BACKGROUND:Ischemic stroke (IS) is a leading cause of death and disability worldwide. Hypercoagulability and thrombus formation play critical roles in its pathogenesis. Leech-derived peptides, especially those obtained from Whitmania pigra, have demonstrated potential antithrombotic and neuroprotective effects. This study aimed to investigate the therapeutic efficacy and underlying mechanisms of a leech peptide in a rat model of cerebral ischemia/reperfusion injury. METHODS:Adult male Sprague-Dawley rats were subjected to middle cerebral artery occlusion followed by reperfusion to establish the IS model. Rats received low-dose or high-dose leech peptide, edaravone as a positive control, or saline. Behavioral assessments, infarct volume measurement, laser speckle imaging for cerebral perfusion, and histopathology were performed. Hemorheology and coagulation parameters were analyzed. Additionally, thrombin levels and fibrinolytic factors were evaluated using ELISA. Biosafety was assessed through hemolysis and histological evaluation of major organs. RESULTS:Leech peptide significantly reduced infarct volume, improved neurological scores, enhanced cerebral perfusion, and preserved brain tissue structure. It modulated thrombin-related parameters, ameliorated coagulation dysfunction, and modulated fibrinolysis-associated factors, thereby contributing to the restoration of coagulation-fibrinolysis homeostasis. Low-dose treatment showed comparable or superior efficacy to the high dose with better safety. No significant toxicity or hemolysis was observed. CONCLUSIONS:Leech peptide derived from Whitmania pigra exerts neuroprotective effects in cerebral ischemia/reperfusion injury through anticoagulation and thrombolysis. These findings support its potential as a novel candidate for IS therapy. TRIAL REGISTRATION:Not applicable. This study did not involve human participants.

Host immune responses to antigens are tightly regulated through the activation and inhibition of synergistic signaling networks that maintain homeostasis. Stimulatory checkpoint molecules initiate attacks on infected or tumor cells, while inhibitory molecules halt the immune response to prevent overreaction and self-injury. Multiple immune checkpoint proteins are grouped into families based on common structural domains or origins, yet the variability within and between these families remains largely unexplored. In this review, we discuss the current understanding of the mechanisms underlying the co-suppressive functions of CTLA-4, PD-1, and other prominent immune checkpoint pathways. Additionally, we examine the IgSF, PVR, TIM, SIRP, and TNF families, including key members such as TIGIT, LAG-3, VISTA, TIM-3, SIRPα, and OX40. We also highlight the unique dual role of VISTA and SIRPα in modulating immune responses under specific conditions, and explore potential immunotherapeutic pathways tailored to the distinct characteristics of different immune checkpoint proteins. These insights into the unique advantages of checkpoint proteins provide new directions for drug discovery, emphasizing that emerging immune checkpoint molecules could serve as targets for novel therapies in cancer, autoimmune diseases, infectious diseases, and transplant rejection.

The popularity of herbal formulas is increasing worldwide. Nevertheless, the effective compound is challenging to identify due to its intricate composition and multiple targets. An integration machine learning-assisted approach was established, whereby the particular action mechanism and direct target were obtained through the correlation of compounds, targets, and metabolites. The association between a compound and an action pathway was selected from the shortest path of the “compound-target-pathway-disease” network, which was analyzed using the Floyd-Warshall algorithm. Subsequently, an investigation was conducted into the relationship between metabolites and action pathways, as well as targets, through the analysis of serum metabolomic profiling and the selection of metabolite biomarkers by random forest. In order to accurately identify the direct acting target as well as the most effective compound, the relationship between the compounds and their targets was investigated using a feature-based prediction model conducted by AdaBoost. The binding mode of the effective compound and the direct-acting target was verified by molecular docking, dynamics simulations, and western blotting. In this study, Baiji Wuweizi Granule (BWG) was employed to elucidate the effective compound against alcoholic liver injury (ALD). BWG exerted an influence on the serum metabolomic, resulting in the identification of seven potential biomarkers. Furthermore, six effective compounds and the PI3K-AKT signalling pathway were identified through a co-analysis with the shortest path from compound to ALD in the “compound-target-pathway-disease” network. It was postulated that the effective compounds would bind with key targets from the PI3K-AKT signaling pathway, as indicated by the prediction model of compound-target interaction (R2 > 0.95). The dominant bonding type for the effective compounds and key targets was hydrogen bond. These results indicated that AKT1 was the notable target for BWG, and that 2,3,4,7-tetramethoxyphenanthrene was the marker compound for BWG against ALD. The present study provides evidence that the protective effect of BWG on ALD can be mediated by the PI3K-AKT signaling pathway. Our findings demonstrate the value of a machine learning-assisted approach in identifying the key compound, target and pathway that underpin the efficacy of an herbal formula. This provides a foundation for future clinical and fundamental research.

Targeting thrombin to screen safe thrombin inhibitors from natural plants and animals is a critical direction in anticoagulant drug development. This study aimed to screen thrombin inhibitors from the nonbloodsucking leech Whitmania pigra (WP) and elucidate the mechanism of anticoagulation through a "computation-guided experimentation" strategy. A peptide library was constructed from WP hydrolysates, and virtual screening was performed using molecular docking and dynamics simulations. A novel thrombin-targeting anticoagulant peptide PEPWP (LRELEDALEQER) was screened out from the peptide library and validated through in vitro/in vivo experiments. PEPWP significantly prolonged thrombin time (TT) and prothrombin time (PT) in a dose-dependent manner in vitro, indicating its role in the common and extrinsic coagulation pathways. Surface plasmon resonance (SPR) analysis then confirmed strong thrombin binding (Kd = 7.242 × 10-6 mol/L). Furthermore, PEPWP prolonged TT while reducing blood viscosity in acute blood stasis rats. Finally, structural analysis revealed that PEPWP bound to Exosite II of thrombin. Arg233 and Arg101 were the key residues for the binding. In conclusion, PEPWP exhibited good anticoagulant activity and significant application potential.

Introduction: Peptide drugs are highly regarded for their therapeutic versatility, safety, and efficacy in treating conditions like cancer, hepatitis, and diabetes. Animal-derived Traditional Chinese Medicines (TCMs) are promising peptide sources, often outperforming plant-based alternatives. The therapeutic effects of leeches, earthworms, and Eupolyphaga sinensis Walkers in thrombosis and blood stasis have been clinically validated, with anticoagulant peptides as key components; however, their pharmacological mechanisms remain unclear. This review categorizes and summarizes anticoagulant peptides from animal-derived TCMs by species. Methods: This study explores the important role of anticoagulant peptides from animal-sourced medicine in treating thrombosis-related diseases. A literature search using keywords like “anticoagulant peptides,” “leech peptides,” and “earthworm peptides” yielded over 120 articles, of which 85 were selected. Priority was given to studies from the past 20 years, while significant historical research was also noted. Articles lacking specific information on the animal sources of these peptides were excluded. Data were obtained from a diverse range of references, including both ancient and modern texts, the Chinese Pharmacopoeia, Web of Science, PubMed, ScienceDirect, Google Scholar, Springer, and CNKI. Conclusion: This study reviews six promising animal-derived anticoagulant drugs currently in clinical use: leeches, earthworms, scorpions, Eupolyphaga sinensis Walkers, and centipedes. Key peptides under development include hirudin (Hirudo nipponica), WP-30 (Whitmania pigra), Lomburkinase (Eisenia foetida). These peptides exhibit potent anticoagulant, antiplatelet, and anti-inflammatory effects, rendering them valuable in the treatment of cardiovascular diseases, stroke, amenorrhea, cerebral thrombosis, and atherosclerosis. Discussion: This study critically evaluates the therapeutic mechanisms of anticoagulant peptides in animal-sourced medicines, emphasizing the potential of peptide-based therapies across different strains of the same species for traditional Chinese medicine and modern clinical use. Despite promising potential, challenges such as low bioavailability, difficulties in peptide identification, pharmacological evaluation, and toxicity assessment persist. Addressing these issues will facilitate deeper exploration and development of peptide-based therapies.

ETHNOPHARMACOLOGICAL RELEVANCE:Several children with pneumonia (especially severe cases) have symptoms of cough and expectoration during the recovery stage after standard symptomatic treatment, which eventually results in chronic lung injury. Danggui yifei Decoction (DGYFD), a traditional Chinese formula, has shown clinical promise for the treatment of chronic lung injury during the recovery stage of pneumonia, however, its mechanism of action is yet to be deciphered.AIM OF THIS STUDY:To investigate the therapeutic mechanism of DGYFD for the treatment of chronic lung injury by integrating network pharmacology and transcriptomics.MATERIALS AND METHODS:BALB/c mice were used to establish the chronic lung injury mouse model by intratracheal instillation of lipopolysaccharide (LPS). Pathological analysis of lung tissue, lung injury histological score, lung index, protein levels in bronchoalveolar lavage fluid (BALF), immunohistochemical staining, blood rheology, inflammatory cytokines, and oxidative stress levels were used to evaluate the pharmacological effects of DGYFD. Chemical components of DGYFD were identified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Integrated network pharmacology together with transcriptomics was used to predict potential biological targets. Western blot analysis was used to verify the results.RESULTS:In this study, we demonstrated that DGYFD could improve lung injury pathological changes, decreases lung index, down-regulate NO and IL-6 levels, and regulate blood rheology. In addition, DGYFD was able to reduce the protein levels in BALF, up-regulate the expression levels of occludin and ZO-1, improve the ultrastructure of lung tissues, and reverse the imbalance of AT I and AT II cells to repair the alveolar-capillary permeability barrier. Twenty-nine active ingredients of DGYFD and 389 potential targets were identified by UPLC-MS/MS and network pharmacology, and 64 differentially expressed genes (DEGs) were identified using transcriptomics. GO and KEGG analysis revealed that the MAPK pathway may be the molecular target. Further, we found that DGYFD inhibits phosphorylation levels of p38 MAPK and JNK in chronic lung injury mouse models.CONCLUSIONS:DGYFD could regulate the imbalance between the excessive release of inflammatory cytokines and oxidative stress, repair the alveolar-capillary permeability barrier and improve the pathological changes during chronic lung injury by regulating the MAPK signaling pathway.

Traditional Chinese medicines (TCMs) have been used to treat cardiovascular diseases (CVDs) with pharmacological effects such as anticoagulation and antithrombosis, and leech known as Hirudo or Shuizhi was the representatives of animal-sourced TCMs. Nonhematophagous leech Whitmania pigra (WP), which is the most widely used leech specie in China, did not secrete the specific thrombin inhibitor hirudin like other hematophagous leeches. To our knowledge, the effective constitutes of Whitmania pigra remains unclear, let alone the anticoagulant mechanism. In this paper, raw and processed non-hematophagous leech Whitmania pigra (WP) were biomimetic enzymatic hydrolyzed by trypsin. The proteins and peptides in the hydrolysate were then identified to establish a Whitmania pigra-derived peptide library containing 160 peptides. Molecular docking and molecular dynamics (MD) simulations were adopted to screen peptide candidates that binds to the thrombin, and the stability of the thrombin-peptide complex and the interaction between them were evaluated. The screened peptide PEPWP (DSYVGDEAQSK) showed anticoagulant properties by PT and TT tests. Based on single-site mutation research on thrombin-PEPWP complex by MD simulation, Arg233 residue in exosite 2 of thrombin was confirmed to account for the major electrostatic interaction between thrombin and PEPWP. These findings not only provide structural insights to understand the anticoagulation mechanism of Whitmania pigra on the thrombin, but also introduce a practical peptide screen strategy for pharmacological research on animal-sourced TCMs.

目的 基于"肺与大肠相表里"理论探讨鼠李糖乳杆菌对过敏性哮喘小鼠细胞外信号调节蛋白激酶1/2(ERK1/2)和p38丝裂原激活蛋白激酶(p38 MAPK)通路及相关免疫细胞的影响.方法 将18只雌性C57 BL/6小鼠随机分为对照组、模型组、鼠李糖乳杆菌组,每组6只.采用卵白蛋白(OVA)致敏加激发方法构建小鼠过敏性哮喘模型,鼠李糖乳杆菌组于雾化激发前给予鼠李糖乳杆菌灌胃,连续7 d,对照组和模型组给予等量生理盐水灌胃.末次灌胃后取各组小鼠肺组织和结肠组织,HE染色进行组织病理观察;ELISA法检测血清OVA特异性IgE含量,流式细胞术检测肺组织中2型固有淋巴样细胞(ILC2)比例,Western blot法检测肺组织中p-ERK1/2和p-p38 MAPK表达情况.结果 与对照组比较,模型组小鼠支气管及血管周围存在明显炎症细胞浸润,部分肺泡结构消失;结肠腺体排列紊乱,隐窝和杯状细胞明显减少,黏膜及黏膜下层炎性细胞浸润;血清OVA特异性IgE含量、肺组织中ILC2比例、肺组织中p-ERK1/2及p-p38 MAPK相对表达量均明显升高(P均<0.05).与模型组比较,鼠李糖乳杆菌组小鼠支气管炎性细胞浸润明显改善;结肠腺体、杯状细胞增多,排列较为整齐,炎细胞浸润明显减少;血清OVA特异性IgE含量、肺组织中ILC2比例、肺组织中p-ERK1/2及p-p38 MAPK相对表达量均明显降低(P均<0.05).结论 鼠李糖乳杆菌可能通过抑制ERK1/2及p38 MAPK磷酸化,减少肺部ILC2,从而减轻过敏性哮喘的肺部炎症.

目的 基于网络药理学和分子对接技术探讨四臣止咳颗粒治疗慢性支气管炎(CB)急性发作的作用机制,并对其进行实验验证.方法 通过TCMSP、TCMID数据库等筛选四臣止咳颗粒的潜在化学成分,采用SwissTargetPrediction数据库预测活性成分的潜在靶点;在OMIM、GeneCards数据库中检索CB急性发作的相关靶点,对四臣止咳颗粒与CB急性发作的靶点进行PPI网络构建,交互处理得到四臣止咳颗粒治疗CB急性发作的关键靶点,进行GO和KEGG分析;利用分子对接技术对成分与关键靶点蛋白进行分子对接,通过多次气道内雾化给药的方式建立脂多糖(LPS)致大鼠CB急性发作模型加以验证.结果 获得四臣止咳颗粒靶点823个,CB急性发作潜在靶点744个,共同靶点180个,关键靶点包括STAT3、AKT1、NFκB1等25个,涉及toll样受体信号通路、NF-κB信号通路等.分子对接结果显示,STAT3、AKT1、MAPK1、NFκB1等可能为四臣止咳颗粒治疗CB急性发作的关键靶点.体内实验验证结果表明,四臣止咳颗粒可明显减轻大鼠肺组织病理程度,肺泡结构紊乱明显好转,炎症明显减轻,WAm/Pbm、WAi/Pbm、WAt/Pbm显著降低(P＜0.01),肺泡灌洗液(BALF)中促炎因子IL-6和TNF-α含量显著降低(P＜0.01),肺组织NF-κBp-p65蛋白表达均降低.结论 四臣止咳颗粒可能通过下调炎症信号通路NF-κB的激活,减轻炎性细胞浸润和气道重塑程度,调节炎症反应进程,从而起到抗CB急性发作的作用,可为深入四臣止咳颗粒治疗CB急性发作分子机制提供理论依据.

目的:探讨炙甘草汤对特发性肺纤维化(Idiopathic pulmonary fibrosis,IPF)小鼠纤维化相关指标的影响,挖掘炙甘草汤治疗IPF的机制.方法:将60只SPF级ICR小鼠随机分为空白组、模型组、吡菲尼酮组和炙甘草汤组,除空白组外,其余组采用气管滴注博莱霉素(5 mg/kg)方法复制JPF小鼠模型,并给予相应的药物治疗.空白组和模型组小鼠灌胃生理盐水,吡菲尼酮组和炙甘草汤组小鼠分别灌胃吡菲尼酮(50mg/kg)和炙甘草汤(25.4 g/kg),各组均连续给药4周后取材,记录各组小鼠的死亡情况,计算各组肺系数;观察肺组织切片病理变化;碱水解法检测肺组织羟脯氨酸(HYP)含量;比色法检测肺组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性;免疫组化、荧光定量PCR检测α-SMA、COL1A蛋白和mRNA的表达水平.结果:炙甘草汤组小鼠死亡数减少,肺系数显著降低(P＜0.01),炎性细胞浸润和胶原沉积面积大量减少,肺泡结构逐渐修复,HYP、MDA含量降低(P＜0.01),SOD活性(P＜0.05)和GSH-Px活性(P＜0.01)显著增强;α-SMA、COL1A蛋白和mRNA表达均降低(P＜0.01).结论:炙甘草汤通过抑制氧化应激反应,从而抑制成纤维细胞活化,减少细胞质基质沉积,从而减缓IPF疾病进程.

Children are susceptible to pneumonia, which affects their growth and development. Immune disorders and unrepaired alveolar mucosal epithelium following pneumonia cause chronic lung injury. The mechanism of chronic lung injury is unknown and lacks animal models for reference. Therefore, we developed a chronic lung injury young mouse model to simulate the pathological process of children. 3-week-old mice were intratracheal instillation of lipopolysaccharide (LPS) every other day for six weeks. Consequently, the histopathology showed damaged integrity of lung tissue, fibrosis, and abnormally distributed alveolar epithelial cells. The total protein concentration in bronchoalveolar lavage fluid (BALF) was increased, alveolar epithelial type (AT) I cells were abnormal distribution, and AT II cells were reduced. The phosphorylation levels of IKBα and the expression levels of NF-κB p65 in lung tissue were up-regulated. In serum and BALF, the IL-6 was oversecretion, nitric oxide (NO) and superoxide dismutase (SOD) were perturbed secretion, oxidative stress imbalance. In addition, blood viscosity, plasma viscosity, and erythrocyte sedimentation rate (ESR) indexes in hemorheology were increased. In conclusion, it is feasible to construct the mouse model of chronic lung injury, and AT I and AT Ⅱ cells were imbalanced, which paves the way for further investigations on the pathogenesis of chronic lung injury and the efficacy of novel treatments.

目的 研究芍药甘草汤对支气管哮喘小鼠肠道菌群结构的调节作用.方法 将50只SPF级BALB/c雌性小鼠随机分为正常组、模型组、醋酸地塞米松组、芍药甘草汤1:1组、芍药甘草汤2:1组.通过卵白蛋白致敏、雾化激发建立肠道菌群紊乱模型.从实验第15天开始,雾化前30 min灌胃,连续灌胃14 d.正常组、模型组给予等量蒸馏水,醋酸地塞米松组(0.676 mg/kg)、芍药甘草汤1:1组(6.01 g/kg)、芍药甘草汤2:1组(6.01 g/kg)给予相应的药物灌胃.实验第29天采集各组结肠组织及粪便样本,观察分析小鼠的一般状态、哮喘典型症状和结肠组织病理改变,各组随机选取6只小鼠进行16S rRNA测序分析肠道菌群多样性和物种丰富度的变化.结果 醋酸地塞米松组、芍药甘草汤1:1组、芍药甘草汤2:1组小鼠的一般状态和哮喘典型症状得到明显改善.病理组织检测显示:正常组结肠组织清晰,结构完整,模型组结肠黏膜及黏膜下层有大量炎性细胞和肿大淋巴结,醋酸地塞米松组充血水肿情况改善,芍药甘草汤1:1组仍可见炎性细胞浸润,芍药甘草汤2:1组结构较完整,黏膜及黏膜下层偶见散在炎性细胞.16S rRNA测序结果显示:与正常组比较,模型组Chao1指数、PD指数降低(P<0.01),observed species指数降低(P<0.05);与模型组比较,醋酸地塞米松组、芍药甘草汤2:1组PD指数升高(P<0.01),Chao1指数、observed species指数升高(P<0.05).肠道菌群菌属水平显示:与正常组相比,模型组毛螺菌属丰度增加(P<0.05),罗斯氏菌属丰度增加(P<0.01),拟普雷沃菌属丰度减少(P<0.05);与模型组相比,芍药甘草汤1:1组毛螺菌属丰度降低(P<0.05);与醋酸地塞米松组相比,芍药甘草汤1:1组拟普雷沃菌属、拟杆菌属、乳杆菌属、罗斯氏菌属丰度增加(P<0.01),芍药甘草汤2:1组罗斯氏菌属丰度降低(P<0.01);与芍药甘草汤1:1组相比,芍药甘草汤2:1组乳杆菌属、罗斯氏菌属丰度降低(P<0.01).结论 芍药甘草汤可改善哮喘小鼠的典型症状、结肠组织病理情况等,其作用机制可能与其恢复肠道菌群的多样性、丰富度及调节菌群结构有关.

目的:探讨麦门冬汤加减方对特发性肺纤维化小鼠转化生长因子-β1(Transforming growth factor-β1,TGF-β1)、平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、胶原 Ⅰ(Collagen type Ⅰ,COL1A)表达的影响以及对 PI3K/AKT/mTOR 通路的调控作用.方法:将120只SPF级ICR小鼠随机分入空白组、模型组、吡菲尼酮组和麦门冬汤加减方组,用博来霉素(5 mg/kg)建立特发性肺纤维化模型,24 h后分别给予相应的药物治疗.吡菲尼酮组和麦门冬汤加减方组小鼠分别灌胃吡菲尼酮和中药麦门冬汤加减方,空白组和模型组小鼠灌胃生理盐水,各组均连续给药3周(21d)后取材.观察指标:各组小鼠的肺系数;肺组织病理变化;肺组织TGF-β1、α-SMA、COL1A的表达量(免疫组化);肺组织中α-SMA、COL1A、p-PI3K、p-AKT、mTOR的蛋白表达量(Western blot);肺组织中TGF-β1、α-SMA、COL1A的mRNA表达量(qPCR).结果:模型组小鼠的肺系数显著增加,麦门冬汤加减方组肺系数显著降低;模型组小鼠肺组织中有较多炎性细胞浸润,胶原沉积明显,肺泡结构破坏严重,麦门冬汤加减方组小鼠肺组织病理改变较模型组明显减轻,胶原沉积大量减少,肺泡结构逐渐修复;麦门冬汤加减方组较模型组α-SMA、COL1A、TGF-β1的蛋白表达量显著降低(P＜0.01);麦门冬汤加减方组较模型组α-SMA、COL1A、p-PI3K、p-AKT、mTOR的蛋白表达量显著下调(P＜0.01);麦门冬汤加减方组较模型组α-SMA、COL1A、TGF-β1的mRNA表达量显著降低(P＜0.01).结论:麦门冬汤加减方能有效改善博来霉素诱导的特发性肺纤维化,降低α-SMA、COL1A、TGF-β1的表达,可能是通过调控PI3K/AKT/mTOR信号通路,抑制上皮间充质转化,减少细胞外基质沉积而发挥作用.

目的:观察诺丽对氢化可的松致肾阳虚模型小鼠的影响,研究诺丽补肾改善记忆的功效,探讨其作用机制.方法:60只昆明小鼠随机分为对照组,模型组,人参组,诺丽高、中、低剂量组,每组10只,除对照组,其他组均灌胃氢化可的松建立肾阳虚模型,观察小鼠给药后记忆行为学变化,尼氏染色观察脑组织海马CA3区锥体细胞形态和尼氏体数量病理改变,ELISA检测小鼠脑组织神经递质、cAMP、cGMP及PKA含量变化.结果:与对照组相比,模型组跳台实验和避暗实验潜伏期明显缩短(P<0.01),错误次数明显增多(P<0.01),消退潜伏期明显缩短(P<0.01),消退错误次数明显增多(P<0.01),大脑海马CA3区尼氏体数量较少(P<0.01),脑组织神经递质5-HT、ACh、DA、NA表达降低(P<0.01),cAMP、PKA含量下降(P<0.01,P<0.05),cAMP/cGMP比值降低(P<0.05).与模型组相比,人参和诺丽高、中剂量能明显延长小鼠跳台实验和避暗实验的潜伏期时间(P<0.01,P<0.05),降低错误次数(P<0.01,P<0.05),延长消退潜伏期时间(P<0.01,P<0.05),降低消退错误次数(P<0.01,P<0.05),增加大脑海马CA3区尼氏体数量(P<0.01),增加脑组织神经递质ACh、5-HT、DA、NA表达(P<0.01,P<0.05),升高cAMP、PKA含量(P<0.01),升高cAMP/cGMP比值(P<0.01).结论:诺丽对氢化可的松致肾阳虚小鼠的记忆损伤有保护作用,其补肾改善记忆是通过调节cAMP/PKA信号通路蛋白,影响下游神经递质,促进神经元蛋白合成实现的.

目的 观察四神方对三硝基苯磺酸(trinitrobenzene sulfonic acid,TNBS)诱导的小鼠实验性炎症性肠病(inflammatory bowel disease,IBD)的免疫调节作用.方法 取56只BALB/c小鼠,随机分为乙醇对照组、模型组、柳氮磺胺吡啶(salicylazosulfapyridine,SASP)组和四神方组.小鼠麻醉后直肠内灌注50％乙醇溶液(含有1.5 mg的TNBS)制备IBD模型.造模2 d后,开始分别使用0.5 g/kgSASP或者2.3 g/kg四神方灌胃给药,连续8 d.观察小鼠的一般状态和疾病活动指数(disease activity index,DAI)评分、结肠组织学、结肠中髓过氧化物 酶(myeloperoxidase,MPO)变化.使用ELISA法检测人重组干扰素γ(IFN-γ)和白细胞介素(IL-12/23 p40)的水平;使用MTT法检测脾脏中以及肠系膜淋巴结(MLNs)中T淋巴细胞增殖;使用流式细胞术检测相关协同刺激因子的表达.结果 四神方有效改善小鼠IBD模型一般状况及DAI评分,缓解结肠局部炎性损伤,减轻炎细胞浸润,减少MPO的产生,降低结肠局部IL-12/23 p40和IFN-γ的水平,引起脾脏中T淋巴细胞数目增加和MLNs中T淋巴细胞数目减少并抑制MLNs中T淋巴细胞协同分子的表达.结论 四神方对TNBS所诱导的小鼠IBD具有显著的治疗作用,可能通过抑制MPO的产生、调节T细胞免疫作用、降低IL-12/23 p40和IFN-γ 的水平缓解结肠中过度的炎性反应.

Viral pneumonia is a common complication caused by Influenza A virus infection and is characterized by severe pulmonary inflammation. A previous study showed that berberine (BBR) significantly ameliorated the pulmonary inflammation in mice with influenza viral pneumonia but its underlying mechanism is not entirely understood. In this study, we reproduced the mouse model of influenza viral pneumonia through intranasal infection of A/Puerto Rico/8/34 (H1N1), to further investigate the anti-inflammatory mechanism of BBR based on nucleotide-binding oligomerization domain-like receptor protein (NLRP) 3 inflammasome activation and Gasdermin D (GSDMD)-mediated pyroptosis. Consistent with MCC950 (10 mg/kg, a specific NLRP3 inflammasome inhibitor), BBR (10 mg/kg) obviously improved the weight loss and survival rate of infected mice, alleviated their pulmonary inflammation, and suppressed the accumulation of tumor necrosis factor and interleukin (IL)-6 in lungs without obvious inhibition on viral multiplication (hemagglutinin titer and nucleoprotein messenger RNA). Moreover, BBR (10 mg/kg) reduced the expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and cysteinyl aspartate-specific proteinase (Caspase)1 (Caspase1 precursor [Pro-caspase1] + Caspase1p20 subunit) and the ratio of Caspase1p20 subunit to Caspase1, thus inhibiting the NLRP3 inflammasome activation and resulting in the decreased contents of mature IL-1 beta and IL-18 in lungs. The GSDMD expression (GSDMD precursor [Pro-GSDMD] + GSDMD-N terminal [NT]) and the ratio of GSDMD-NT to GSDMD were also declined by BBR (10 mg/kg). These evidence indicate that BBR may ameliorate pulmonary inflammation in mice with influenza viral pneumonia through inhibiting NLRP3 inflammasome activation, as well as depressing GSDMD-mediated pyroptosis via declining GSDMD expression and restraining NLRP3 inflammasome-mediated GSDMD activation.

Objective Ulcerative colitis (UC) as one of the intractable diseases in gastroenterology seriously threatens human health. Respiratory pathology is a representative extraintestinal manifestation of UC affecting the quality of life of patients. Gegen Qinlian Decoction (GQD) is a classical traditional Chinese medicine prescription for UC or acute lung injury. This study was aimed to reveal the therapeutic effect of GQD on UC and its pulmonary complications and uncover its molecular mechanism mediated by myeloid cells and microbiota. Methods Mice with DSS-induced colitis were orally administrated with GQD. Overall vital signs were assessed by body weight loss and disease activity index (DAI). Pulmonary general signs were evaluated by pulmonary pathology and lung function. The mechanism of GQD relieving UC was characterized by detecting myeloid cells (neutrophils, macrophages, inflammatory monocytes, and resident monocytes) in colonic and lung tissues, related inflammatory cytokines, as well as the microbiota in bronchoalveolar lavage fluid (BALF) and feces. Results GQD significantly reduced weight loss, DAI scores, and lung injury but improved the lung function of colitis mice. The DSS-induced colonic and concurrent pulmonary inflammation were also alleviated by GQD, as indicated by the down-regulated expressions of inflammatory cytokines (TNF-α, IL-1β, IL-6, CCR2, and CCL2) and the suppressed recruitment of neutrophils and inflammatory monocytes. Meanwhile, GQD greatly improved intestinal microbiota imbalance by enriching Ruminococcaceae UCG-013 while decreasing Parabacteroides, [Eubacterium]_fissicatena_group, and Akkermansia in the feces of colitis mice. Expectantly, GQD also restored lung microbiota imbalance by clearing excessive Coprococcus 2 and Ochrobactrum in the BALF of colitis mice. Finally, significant correlations appeared between GQD-mediated specific bacteria and inflammatory cytokines or immune cells. Conclusion GQD could alleviate UC by decreasing excessive inflammatory myeloid cells and cytokines, and reshaping the microbiota between the colon and lung, which contributes to clarifying the mechanism by which GQD ameliorates colitis-associated pulmonary inflammation.

目的 基于ERK1/2和p38 MAPK信号通路探讨驴食草酚对溃疡性结肠炎小鼠的干预作用.方法 将32只雄性C57BL/6小鼠随机分为正常组、模型组、驴食草酚组和柳氮磺吡啶组,每组8只.模型组、驴食草酚组和柳氮磺吡啶组小鼠均采用饮水中添加3％葡聚糖硫酸钠溶液方法构建溃疡性结肠炎模型,连续7 d.从实验第1天起,驴食草酚组给予驴食草酚10 mg/kg灌胃,柳氮磺吡啶组给予柳氮磺吡啶0.5 g/kg灌胃,正常组和模型组灌喂等量生理盐水,均连续7 d.干预期间观察各组小鼠体重、粪便性状及便血情况,计算疾病活动指数(DAI).末次灌胃后处死小鼠,测量结肠长度,HE染色观察结肠组织病理形态,酶联免疫吸附法检测血清白细胞介素(IL)-17 A含量,流式细胞术检测结肠组织中中性粒细胞比例,Western blot法检测结肠组织中ERK1/2、p-ERK1/2、p38 MAPK、p-p38 MAPK蛋白表达情况.结果 模型组小鼠体重明显低于正常组(P<0.05),DAI明显高于正常组(P<0.05),结肠长度明显短于正常组(P<0.05),驴食草酚组和柳氮磺吡啶组各指标均较模型组明显改善(P均<0.05).模型组小鼠结肠黏膜上皮组织破损严重,黏膜下层有大量的炎性细胞浸润,驴食草酚组和柳氮磺吡啶组结肠组织损伤程度明显减轻.模型组小鼠血清IL-17 A含量、结肠组织中中性粒细胞比例、结肠组织中p-ERK1/2/ERK1/2和p-p38 MAPK/p38 MAPK比值均明显高于正常组(P均<0.05),驴食草酚组和柳氮磺吡啶组各指标均明显低于模型组(P均<0.05).结论 驴食草酚可能通过下调ERK1/2和p38 MAPK的磷酸化表达,抑制IL-17 A分泌,降低结肠组织中性粒细胞比例,减轻溃疡性结肠炎炎性损伤.

This study explored whether Sagittaria sagittifolia polysaccharides(SSP) activates the nuclear factor erythroid-2-related factor2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway to protect against liver damage jointly induced by multiple heavy metals. First, based on the proportion of dietary intake of six heavy metals in rice available in Beijing market, a heavy metal mixture was prepared for inducing mouse liver injury and HepG2 cell injury. Forty male Kunming mice were divided into five groups: control group, model group, glutathione positive control group, and low-and high-dose SSP groups, with eight mice in each group. After 30 days of intragastric administration, the liver injury in mice was observed by HE staining. In the in vitro experiment, MTT assay was conducted to detect the effects of SSP at 0.25, 0.5, 1, and 2 mg·mL~(-1) on HepG2 cell survival at different time points. The content of alanine transaminase(ALT) and aspartate aminotransferase(AST) in the 48-h cell culture fluid was measured using micro-plate cultivation method, followed by the detection of the change in reactive oxygen species(ROS) content by flow cytometry. The mRNA expression levels of Nrf2 and HO-1 in cells were determined by RT-PCR, and their protein expression by Western blot. HE staining results showed that compared with the model group, the SSP administration groups exhibited significantly alleviated inflammatory cell infiltration and fatty infiltration in the liver, with better outcomes observed in the high-dose SSP group. In the in vitro MTT assay, compared with the model group, SSP at four concentrations all significantly increased the cell survival rate, decreased the ALT, AST, and ROS content(P<0.05), and down-regulated Nrf2 and HO-1 mRNA and protein expression(P<0.05). SSP significantly improves inflammatory infiltration in the liver tissue of mice exposed to a variety of heavy metals and corrects the liver fat degeneration, which may be related to its regulation of the Nrf2/HO-1 signaling pathway, reduction of ROS, and alleviation of oxidative damage.

Pneumonia is not only an acute lung infection but also a chronic disease that is susceptible and recurrent. Children are susceptible to pneumonia, which affects their growth and development. Immune disorders and unrepaired alveolar mucosal epithelium following pneumonia cause chronic lung injury. The mechanism of chronic lung injury is unknown and lacks animal models for reference. Therefore, we aim to develop the chronic lung injury mouse model to simulate the pathological process of children. 3-week-old mice were intratracheal instillation of lipopolysaccharide (LPS) every other day for six weeks to induce chronic lung injury. Consequently, the histopathology showed damaged integrity of lung tissue, fibrosis, and abnormally distributed alveolar epithelial cells. The total protein concentration in Bronchoalveolar lavage fluid (BALF) was increased, which indicated the destruction of the alveolar epithelial-capillary barrier. The phosphorylation levels of IKBα and the expression levels of NF-κB p65 in lung tissue were up-regulated. In serum and BALF, the IL-6 was oversecretion, NO and SOD were perturbed secretion, oxidative stress imbalance. In addition, blood viscosity, plasma viscosity, HCT, and ESR indexes in hemorheology were increased. In conclusion, it is feasible to construct the mouse model of chronic lung injury, which paves the way for further investigations on the pathogenesis of chronic lung injury and the efficacy of novel treatments.