Vitamin K2 (MK-7) attenuates LPS-induced acute lung injury via inhibiting inflammation, apoptosis, and ferroptosis

Abstract

Acute lung injury (ALI) is a life-threatening disease that has received considerable critical attention in the field of intensive care. This study aimed to explore the role and mechanism of vitamin K2 (VK2) in ALI. Intraperitoneal injection of 7 mg/kg LPS was used to induce ALI in mice, and VK2 injection was intragastrically administered with the dose of 0.2 and 15 mg/kg. We found that VK2 improved the pulmonary pathology, reduced myeloperoxidase (MPO) activity and levels of TNF-α and IL-6, and boosted the level of IL-10 of mice with ALI. Moreover, VK2 played a significant part in apoptosis by downregulating and upregulating Caspase-3 and Bcl-2 expressions, respectively. As for further mechanism exploration, we found that VK2 inhibited P38 MAPK signaling. Our results also showed that VK2 inhibited ferroptosis, which manifested by reducing malondialdehyde (MDA) and iron levels, increasing glutathione (GSH) level, and upregulated and downregulated glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HO-1) expressions, respectively. In addition, VK2 also inhibited elastin degradation by reducing levels of uncarboxylated matrix Gla protein (uc-MGP) and desmosine (DES). Overall, VK2 robustly alleviated ALI by inhibiting LPS-induced inflammation, apoptosis, ferroptosis, and elastin degradation, making it a potential novel therapeutic candidate for ALI.

Fig 1. VK2 attenuated LPS-induced acute lung injury.

(A) Mice were pre-administered intragastrically solvent or VK2 (0.2 and 15 mg/kg respectively) and subsequent intraperitoneal injection of LPS (7 mg/kg). (B) Histological analysis of lung tissue sections by HE staining (original multiples: 200 ×, scale = 75 μm). (C) Lung tissue injury was assessed by histological scores in all groups. (D) Determination of the myeloperoxidase (MPO) activity in lung homogenates. Values represent means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 2. Effects of VK2 on LPS-induced inflammation.

(A) TNF-α, (B) IL-6 and (C) IL-10 were measured with ELISA in mouse serum. (D) The protein expression levels of TLR4, p-P38 MAPK, and P38 MAPK were evaluated by western blotting. (E, F) Quantitative analysis of TLR4 and the ratio of p-P38/P38 normalized with GAPDH were performed using Image J software. (G) The protein expression levels of iNOS and IL-6 were evaluated by western blotting. (H, I) Quantitative analysis of iNOS and IL-6 normalized with GAPDH. Values represent means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 3. VK2 inhibits apoptosis in LPS-induced ALI.

(A) The protein expression levels of Caspase-3 and Bcl-2 were evaluated by western blotting. (B, C) Quantitative analysis of Caspase-3 and Bcl-2 normalized with GAPDH were performed using Image J software. Values represent means ± SEM, *p < 0.05 and **p < 0.01.

Fig 4. Role of VK2 in ferroptosis during LPS-mediated injury.

(A) GSH, (B) MDA, and (C) Total iron levels in lung tissues. (D) GPX4 and HO-1 protein expression were measured by western blotting. (E, F) Quantitative analysis of GPX4 and HO-1 normalized with GAPDH were performed using Image J software. Values represent means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 5. VK2 alleviates LPS-induced lung elastin degradation.

(A) uc-MGP levels in serum. (B) DES levels in lung tissues. Values represent means ± SEM, *p < 0.05 and **p < 0.01.

Fig 6. Schematic representation of VK2 action on LPS-induced ALI.