Unveiling the potent effect of vitamin D: harnessing Nrf2/HO-1 signaling pathways as molecular targets to alleviate urban particulate matter-induced asthma inflammation
Authors: Ge Dandan , Chen Qihong , Xie Xiaohua , Li Qiyuan , Yang Yungang 
Published: January 2024

Abstract
Background
Asthma is the most common allergic disease characterized by an inflammatory response in the airways. Mechanismly, urban particulate matter (PM) is the most widely air pollutant associated with increased asthma morbidity and airway inflammation. Current research found that vitamin D is an essential vitamin with anti-inflammatory, antioxidant and other medical efficacy. Inadequate or deficient vitamin D often leads to the pathogenesis and stability of asthma. NGF exacerbates airway inflammation in asthma by promoting smooth muscle cell proliferation and inducing the Th2 immune response. Activation of the Nrf2/HO-1 signaling pathway can exert a protective effect on the inflammatory response in bronchial asthma. However, the specific mechanism of this pathway in PM-involved asthmatic airway smooth muscle cells remains unclear.

Methods
Mice were sensitized and challenged with Ovalbumin (OVA) to establish an asthma model. They were then exposed to either PM, vitamin D or a combination of both, and inflammatory responses were observed. Including, acetylcholine stimulation at different concentrations measured airway hyperresponsiveness in mice. Bronchoalveolar lavage fluid (BALF) and serum were collected for TNF-α, IL-1β, IL-6, and Nerve growth factor (NGF) analysis. Additionally, lung tissues underwent histopathological examination to observe alveolar structure and inflammatory cell infiltration. Specific ELISA kits were utilized to determine the levels of the inflammatory factors TNF-α, IL-1β, IL-6, and Nerve growth factor (NGF). Nrf2/HO-1 signaling pathways were examined by western blot analysis. Meanwhile, we constructed a cell system with low HO-1 expression by lentiviral transfection of airway smooth muscle cells. The changes of Nrf2, HO-1, and NGF were observed after the treatment of OVA, PM, and Vit D were given.

Results
The in vivo results showed that vitamin D significantly alleviated pathological changes in lung tissue of PM-exposed mice models. Mechanismly, vitamin D decreased substantial inflammatory cell infiltration in lung tissue, as well as the number of inflammatory cells in BALF. Furthermore, vitamin D reduced the heightened inflammatory factors including of TNF-α, IL-1β, IL-6, and NGF caused by PM exposure, and triggered the activity of nucleus Nrf2 and HO-1 in PM-exposed asthmatic mice. Notably, knockdown HO-1 weakens the Vitamin D- mediated inhibition to pollution toxicity in asthma. Importantly, in vitro experiments on OVA-stimulated mice airway smooth muscle cells, the results showed that OVA and PM, respectively, reduced Nrf2/HO-1 and increased NGF’s expression, while vitamin D reversed the process. And in the HO-1 knockdown cell line of Lenti-si-HO-1 ASMCs, OVA and PM reduced Nrf2’s expression, while HO-1 and NGF’s expression were unchanged.

Conclusions
The above results demastrate that vitamin D downregulated the inflammatory response and the expression of NGF by regulating the Nrf2/HO-1 signaling pathways in airway smooth muscle cells, thereby showing potent anti-inflammatory activity in asthma.