研究背景：
特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）是慢性进行性、不可逆性间质性肺疾病，目前病因未明，治疗手段有限，预后极差。近年来研究发现核苷酸结合寡聚化结构域样受体家族含 pyrin 结构域蛋白 3（NLRP3）炎性小体的持续激活在 IPF 的发病机制中发挥重要作用。人参皂苷 Rb1（ginsenoside Rb1，G-Rb1）作为传统中草药人参中含量最多的活性成分，具有抗炎和抗纤维化作用。已经有研究证实，G-Rb1 可以缓解肝、肾纤维化，并可以抑制脂多糖（Lipopolysaccharide，LPS）诱导的急性肺损伤，但 G-Rb1 能否缓解肺间质纤维化尚未有研究。
研究目的：
本研究通过体内和体外实验探讨了 G-Rb1 是否通过抑制 NLRP3 炎性小体和上游 NF-κB 信号通路，进而缓解博来霉素（bleomycin, BLM）诱导的肺纤维化。
研究方法：
体内实验：
1. 48 只雄性 C57BL/6J 小鼠随机分为 4 组（12 只/组）：正常组（control 组）、博来霉素模型组（BLM 组）、博来霉素+人参皂苷 Rb1 组（BLM+G-Rb1 组）、人参皂苷 Rb1 组（G-Rb1 组），经气道内滴注 BLM 构建小鼠肺纤维化模型，每组 6 只小鼠分别在第 3 天和第 21 天安乐死。
2. 将小鼠肺组织进行苏木精-伊红染色（Hematoxylin-Eosin Staining, HE）、马松（Masson）染色、天狼星红（Sirius Red）染色，进行病理学评估。检测肺纤维化相关指标如 α-平滑肌肌动蛋白（alpha-smooth muscle actin, α-SMA）、胶原 I（collagen
I, col I）、羟脯氨酸（hydroxyproline, HYP），评估各组小鼠肺纤维化程度。
3. 通过酶联免疫吸附测定（Enzyme linked immunosorbent assay, Elisa）检测肺泡灌洗液（bronchoalveolar lavage fluid, BALF）中细胞因子水平。
4. 通过 western blot 检测小鼠肺组织内 NLRP3 炎性小体相关蛋白和上游 NF-κB 信号通路相关蛋白表达。
5. 通过双重组织免疫荧光，明确 NLRP3 炎性小体的主要细胞来源。
体外实验：
1. 通过佛波醇 12-十四酸酯 13-乙酸酯（phorbol-12-myristate-13-acetate, PMA）诱导人急性单核细胞白血病细胞（THP-1 细胞）分化为巨噬细胞，使用不同浓度 G-Rb1刺激巨噬细胞，使用MTT试剂盒检测不同浓度G-Rb1对巨噬细胞活力的影响，并选取出最合适的 G-Rb1 浓度。
2. 构建体外细胞模型，采用 LPS 和三磷酸腺苷（adenosine triphosphate, ATP）刺激巨噬细胞以激活 NLRP3 炎性小体。将巨噬细胞分为四组：正常组，LPS+ATP组，LPS+ATP+G-Rb1 组，G-Rb1 组。
3. 通过western blot、real-time PCR检测NLRP3炎性小体相关蛋白的表达情况；通过 western blot、细胞免疫荧光检测 NF-κB 信号通路相关蛋白。
4. 通过 ELISA 检测细胞上清中白介素-1β（interleukin-1 beta, IL-1β）和白介素-18（interleukin-18, IL-18）水平。
5. 收集不同组巨噬细胞的上清液制作条件培养基，将人胚肺成纤维细胞（MRC-5 细胞）在不同条件培养基中培养，检测细胞中 α-SMA 和 col I 蛋白水平。
6. 使用重组人 IL-1β（recombinant human IL-1 beta, rhIL-1β）刺激 MRC-5 细胞，检测细胞中 α-SMA 和 col I 蛋白水平。
7. 分别使用 control siRNA、NLRP3 siRNA 转染巨噬细胞后，给予 LPS+ATP 刺激，通过 ELISA 检测上清液中 IL-1β 水平；并制作条件培养基培养 MRC-5 细胞，观察细胞中 α-SMA 蛋白表达。
研究结果：
体内实验结果：
1. 根据病理学评估，BLM 处理后第 3 天能够引起小鼠急性肺损伤，小鼠肺组织内可见严重肺泡炎症，大量炎性细胞浸润和明显肺水肿。BLM 处理后第 21 天可以导致小鼠肺组织出现纤维化，肺组织结构遭到严重破坏、肺泡结构消失、肺泡间隔明显增厚、成纤维细胞显著增生和细胞外基质异常沉积。G-Rb1 干预能够缓解BLM 诱导的小鼠急性肺损伤和肺纤维化。
2. G-Rb1 能够减轻 BLM 诱导的小鼠 BALF 中细胞因子如：IL-1β、IL-18、白介素-6（interleukin-6, IL-6）、肿瘤坏死因子 α（tumor necrosis factor α, TNF-α）和转化生长因子 β1（transforming growth factor-β1, TGF-β1）的释放。（p＜0.01）
3. BLM 能够诱导小鼠肺组织中 NLRP3 炎性小体的激活以及下游细胞因子的成熟，并且根据组织免疫荧光结果提示，激活的 NLRP3 炎性小体主要存在于肺泡巨噬细胞中，G-Rb1 干预能够抑制肺泡巨噬细胞中 NLRP3 炎性小体的激活。
4. BLM 刺激后可以促进 NF-κB p65 和 IκBα 磷酸化以激活 NF-κB 信号通路，G-Rb1 干预能够通过抑制 NF-κB p65 和 IκBα 磷酸化阻止 NF-κB 信号通路的激活。
体外实验结果：
1. 根据 MTT 结果，我们选取 G-Rb1 最适宜的浓度为 20μM。
2. LPS+ATP 能够激活巨噬细胞内 NLRP3 炎性小体，G-Rb1 干预能够抑制NLRP3 炎性小体的激活。
3. LPS+ATP 可以激活巨噬细胞内 NF-κB 信号通路，G-Rb1 通过抑制 NF-κB p65和 IκBα 磷酸化以及 NF-κB p65 入核，进而阻断 NF-κB 信号通路。
4. 巨噬细胞中 NLRP3 炎性小体激活，促进 IL-1β 成熟和释放，进而诱导肺成纤维细胞分化为肌成纤维细胞，G-Rb1可以抑制巨噬细胞引导的肌成纤维细胞活化。
5. rhIL-1β 刺激 MRC-5 细胞，促进细胞内 α-SMA 和 col I 蛋白表达。NLRP3 siRNA 转染巨噬细胞后，细胞上清中 IL-1β 水平显著下降；并且使用 NLRP3 siRNA条件培养基中的 MRC-5 细胞内 α-SMA 水平较 control siRNA 条件培养基组明显下降。
研究结论：
1. BLM 能够导致小鼠肺组织内大量炎症细胞浸润、肺泡结构破坏、成纤维细胞显著增生以及细胞外基质沉积，引起急性肺损伤和晚期肺纤维化。同时 BLM 还可以激活肺泡巨噬细胞中 NLRP3 炎性小体和上游 NF-κB 信号通路。
2. G-Rb1 可以延缓 BLM 诱导的小鼠急性肺损伤和肺纤维化，其机制可能是通过抑制肺泡巨噬细胞中 NLRP3 炎性小体和上游 NF-κB 信号通路的激活。
3. 巨噬细胞可以诱导肺成纤维细胞分化为肌成纤维细胞，主要是通过激活NLRP3-IL-1β 通路实现。
4. G-Rb1 抑制巨噬细胞-成纤维细胞之间的相互作用，其机制可能是通过抑制巨噬细胞内 NLRP3 炎性小体的激活和下游 IL-1β 的成熟。

Background:
Idiopathic pulmonary fibrosis (IPF) is chronic, progressive, and irreversible fibrotic disease of unknown etiology. Patients with IPF have an overall poor prognosis and limited treatment options. Emerging evidence suggests that the continuous activation of the central nucleotide-binding oligomerization-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome is involved in the pathogenesis of pulmonary fibrosis. Ginsenoside Rb1 (G-Rb1) is the most abundant component in the traditional Chinese herb ginseng, with anti-inflammatory and anti-fibrotic effects. Studies have demonstrated that G-Rb1 exerts anti-fibrotic effects on the liver and kidney. In addition, G-Rb1 attenuates lipopolysaccharide (LPS)-induced acute lung injury though the NF-κB pathway. However, the efficacy of G-Rb1 in pulmonary fibrosis has not been reported.
Objective:
The purpose of this study was to explore whether G-Rb1 exerts anti-inflammatory and anti-fibrotic activities in vivo and in vitro by suppressing the activation of NLRP3 inflammasome and the upstream NF-κB pathway.
Methods:
In vivo:
1. Forty-eight mice were randomly divided into four groups (12mice per group): Control group (control); bleomycin group (BLM); BLM plus ginsenoside Rb1 group (BLM+G-Rb1); ginsenoside Rb1 group (G-Rb1). Pulmonary fibrosis was initiated via an intratracheal injection with BLM. Six mice from each group were euthanized on day 3 and day 21.
2. The lung samples were cut into 5-μm sections and stained with hematoxylin-eosin solution (HE) staining, Masson’s Trichrome staining, and Sirius Red staining for pathological evaluation. The pulmonary fibrosis-related indicators such as alpha-smooth muscle actin (α-SMA), collagen 1 (col I), and hydroxyproline (HYP) were detected to assess the degree of pulmonary fibrosis.
3. The levels of IL-1β, IL-18, IL-6, tumor necrosis factor α (TNF-α), and transforming growth factor-β1 (TGF-β1) in mice BALF were measured by ELISA kit.

4. The expressions of NLRP3 inflammasome and upstream NF-κB signaling pathway in lung tissues were measured by western blotting.
5. To further elucidate the cellular source of the NLRP3 inflammasome in the BLM-induced fibrotic lungs, we detected by double immunofluorescence.
In vitro:
1. THP-1 cells were made to differentiate into macrophages by adding phorbol-12-myristate-13-acetate (PMA), then used in experiments. The cells were treated with vehicle control and G-Rb1 at different doses for 24h. The cells were tested by a thiazolyl blue tetrazolium bromide assay kit to calculate cell viability and select the most suitable concentration of G-Rb1.
2. The macrophages injury model induced by Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) were divided into four group: control group; LPS+ATP
group; LPS+ATP+G-Rb1 group; G-Rb1 group.
3. The expressions of NLRP3 inflammasome in macrophages were evaluated by western blotting and real time PCR. The expressions of NF-κB pathway in macrophages were detected by western blotting and immunofluorescence.
4. The levels of IL-1β and IL-18 in cell supernatants detected using ELISA.
5. The conditioned medium from the treated macrophages supernatants was gathered, centrifuged, and mixed with fresh culture medium. The conditioned media were applied to MRC-5 cells; the cells were collected for detecting the levels of α-SMA and col I.
6. MRC-5 cells were stimulated by recombinant human IL-1β (rhIL-1β), and the α-SMA and col I levels in the cells were detected by western bloting and immunofluorescence.
7. The macrophages were transfected with control siRNA or NLRP3 siRNA using lipofectamine 3000. The control or NLRP3 siRNA-transfected cells were stimulated by LPS and ATP. The IL-1β level in cell supernatants were detected by ELISA. Moreover, collected cell supernatants to make conditioned medium for culturing the MRC-5 cells, then detected the α-SMA levels in the MRC-5 cells.
Result:
In vivo:
1. According to histological evaluation, BLM induced severe alveolitis, pulmonary edema, and serious inflammatory cell infiltration in mouse model on day 3. Moreover, BLM induced vascular hemorrhage, excessive collagen accumulation, and alveolar structure chaos in mouse model on day 21. Whereas, G-Rb1 administration had obviouseffects on reducing the intensity of alveolitis, inflammatory cell accumulation, alveolar
chaos, and collagen accumulation.
2. G-Rb1 treatment decreased the levels of IL-1β, IL-18, IL-6, TNF-α, and TGF-β1 in the BALF in mice.
3. BLM stimulation can trigger the activation of NLRP3 inflammasome and promote the maturation of downstream cytokines in the lung tissue. And according to the results of
double immunofluorescence, the NLRP3 inflammasome was mainly expressed in alveolar macrophages, which was blocked by G-Rb1.
4. Upon BLM stimulation, the phosphorylation of IκBα and NF-κB p65 increased, and the G-Rb1 treatment inhibited phosphorylation compared with the BLM treatment.
In vitro:
1. G-Rb1 was not cytotoxic to the macrophages at doses below 20μM but was slightly toxic at dosed above 40μM. Therefore, the G-Rb1 concentration of 20μM was selected for further experiments.
2. Upon LPS and ATP stimulation, the NLRP3 inflammasome was activated in the macrophages, whereas the G-Rb1 treatment can inhibit the activation of NLRP3 inflammasome.
3. NF-κB signaling pathway was activated in the macrophages upon LPS/ATP stimulation and G-Rb1 pretreatment significantly suppressed the NF-κB pathway by inhibiting the phosphorylated IκBα and NF-κB p65, and reducing the nuclear translocation of NF-κB p65 from the cytoplasm.
4. The NLRP3-IL-1β axis participated in the progression of macrophage-initiated fibroblast differentiation, and G-Rb1 inhibited the crosstalk between macrophages and fibroblasts.
5. The α-SMA and col I levels increased more in the rhIL-1β group than that in the control group. IL-1β expression decreased significantly after NLRP3 siRNA treatment, and
the α-SMA levels in MRC-5 cells cultured by NLRP3 siRNA-conditioned medium significantly lower than that cultured by control siRNA-conditioned medium.
Conclusion:
1. Serious inflammatory cell infiltration, alveolar structure chaos, and excessive collagen accumulation were observed in the BLM induced mice model. Furthermore, BLM stimulation triggered the NLRP3 inflammasome and NF-κB signaling pathway in alveolar macrophages.
2. G-Rb1 ameliorated BLM-induced pulmonary inflammation and fibrosis in mice by suppressing NLRP3 inflammasome activation and the NF-κB pathway.
3. Macrophage can initiate fibroblast differentiation by activating the NLRP3-IL-1β axis.
4. G-Rb1 can disturb the crosstalk between macrophages and fibroblasts, and possibly by inhibiting the activation of NLRP3 inflammasome and the downstream IL-1β maturation.