炙甘草汤通过抗氧化抑制博莱霉素诱导的小鼠肺纤维化

摘要 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IPF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(50 mg/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。     

GSK3β/eEF2K信号通路对小鼠肺纤维化过程的影响*

目的: 探讨糖原合成酶激酶-3(GSK3β)/真核延伸因子激酶2(eEF2K)信号通路对肺纤维化进程的影响,为临床治疗肺纤维化寻找新的思路。方法: 采用一次性气管注射法构建C57BL/6雄性小鼠博莱霉素肺纤维化模型,造模14 d后将动物分成模型组、阴性抑制组与抑制组(n=5),另设空白组不作处理。抑制组使用腹腔注射TDZD-8(4 mg/kg),阴性抑制组腹腔注射二甲基亚砜(DMSO)溶液,28 d后处死采集指标。采用苏木精-伊红染色法检测小鼠肺脏病变情况;试剂盒水解法检测肺组织中羟脯氨酸(Hyp)的含量;采用Western blot法检测肺脏中GSK3β、磷酸化GSK3β(p-GSK3β)、eEF2K、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、基质金属蛋白酶-2前体蛋白(pro-MMP-2)、基质金属蛋白酶-2(MMP-2)蛋白表达水平,使用免疫组织化学法检测肺脏中MMP-2、胶原蛋白I(Col I)、胶原蛋白Ⅲ(Col Ⅲ)、α-平滑肌蛋白(α-SMA)的表达。结果: 与空白组相比,模型组中GSK3β、p-GSK3β、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、pro-MMP-2、MMP-2、Col I、Col Ⅲ、α-SMA蛋白表达水平升高,eEF2K蛋白表达水平降低(P＜0.05);与模型组相比,抑制组GSK3β、p-GSK3β、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、pro-MMP-2、MMP-2、Col I、Col Ⅲ、α-SMA蛋白表达降低,eEF2K蛋白表达升高(P＜ 0.05)。结论: GSK3β能通过Ser70、Ser392、Ser470这3个位点磷酸化激活eEF2K,增加纤维化指标含量,促进肺纤维化形成,加重肺组织病变。     

Autophagy protects against redox-active trace metal-induced cell death in rabbit synovial fibroblasts through Toll-like receptor 4 activation

Reactive oxygen species (ROS) are implicated to play a role in initiating rheumatoid arthritis (RA) pathogenesis. We have investigated the mechanism(s) by which essential redox-active trace metals (RATM) may induce cell proliferation and cell death in rabbit synovial fibroblasts. These fibroblast-like synovial (FLS) cells, which express Toll-like receptor 4 (TLR4), were used as a model system that plays a role in potentially initiating RA through oxidative stress. Potassium peroxychromate (PPC, [Cr⁵⁺]), ferrous chloride (FeCl₂, [Fe²⁺]), and cuprous chloride (CuCl, [Cu⁺]) in the indicated valency states were used as exogenous pro-oxidants that can induce oxidative stress through TLR4 coupled activation that also causes HMGB1 release. We measured the proliferation index (PI) of FLS, and examined the effect of RATM oxidants on apoptosis and autophagy by fluorescence cell-sorting flow cytometry (FC). Cell cycle was analysed by FC and autophagy-related protein expression levels were measured by western blot. Our data showed that as RATM as prooxidants increased intracellular ROS (iROS) that can induce oxidative stress. Whereas iROS increased PI in FLS, these reactive species also protected cells against apoptosis by inducing autophagy. Our results indicate that ROS/TLR4-coupled activation may contribute to the pathogenesis of RA in FLS by induction of autophagy. The signalling pathway by which inflammation and its tissue destructive sequel may occur in RA underlies the need for developing therapeutic agents that can inhibit release of tissue-damaging high mobility group box 1 (HMGB1), cytokines, and possess both trace metal chelating capacity and oxidant scavenging properties in a directed combinatorial therapy for RA.     

RGS1 silencing inhibits the inflammatory response and angiogenesis in rheumatoid arthritis rats through the inactivation of Toll-like receptor signaling pathway

Emerging evidence shows that rheumatoid arthritis (RA) progression can be induced by the activation of Toll-like receptor (TLR) signaling pathway. Regulator of G-protein signaling 1 (RGS1) is observed to be a candidate biomarker for arthritis. Accordingly, the present study aims to determine the potential effects of RGS1 mediating TLR on RA. A rat model of collagen-induced arthritis (CIA) was established to mimic the features of RA by injection of bovine type II collagen. The rats with CIA were treated with short hairpin RNA (shRNA) against RGS1 or TLR pathway activator Poly I:C to elucidate the role of RGS1 in RA progression. The inflammatory factors were measured, and the thoracic gland and spleen indexes as well as the vascular density were determined. The expression levels of RGS1, TLR3, vascular endothelial growth factor (VEGF), metalloproteinase-2 (MMP-2), MMP-9, and interleukin 1 receptor-associated kinase-4 (IRAK4) were determined. RGS1 was robustly increased in RA. The TLR signaling pathway was suppressed by RGS1 silencing. shRNA-mediated depletion of RGS1 was shown to significantly enhance thoracic gland index and inhibit the serum levels of TNF-α, IL-1β, and IL-17, spleen index, vascular density, and the expression levels of TLR3, VEGF, MMP-2, MMP-9, and IRAK4. However, when the rats with CIA were treated with Poly I:C, the trend of effects was opposite. These findings highlight that functional suppression of RGS1 inhibits the inflammatory response and angiogenesis by inactivating the TLR signaling pathway in rats with CIA, thereby providing a novel therapeutic target for RA treatment.     

Identification of TAF1, HNF4A,and CALM2 as potential therapeutic target genes for liver fibrosis

The molecular mechanism of liver fibrosis caused by hepatitis C virus (HCV) is not clear. The aim of this study is to understand the molecular mechanism of liver fibrosis induced by HCV and to identify potential therapeutic targets for hepatic fibrosis. We analyzed gene expression patterns between high liver fibrosis and low liver fibrosis samples, and identified genes related to liver fibrosis. We identified TAF1, HNF4A, and CALM2 were related to the development of liver fibrosis. HNF4A is important for hepatic fibrogenesis, and upregulation of HNF4A is an ideal choice for treating liver fibrosis. The gene expression of CALM2 is significantly lower in liver fibrosis samples than nonfibrotic samples. TAF1 may serve as a biomarker for liver fibrosis. The results were further validated by an independent data set GSE84044. In summary, our study described changes in the gene expression during the occurrence and development of liver fibrosis. The TAF1, HNF4A, and CALM2 may serve as novel targets for the treatment of liver fibrosis.     

Romina: A powerful strawberry with in vitro efficacy against uterine leiomyoma cells

Uterine leiom yomas are benign tumors highly prevalent in reproductive women. In thecurrent study, initially, we aimed to screen five different strawberry cultivars (Alba, Clery, Portola, Tecla, and Romina) to identify efficient cultivars in terms of phytochemical characterization and biological properties by measuring phenolic and anthocyanin content as well as antioxidant capacity, and by measuring apoptotic rate and reactive oxygen species (ROS) production in uterine leiomyoma cells. Next, we focused on the most efficient ones, cultivar Alba (A) and Romina (R) as well as Romina anthocyanin (RA) fraction for their ability to regulate oxidative phosphorylation (oxygen consumption rate [OCR]) glycolysis (extracellular acidification rate [ECAR]), and also fibrosis. Leiomyoma and myometrial cells were treated with a methanolic extract of A and R (250 μg/ml) or with RA (50 μg/ml) for 48 hr to measure OCR and ECAR, as well as gene expression associated with fibrosis. In the leiomyoma cells, RA was more effective in inducing apoptosis and increasing intracellular ROS levels, followed by R and A. In myometrial cells, all strawberry treatments increased the cellular viability and decreased ROS concentrations. Leiomyoma cells showed also a significant decrease in ECAR, especially after RA treatment, while OCR was slightly increased in both myometrial and leiomyoma cells. R and RA treatment significantly decreased collagen 1A1, fibronectin, versican, and activin A messenger RNA expression in leiomyoma cells. In conclusion, this study suggests that Romina, or its anthocyanin fraction, can be developed as a therapeutic and/or preventive agent for uterine leiomyomas, confirming the healthy effects exerted by these fruits and their bioactive compounds.     

C1q/tumor necrosis factor-related protein-3 improves renal fibrosis via inhibiting notch signaling pathways

C1q/tumor necrosis factor-related protein-3 (CTRP3) has been extensively reported as an important role involved in antifibrosis, antiapoptosis, and anti-inflammation. However, the role of CTRP3 involved in renal fibrosis remains unclear. Our current study explored the role of CTRP3 in renal fibrosis and its underlying mechanisms by using serums and renal biopsy specimens from renal fibrosis patients and control subjects, rats models with the surgery of unilateral ureteral obstruction (UUO) and human renal proximal tubular epithelial cells (HRPTEpiCs). We found that circulating levels of CTRP3 had no significant difference between renal fibrosis patients and healthy subjects; however, renal CTRP3 expression was markedly downregulated in the fibrotic region with an abundant expression of collagen-I. In UUO rat models, circulating levels of CTRP3 have not changed with the prolonged obstruction of the kidney; renal CTRP3 expression was decreased with the severity of renal fibrosis; adenovirus-mediated CTRP3 treatment inhibited renal interstitial fibrosis. In vitro experiments revealed that CTRP3 attenuates TGF-β1 induced tubular epithelial cells fibrotic changes; CTRP3 knockdown facilitates the expression of fibrotic markers in TGF-β1-induced HRPTEpiCs; recombinant CTRP3 or adenovirus-mediated CTRP3 overexpression significantly inhibited the Notch signaling pathway-associated factors, and knockdown of CTRP3 increased TGF-β1-mediated activation of the Notch signaling pathways. Collectively, our current study found that CTRP3 could improve renal fibrosis, to some extent, through inhibiting the Notch pathway.