目的：放射性肺纤维化（RIPF）是胸部肿瘤（如肺癌、食管癌、乳腺癌等）放射治疗后的一种严重并发症。患者主要表现为进行性呼吸困难，严重者可发展为呼吸衰竭，显著降低患者的生活质量及远期生存率。目前，RIPF的临床治疗手段有限，疗效不理想。因此，深入研究其发病机制并开发新型治疗策略具有重要的临床意义。Ⅱ型肺泡上皮细胞（AECⅡ）的衰老在肺纤维化发生发展中起着重要作用，而牙龈间充质干细胞（GMSCs）因其独特的干细胞特性被认为具有潜在的治疗价值。在我们的前期研究中发现GMSCs能够减轻AECⅡ的衰老，从而缓解肺纤维化的病理过程。本研究拟进一步研究GMSCs中抗衰老作用的关键活性物质及其具体机制，可能为RIPF的防治提供新的理论依据和治疗靶点。

方法：本研究选用小鼠和大鼠Ⅱ型肺泡上皮细胞（MLE12和RLE-6TN），通过γ射线高剂量单次照射（6 Gy）和低剂量多次累积照射（2 Gy×10）构建辐射诱导的细胞衰老模型；通过细胞形态、增殖能力、衰老相关分泌表型因子（SASP）的mRNA及分泌水平、衰老标志物p16、p21的mRNA及蛋白表达水平、衰老相关β-半乳糖苷酶（SA-β-gal）染色等指标评估细胞衰老水平；通过蛋白组学分析GMSCs培养上清，富集衰老相关通路，筛选潜在功能因子；构建稳定敲降PTX3的GMSCs细胞系（GMSCs_lenti sgPTX3），加入PTX3中和抗体（PTX3-NA）和添加真核来源的PTX3重组蛋白（rPTX3（HEK293））的方式，验证PTX3在GMSCs缓解AECⅡ细胞衰老中的作用；采用X射线对C57BL/6N小鼠行单侧右肺照射（总剂量20 Gy），构建RIPF模型；采取腹腔注射形式给予RIPF小鼠以原核表达系统纯化的PTX3重组蛋白（rPTX3（E.coli））；通过小鼠体重和肺部形态改变、脏器比、羟脯氨酸含量以及H&E和Masson染色评估PTX3对RIPF小鼠纤维化的影响；通过检测SASP的mRNA及分泌水平，流式分析pro-SPC与p16/p21的共标记，评估肺内AECⅡ细胞衰老状态；通过血常规、血生化检测，评估rPTX3（E.coli）的安全性。在机制研究中，通过敲降和过表达FGFR，验证rPTX3是否通过与FGF2竞争性结合FGFR发挥作用；通过排除培养基血清补体的影响，探究PTX3是否通过补体调控缓解衰老；通过RIPF小鼠单细胞测序数据库分析，衰老AECⅡ和RIPF小鼠中β-catenin的mRNA及蛋白表达水平变化，β-catenin敲降和过表达，来评估β-catenin对PTX3抗衰老作用的影响；通过抑制蛋白酶体降解（CHX和MG132）降解途径结合蛋白免疫印迹实验，检测PTX3是否促进β-catenin降解；通过IP实验验证PTX3对β-catenin泛素化水平的影响。

结果：在GMSCs培养上清的蛋白组学分析中，PTX3呈现相对高表达，其生物学功能特性表明其参与炎症调节及细胞外基质重塑。体外实验中，与GMSCs相比，GMSCs_lenti sgPTX3在共培养或上清干预实验中，对MLE12细胞衰老的缓解作用显著减弱。GMSCs培养上清中加入PTX3-NA后，其缓解衰老的效果也受到明显抑制；在GMSCs_lenti sgPTX3培养上清基础上添加rPTX3（HEK293）可显著降低MLE12衰老表型。在原核系统表达纯化的rPTX3（E.coli）经与rPTX3（HEK293）对比，其缓解MLE12衰老的能力具有高度一致性。在体内实验中，rPTX3（E.coli）干预治疗能够提升RIPF小鼠基础体重，提高小鼠生存质量。rPTX3（E.coli）改善了小鼠肺部猪肝色病理表观，降低肺内实质性沉积下调肺部脏器比，并减少了肺泡塌陷、胶原蛋白沉积、羟脯氨酸含量等病理指征，且rPTX3（E.coli）有效减少RIPF肺内AECⅡ的衰老。

在机制探究中FGFR的敲降虽有效下调MLE12辐射后的衰老，但并未影响rPTX3（HEK293）进一步对衰老的缓解；外源性补体的清除也能够有效减少衰老的MLE12，但rPTX3（HEK293）的加入仍能在此基础上降低细胞的衰老指标。基于RIPF单细胞测序公共数据库分析，β-catenin基因在RIPF小鼠肺内的AECⅡ中显著高表达。实验证实，β-catenin的过表达进一步促进了辐射诱导的MLE12衰老，而其敲降则显著减轻MLE12衰老表型。在体内和体外实验中，rPTX3均能有效下调β-catenin的蛋白水平；β-catenin的荧光亚细胞定位表明，PTX3主要促进其在衰老细胞胞质中的降解。在CHX干预基础上PTX3加速了β-catenin的蛋白降解速率，而蛋白酶体抑制剂MG132则阻断了PTX3对β-catenin的降解作用。此外，与未处理的衰老MLE12细胞相比，PTX3处理显著增强了β-catenin的泛素化修饰水平。

结论：

1. GMSCs通过分泌PTX3缓解AECⅡ细胞的衰老。

2. PTX3重组蛋白可缓解辐射诱导的AECⅡ细胞衰老。

3. PTX3缓解衰老的能力不依赖其糖基化修饰或天然八聚体结构。

4. PTX3重组蛋白可显著减轻RIPF小鼠的肺组织纤维化程度，并减轻纤维化内衰老AECⅡ且未对血液指标及肝肾功能产生明显影响。

5. PTX3不依赖FGF2-FGFR途径调节AECⅡ细胞衰老。

6. PTX3不以细胞外补体系统依赖的方式缓解AECⅡ细胞衰老。

7. β-catenin参与辐射诱导的AECⅡ衰老进程。

8. PTX3可通过促进β-catenin的泛素化降解途径，下调β-catenin的蛋白水平。

Objective: Radiation-induced pulmonary fibrosis (RIPF) is a severe complication following radiotherapy for thoracic tumors such as lung, esophageal, and breast cancer. Patients typically present with progressive dyspnea, which can lead to respiratory failure, significantly reducing quality of life and long-term survival rates. Current clinical treatments for RIPF are limited and ineffective. Therefore, exploring the pathogenesis of RIPF and developing novel therapeutic strategies is of great clinical significance. The senescence of typeⅡalveolar epithelial cells (AECⅡ) plays a crucial role in the development of pulmonary fibrosis, and gingival mesenchymal stem cells (GMSCs), with their unique stem cell properties, are considered to have potential therapeutic value. Our previous studies have shown that GMSCs can alleviate the senescence of AECⅡ, thereby mitigating the pathological process of pulmonary fibrosis. This study aims to further investigate the key active substances in GMSCs that exert anti-senescence effects and their specific mechanisms, which may provide new theoretical evidence and therapeutic targets for the prevention and treatment of RIPF.

Methods: This study used mouse and rat type II alveolar epithelial cells (MLE12 and RLE-6TN) to construct cellular senescence models via high-dose single irradiation (6 Gy) and low-dose fractionated irradiation (2 Gy × 10) with γ-rays. Cellular senescence was evaluated by cell morphology, proliferation capacity, mRNA and secretion levels of senescence-associated secretory phenotype (SASP) factors, mRNA and protein expression levels of senescence markers p16 and p21, and senescence-associated β-galactosidase (SA-β-gal) staining. Proteomic analysis of GMSCs culture supernatants was performed to enrich senescence-related pathways and screen potential functional factors. A stable GMSCs cell line with PTX3 knockdown (GMSCs_lenti sgPTX3) was constructed, and the role of PTX3 in alleviating AECII cell senescence was verified by adding PTX3 neutralizing antibody (PTX3-NA) and eukaryotic-derived PTX3 recombinant protein (rPTX3 (HEK293)). A RIPF model was established by unilateral right lung irradiation (total dose 20 Gy) with X-rays in C57BL/6N mice, and RIPF mice were treated with intraperitoneal injections of prokaryotically expressed and purified PTX3 recombinant protein (rPTX3 (E.coli)). The effects of PTX3 on RIPF in mice were assessed by body weight, pulmonary morphological changes, organ ratios, hydroxyproline content, and H&E and Masson staining. The senescence state of AECII cells in the lungs was evaluated by detecting mRNA and secretion levels of SASP factors and flow cytometry analysis of pro-SPC co-marked with p16 or p21. The safety of rPTX3 (E.coli) was assessed by blood routine and biochemical tests. In mechanistic studies, the role of rPTX3 in competing with FGF2 for binding to FGFR was verified by FGFR knockdown and overexpression. The effect of PTX3 on senescence through complement regulation was explored by excluding the influence of serum complement in the culture medium. Analysis of single-cell sequencing databases from RIPF mice and changes in β-catenin mRNA and protein expression levels in senescent AECⅡ and RIPF mice were used to assess the impact of β-catenin on the anti-senescence effects of PTX3. The effect of PTX3 on β-catenin degradation was detected by inhibiting proteasomal degradation (CHX and MG132) combined with Western blot experiments, and the effect of PTX3 on β-catenin ubiquitination levels was verified by IP experiments.

Results: Proteomic analysis of GMSCs culture supernatants showed relatively high expression of PTX3, which is involved in inflammation regulation and extracellular matrix remodeling. In vitro experiments showed that compared to GMSCs, GMSCs_lenti sgPTX3 had significantly reduced alleviation of MLE12 cell senescence in co-culture or supernatant intervention experiments. The alleviation of senescence by GMSCs culture supernatants was significantly inhibited after adding PTX3-NA, and adding rPTX3 (HEK293) to GMSCs_lenti sgPTX3 culture supernatants significantly reduced the senescent phenotype of MLE12 cells. Recombinant PTX3 (E.coli) expressed and purified in a prokaryotic system showed high consistency with rPTX3 (HEK293) in alleviating MLE12 senescence. In vivo experiments showed that rPTX3 (E.coli) intervention improved the body weight and survival quality of RIPF mice, improved pathological appearance of the lungs, reduced organ ratios, and decreased pathological indicators such as alveolar collapse, collagen deposition, and hydroxyproline content. rPTX3 (E.coli) also effectively reduced the senescence of AECⅡin RIPF lungs. Mechanistic studies showed that although FGFR knockdown effectively downregulated MLE12 senescence after radiation, it did not affect the further alleviation of senescence by rPTX3 (HEK293). The clearance of exogenous complement also effectively reduced the senescence of MLE12, but the addition of rPTX3 (HEK293) further lowered cellular senescence indicators. Analysis of public single-cell sequencing databases for RIPF showed significantly high expression of β-catenin gene in AECⅡof RIPF mouse lungs. Experiments confirmed that overexpression of β-catenin further promoted radiation-induced MLE12 senescence, while its knockdown significantly reduced the senescent phenotype of MLE12. In vitro and in vivo experiments showed that rPTX3 effectively downregulated β-catenin protein levels. Fluorescent subcellular localization of β-catenin indicated that PTX3 mainly promoted its degradation in the cytoplasm of senescent cells. PTX3 accelerated the degradation rate of β-catenin under CHX intervention, and the proteasome inhibitor MG132 blocked the degradative effect of PTX3 on β-catenin. Additionally, compared to untreated senescent MLE12 cells, PTX3 treatment significantly enhanced the ubiquitination modification level of β-catenin.

Conclusions:

1. GMSCs alleviate the senescence of AECⅡcells through the secretion of PTX3.

2. PTX3 recombinant protein can alleviate radiation-induced senescence of AECⅡcells.

3. The senescence-alleviating ability of PTX3 does not depend on its glycosylation modification or native octameric structure.

4. PTX3 recombinant protein significantly reduces pulmonary fibrosis in RIPF mice and alleviates senescent AECⅡwithin the fibrosis without significantly affecting blood indicators and liver and kidney functions.

5. PTX3 does not regulate AECⅡcell senescence through the FGF2-FGFR pathway.

6. PTX3 does not alleviate AECⅡcell senescence in a complement-dependent manner.

7. β-catenin is involved in the process of radiation-induced AECII senescence.

8. PTX3 can downregulate β-catenin protein levels by promoting its ubiquitination degradation pathway.