Table_1_Ma Xing Shi Gan Decoction Attenuates PM2.5 Induced Lung Injury via Inhibiting HMGB1/TLR4/NFκB Signal Pathway in Rat.docx

Ma Xing Shi Gan Decoction (MXD), a classical traditional Chinese medicine prescription, is widely used for the treatment of upper respiratory tract infection. However, the effect of MXD against particulate matters with diameter of less than 2.5 μm (PM2.5) induced lung injury remains to be elucidated. In this study, rats were stimulated with PM2.5 to induce lung injury. MXD was given orally once daily for five days. Lung tissues were harvested to assess pathological changes and edema. Myeloperoxidase (MPO) activity and malonaldehyde (MDA) content in lung were determined to evaluate the degree of injury. To assess the barrier disruption, the bronchoalveolar lavage fluid (BALF) was collected to determine the total protein content and count the number of neutrophils and macrophages. For evaluating the activation of macrophage in lung tissue, CD68 was detected using immunohistochemistry (IHC). The levels of inflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6) in BALF and serum were measured. In vitro, a PM2.5-activated RAW 264.7 macrophages inflammatory model was introduced. To evaluate the protective effect of MXD-medicated serum, the cell viability and the release of inflammatory factors were measured. The effects of MXD on the High mobility group box-1/Toll-like receptor 4/Nuclear factor-kappa B (HMGB1/TLR4/NFκB) pathway in lung tissue and RAW 264.7 cells were assessed by Western blot. For further confirming the protective effect of MXD was mediated by inhibiting the HMGB1/TLR4/NFκB pathway, RAW 264.7 cells were incubated with MXD-medicated serum alone or MXD-medicated serum plus recombinant HMGB1 (rHMGB1). MXD significantly ameliorated the lung injury in rats, as evidenced by decreases in the pathological score, lung edema, MPO activity, MDA content, CD68 positive macrophages number, disruption of alveolar capillary barrier and the levels of inflammatory factors. In vitro, MXD-medicated serum increased cell viability and inhibited the release of inflammatory cytokines. Furthermore, MXD treatment was found to inhibit HMGB1/TLR4/NFκB signal pathway both in vivo and in vitro. Moreover, the protection of MXD could be reversed by rHMGB1 in RAW 264.7. Taken together, these results suggest MXD protects rats from PM2.5 induced acute lung injury, possibly through the modulation of HMGB1/TLR4/NFκB pathway and inflammatory responses.

马杏石甘汤（MXD），乃经典之传统中医药方，广泛用于治疗上呼吸道感染。然而，MXD对于直径小于2.5微米（PM2.5）之颗粒物引起的肺损伤的疗效尚需进一步阐明。本研究中，通过向大鼠注入PM2.5以诱导肺损伤。MXD每日口服一次，连续五日。收集肺组织以评估病理变化及水肿。通过测定肺中髓过氧化物酶（MPO）活性和丙二醛（MDA）含量，以评估损伤程度。为评估屏障破坏，收集支气管肺泡灌洗液（BALF）以确定总蛋白含量，并计数中性粒细胞和巨噬细胞。通过免疫组织化学（IHC）检测CD68以评估肺组织中巨噬细胞的活化。在BALF和血清中测定包括肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）在内的炎症因子水平。体外构建了PM2.5激活的RAW 264.7巨噬细胞炎症模型。为评估MXD药物治疗血清的保护作用，测量细胞活力和炎症因子的释放。通过Western blot评估MXD对肺组织和RAW 264.7细胞中高迁移率族蛋白盒-1/ Toll样受体4/ 核因子-κB（HMGB1/TLR4/NFκB）通路的影响。为进一步确认MXD的保护作用是通过抑制HMGB1/TLR4/NFκB通路介导的，将RAW 264.7细胞与MXD药物治疗血清或MXD药物治疗血清加上重组高迁移率族蛋白盒-1（rHMGB1）共同孵育。MXD显著改善了大鼠的肺损伤，表现为病理评分、肺水肿、MPO活性、MDA含量、CD68阳性巨噬细胞数量、肺泡毛细血管屏障破坏以及炎症因子水平的降低。体外实验中，MXD药物治疗血清增加了细胞活力并抑制了炎症细胞因子的释放。此外，MXD治疗被发现可抑制体内和体外HMGB1/TLR4/NFκB信号通路。更进一步，MXD的保护作用可被重组HMGB1逆转。综上所述，这些研究结果提示MXD可能通过调节HMGB1/TLR4/NFκB通路和炎症反应，保护大鼠免受PM2.5引起的急性肺损伤。