masson staining indicated reduced collagen deposition, with a significantly lower CVF(11.16%±1.43%, P<0.01). Serum GSH and SOD levels were elevated((74.75±6.26)μmol/L and(20.59±1.42)U/mL, P<0.01), while MDA levels decreased((12.26±0.97)nmol/mL, P<0.01). BALF total protein content and peripheral leukocyte/lymphocyte counts were significantly reduced((0.58±0.08)mg/mL, (1.43±0.40) 10~9/L, (0.90±0.36)10~9/L, P<0.05). Serum HYP levels declined((15.01±0.34)μg/mL, P<0.01), accompanied by restored E-cadherin(0.65±0.02) expression and suppressed COL-I(1.08±0.01), IP_3R(1.09±0.03), VDAC(0.68±0.02), and MCU(0.51±0.09) protein levels in lung tissues(P<0.01).

CONCLUSION: carotene may exert a protective effect against PM_(2.5)-induced lung injury in mice, potentially by modulating the function of mitochondria-associated membranes to alleviate PM_(2.5)-induced pulmonary fibrosis. Further studies are warranted to elucidate the precise mechanisms.