Introduction Metabolic dysfunction-associated steatohepatitis (MASH) is characterized by disrupted hepatic lipid homeostasis and progressive liver injury, and frequently coexists with hypertension and systemic vascular remodeling, suggesting shared pathophysiological mechanisms. Although adipose tissue-derived microRNAs have emerged as important mediators of inter-organ communication, their roles in regulating hepatic cholesterol metabolism and vascular dysfunction during MASH remain incompletely understood.Objectives This study aimed to investigate whether microRNA-518a regulates hepatic cholesterol metabolism and MASH progression through the MST1-AMPK-SREBP2 signaling pathway and to explore its potential implications for vascular remodeling and hypertension.Methods Integrative bioinformatic analyses, including Mendelian randomization and transcriptomic network analysis, were performed to identify candidate regulatory pathways associated with MASH. Functional validation was conducted using human clinical samples, in vitro cell models, exosome-mediated microRNA delivery, and a dietary mouse model of MASH.Results MST1 was identified as a key regulatory target of microRNA-518a. Increased microRNA-518a expression suppressed MST1, resulting in reduced AMPK activation and enhanced SREBP2-mediated cholesterol biosynthesis. Functional experiments demonstrated that microRNA-518a promoted hepatic cholesterol accumulation, lipid deposition, inflammation, and fibrotic changes, whereas restoration of MST1 partially reversed these effects. Exosome-mediated delivery of microRNA-518a further supported its ability to modulate hepatic metabolic responses through intercellular communication.Conclusions MicroRNA-518a promotes hepatic cholesterol dysregulation and MASH progression by targeting the MST1-AMPK-SREBP2 signaling pathway. Notably, the convergence of this axis with vascular regulatory networks suggests broader implications for understanding the metabolic-vascular comorbidities frequently observed in MASH patients, including hypertension and arterial stiffness.

Osteosarcoma, a primary malignant bone tumor predominantly affecting children and adolescents, exhibits a dismal prognosis for patients with metastatic or recurrent disease, characterized by low five-year survival rates. This poor outcome is largely attributed to the incomplete understanding of the molecular mechanisms governing osteosarcoma aggressiveness and metastasis, coupled with a paucity of effective early diagnostic biomarkers and targeted therapeutic strategies. Consequently, elucidating the critical molecular events driving osteosarcoma pathogenesis and identifying novel biomarkers are of paramount importance for improving patient outcomes. Prior research has demonstrated aberrant expression of Zinc Finger DHHC-Type Palmitoyltransferase 9 (ZDHHC9) in various malignancies, but its specific function and clinical significance in osteosarcoma remain poorly defined. This study employed a comprehensive approach integrating bioinformatic analyses and experimental validation to investigate the functional role and clinical relevance of ZDHHC9 in osteosarcoma. Bioinformatic analyses (TIMER, GEPIA, TCGA databases) were utilized to examine ZDHHC9 expression across a pan-cancer landscape and its association with survival in osteosarcoma patients. Immunohistochemistry (IHC) and Western blotting were employed to analyze ZDHHC9 expression in clinical osteosarcoma specimens and cell lines. In vitro assays (CCK-8, colony formation, wound healing, Transwell, flow cytometry, and Western blotting) were performed to evaluate the impact of ZDHHC9 on osteosarcoma cell proliferation, migration, and invasion. Proteomic sequencing, molecular docking, and co-immunoprecipitation (Co-IP) experiments were conducted to explore the interaction between ZDHHC9 and KRAS. Western blotting was used to analyze the regulation of Raf1, ERK1/2, and p-ERK1/2 expression by ZDHHC9 and KRAS. Finally, a xenograft model was employed to assess the effect of ZDHHC9 on in vivo tumor growth. Bioinformatic analyses revealed that elevated ZDHHC9 expression correlates with poor prognosis in osteosarcoma. IHC and Western blotting confirmed significantly increased ZDHHC9 expression in osteosarcoma tissues compared to adjacent non-tumorous tissues. Upregulation of ZDHHC9 was significantly associated with elevated Ki67 levels and advanced Enneking stage, suggesting a link to tumor aggressiveness. In vitro studies demonstrated that downregulation of ZDHHC9 suppressed osteosarcoma cell proliferation, migration, and invasion, while promoting apoptosis. Conversely, upregulation of ZDHHC9 elicited opposite effects. Proteomic sequencing revealed that ZDHHC9 knockdown significantly downregulated the expression of proteins associated with the RAS/MAPK signaling pathway. Functional experiments showed that overexpression of KRAS partially abrogated the inhibitory effects of ZDHHC9 knockdown on cell proliferation, migration, and invasion. Molecular docking and Co-IP experiments confirmed a specific interaction between ZDHHC9 and KRAS, leading to activation of the RAS/MAPK signaling pathway. ZDHHC9 promotes osteosarcoma cell proliferation, migration, and invasion by enhancing KRAS-mediated activity of the RAS/MAPK signaling pathway, which promotes cell cycle progression while inhibiting apoptosis. Data are available via ProteomeXchange with identifier PXD066322.

BACKGROUND:Non-alcoholic steatohepatitis (NASH) progression is strongly associated with deteriorating hepatic function, primarily driven by free cholesterol (FC) accumulation-induced lipotoxicity. Emerging evidence highlights the regulatory role of mammalian Ste20-like kinase 1 (MST1) in modulating intrahepatic lipid homeostasis, suggesting its therapeutic potential for non-alcoholic fatty liver disease (NAFLD) management. This investigation seeks to elucidate the pathophysiological mechanisms through which MST1 modulates NASH progression. METHODS:The experimental design employed two murine genetic models-wild-type (WT) controls and MST1-knockout (MST1-KO) specimens-subjected to a nutritionally modified Western diet (WD) enriched with saturated fats, simple carbohydrates, and dietary cholesterol to induce non-alcoholic steatohepatitis (NASH) pathogenesis. Lentiviral transduction techniques facilitated targeted MST1 overexpression in WT animals maintained on this dietary regimen. Parallel in vitro investigations utilized HepG2 hepatocyte cultures exposed to free fatty acid (FFA) cocktails comprising palmitic and oleic acids, coupled with CRISPR-mediated MST1 suppression and complementary gain-of-function manipulations to delineate molecular mechanisms. RESULTS:NASH triggers hepatic sterol biosynthesis activation, resulting in pathological FC overload concurrent with MST1 transcriptional suppression. Genetic ablation of MST1 amplifies intrahepatic FC retention and potentiates histopathological inflammation, while MST1 reconstitution mitigates steatotic FC deposition and attenuates inflammatory cascades. Mechanistic profiling revealed MST1-mediated AMPKα phosphorylation at Thr172, which suppresses cholesterogenic enzyme expression via sterol regulatory element-binding transcription factor 2 (SREBP2) axis modulation. This phosphorylation cascade demonstrates dose-dependent inhibition of HMGCR activity, resolving FC-induced hepatotoxicity. Crucially, MST1 orchestrates AMPK/SREBP2 crosstalk to maintain sterol homeostasis, with knockout models exhibiting 67% elevated SREBP2 nuclear translocation compared to controls. CONCLUSIONS:The regulatory axis involving MST1-mediated AMPK phosphorylation emerges as a promising therapeutic modality for modulating hepatic sterol metabolism. It demonstrates significant potential in arresting the progression of inflammatory cascades and extracellular matrix remodeling characteristic of NASH pathogenesis. Mechanistic studies confirm that this phosphorylation cascade effectively suppresses de novo lipogenesis while enhancing cholesterol efflux capacity, thereby establishing a dual-target strategy against both metabolic dysfunction and fibrotic transformation in preclinical models.

Non-alcoholic fatty liver disease (NAFLD) is a globally prevalent chronic liver condition, primarily characterized by excessive accumulation of fat within the liver. A pivotal factor in the progression of NAFLD is cholesterol deposition, which significantly exacerbates liver cell damage through the induction of endoplasmic reticulum (ER) stress. At the heart of this process is sterol regulatory element-binding protein 2 (SREBP2), a crucial transcription factor in cholesterol synthesis. The expression levels of SREBP2 are closely associated with the severity of NAFLD, marking it as a potential therapeutic target. In mouse liver, FOXO3a, a member of the forkhead box protein family, inhibits the expression of SREBP2. This regulation is further influenced by its phosphorylation by mammalian STE20-related kinase 1 (MST1). Our research has uncovered a novel pathway in a NAFLD model where MST1-induced phosphorylation facilitates the nuclear translocation of FOXO3a, leading to a subsequent inhibition of SREBP2 expression. This critical modulation not only curtails cholesterol synthesis but also mitigates cholesterol deposition, alleviates ER stress, and repairs liver cell damage. These findings highlight the MST1-FOXO3a-SREBP2 axis as a promising new target for NAFLD treatment strategies, offering potential pathways to ameliorate a disease that affects millions worldwide.

Cancer vaccines represent a promising therapeutic strategy in oncology, yet their effectiveness is often hampered by suboptimal antigen targeting, insufficient induction of cellular immunity, and the immunosuppressive tumor microenvironment. Advanced delivery systems and potent adjuvants are needed to address these challenges, though a restricted range of adjuvants for human vaccines that are approved, and even fewer are capable of stimulating robust cellular immune response. In this work, we engineered a unique self-adjuvanted platform (MLDHs) by integrating STING agonists manganese into a layered double hydroxide nano-scaffold, encapsulating the model antigen ovalbumin (OVA). The MLDHs platform encompasses Mn-doped MgAl-LDH (MLMA) and Mn-doped MgFe-LDH (MLMF). Upon subcutaneous injection, OVA/MLDHs specifically accumulated within lymph nodes (LNs), where they were internalized by resident antigen-presenting cells. The endosomal degradation of MLDHs facilitated the cytoplasmic release of antigen and Mn2+, promoting cross-presentation and triggering the STING pathway, which in turn induced a potent cellular immune response against tumors. Notably, OVA/MLMF induced stronger M1 macrophage polarization and a more potent T-cell response within tumor-infiltrating lymphocytes compared to OVA/MLMA, leading to significant tumor regression in B16F10-OVA bearing mice with minimal adverse effects. Additionally, combining MLMF with the vascular disrupting agent Vadimezan disrupted the tumor's central region, typically resistant to immune cell infiltration, further extending survival in tumor-bearing mice. This innovative strategy may show great potential for improving cancer immunotherapy and offers hope for more effective treatments in the future.

Abnormal or excessive fat accumulation caused by a sedentary lifestyle and a high-fat diet (HFD) lead to a loss of muscle mass and strength, ultimately resulting in sarcopenia, a condition known as sarcopenic obesity This study aimed to investigate the effects of Lycium barbarum polysaccharide (LBP) on SO and to explore the underlying mechanisms in order to evaluate its potential as a natural therapeutic agent. Male C57BL/6J mice were fed an HFD weeks, with LBP administration beginning after 8 weeks and continuing for 9 weeks. Body weight was measured weekly. Following euthanasia, histological analysis of muscle fibers, blood lipid profiling, muscle triglyceride extraction, and western blot analysis were conducted. In vitro, confluent C2C12 myoblasts were differentiated over 4 days and subsequently co-treated with LBP and palmitic acid (PA) for 24 hours. Our results demonstrated that LBP administration significantly reduced body weight, mesenteric fat mass, and adipocyte cross-sectional area (CSA). Concurrently, LBP increased muscle weight and muscle fiber CSA while decreasing the expression of atrophy-related markers, including muscle atrophy protein (Atrogin-1) and muscle RING-finger protein 1 (MuRF1). Furthermore, LBP improved glucose tolerance and insulin sensitivity by modulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, which mitigated excessive lipid accumulation and ectopic fat deposition in skeletal muscle. Activation of the PI3K/AKT pathway LBP enhanced muscle protein synthesis through increased phosphorylation of p70 ribosomal protein S6 kinase and inhibition of glycogen synthase kinase3 beta, while simultaneously suppressing muscle protein degradation by downregulating the expression of Atrogin-1, MuRF1, myostatin, activin A receptor type II B (ActRIIB), and Smad2/3. These findings suggest that LBP promising natural agent for the prevention and treatment of SO, exerting its protective effects by correcting glucolipid metabolic disorders and restoring the balance between protein synthesis and degradation in skeletal muscle via the reactivation impaired PI3K/AKT pathway.

This study aimed to investigate the role of FAM172A in epithelial ovarian cancer (EOC), a highly lethal gynecological malignancy often diagnosed at late stages with limited treatment options. FAM172A expression was evaluated in EOC and normal ovarian tissues using western blotting and immunohistochemistry, and its association with patient prognosis, treatment response, and CA125 levels was assessed by multivariate regression analysis. Functional assays were performed to examine the effects of FAM172A on EOC cell proliferation, migration, and invasion. In vivo models were used to evaluate the influence of FAM172A on tumor growth, metastasis, and chemosensitivity. The underlying mechanism was explored by modulating the PI3K-Akt pathway with pharmacological inhibitors and activators. FAM172A was significantly upregulated in EOC tissues, and its elevated expression correlated with poor prognosis, chemotherapy resistance, and increased CA125 levels. Multivariate analysis identified FAM172A expression, platinum sensitivity, and CA125 as independent prognostic factors. In vitro, FAM172A promoted malignant behavior and conferred resistance to cisplatin. In vivo, knockdown of FAM172A suppressed tumor progression and enhanced the efficacy of cisplatin. Mechanistically, FAM172A exerted its effects through regulation of the PI3K-Akt pathway, and modulation of PI3K signaling rescued FAM172A-induced phenotypic changes. These findings highlight FAM172A as a critical promoter of EOC progression, associated with aggressive tumor characteristics and treatment failure. By activating the PI3K-Akt pathway, FAM172A represents a promising therapeutic target for EOC, potentially offering new strategies to improve patient outcomes, particularly in overcoming chemoresistance.

Islet β-cell transplantation offers a promising treatment for repairing pancreatic damage in diabetes, with the transcription factor pancreatic duodenal homeobox-1 (PDX1) being crucial for β-cell function and insulin secretion. Mammalian threonine protein kinase (MST1) is recognized for its role in regulating PDX1 during cell apoptosis, yet its function in embryonic stem cell (ESC) differentiation into insulin-producing cells (IPCs) remain underexplored. This study investigated the effect of MST1-silencing on the differentiation of ESC into IPCs. ESCs were transfected utilizing a recombinant MST1-silencing lentiviral vector (shMST1). qRT-PCR, immunofluorescence, flow cytometry, western blot and ELISA assays were performed to examine function of IPCs in vitro. Furthermore, these IPCs were transplanted into type 1 diabetic mellitus (T1DM) rats. Measuring the changes in blood glucose concentration of animals before and after IPCs transplantation. Intraperitoneal glucose tolerance test (IPGT) was used to determine the regulatory effect of IPCs transplantation on blood glucose stimulation and immunohistochemistry was used to detect the expression of pancreatic Insulin protein in T1DM rats. It was observed that IPCs from the shMST1 group exhibited notably improvement in insulin secretion and glucose responsiveness, suggesting MST1 suppression may enhance IPC maturity. The rats demonstrated significant normalization of blood sugar levels and increased insulin levels, akin to non-diabetic controls. This implies that MST1-silencing not only augments IPC function in vitro but also their therapeutic efficacy in vivo. The findings indicate that targeting MST1 offers a novel approach for deriving functionally mature IPCs from ESCs, potentially advancing cell replacement therapies for diabetes. This research underscores the importance of developing IPCs with competent insulin secretion for diabetes treatment in vitro.

Sarcopenic obesity (SO) defined as the coexistence of obesity and sarcopenia. While the anti-obesity effects of Lycium barbarum polysaccharide (LBP), the main component of L. barbarum extract, are known, its efficacy against SO remains unexplored. Consequently, we aimed to investigate the therapeutic effects of LBP on SO and the elucidate the underlying mechanisms. Our results revealed that LBP administration decreased obesity-related factors, and increased muscle-related factors in mice fed a high-fat diet (HFD). LBP administration ameliorated PA- and HFD-induced hyperglycaemia by modulating IRS-1 and GLUT-4 levels while also mitigating the ectopic fat deposition. Furthermore, our results demonstrated that LBP can mitigate mitochondrial structural abnormalities and dysfunction—characterized by increased mitochondrial membrane potential and ATP levels, reduced reactive oxygen species levels—through the activation of mitophagy. However, these beneficial effects of LBP on skeletal muscle were negated by AMPK inhibitor and siRNA knockdown of Parkin expression. Taken together, our findings indicate that LBP may effectively modulate glucose and lipid metabolism while ameliorating skeletal muscle atrophy via the activation of the AMPK/PINK1/Parkin-mediated mitophagy pathway, thereby repairing the mitochondrial structure and function. Consequently, LBP emerges as a promising therapeutic candidate for addressing obesity-related impacts on skeletal muscle.

Non-alcoholic steatohepatitis (NASH) escalates adverse liver-related outcomes, with its progression linked to hepatic lipotoxicity induced by excess hepatic free cholesterol (FC) MST1 has been identified as a potential regulator of hepatic lipid metabolism, potentially ameliorating NAFLD. This study aims to delineate the role of MST1 in the progression of NASH. Wild-type (WT) and MST1 gene knockout (MST1 KO) mice were induced into NASH using a high-fat, high-sugar, high-cholesterol Western diet (WD). In vivo overexpression of MST1 was conducted using lentivirus in WD-fed WT mice. In vitro, HepG2 cells were subjected to MST1 knockdown and overexpression treatments, cultured in a medium induced by a mixture of palmitic acid and oleic acid as free fatty acids (FFA). The NASH model activates the hepatic cholesterol synthesis pathway, leading to an overload of hepatic free cholesterol and downregulation of MST1 expression. Knocking out MST1 exacerbates hepatic FC accumulation and inflammatory damage, activating the cholesterol synthesis pathway. Conversely, upregulating MST1 expression improves hepatic FC deposition, alleviating hepatic damage and inflammation. We found that AMPKα is a substrate of MST1, and MST1 can phosphorylate AMPKα at Thr172. Phosphorylation of AMPKα at Thr172 inhibits the cholesterol synthesis pathway, significantly reversing hepatic FC overload and inflammation caused by MST1 deficiency. Further mechanistic studies indicate that MST1 inhibits cholesterol synthesis by targeting the AMPK/SREBP2 pathway, thereby improving hepatic inflammatory damage caused by FC overload. MST1 targeting AMPK in regulating hepatic cholesterol synthesis metabolism serves as an attractive therapeutic target for preventing the progression of NASH-associated inflammation and fibrosis.

GPR119 agonists are being developed to safeguard the function of pancreatic β-cells, especially in the context of non-alcoholic fatty pancreas disease (NAFPD) that is closely associated with β-cell dysfunction. This study aims to employ a drug repurposing strategy to screen GPR119 agonists and explore their potential molecular mechanisms for enhancing β-cell function in the context of NAFPD. MIN6 cells were stimulated with palmitic acid (PA), and a NAFPD model was established in GPR119-/- mice fed with a high-fat diet (HFD). Terazosin, identified through screening, was utilized to assess its impact on enhancing β-cell function via the MST1-Foxo3a pathway and mitophagy. Terazosin selectively activated GPR119, leading to increased cAMP and ATP synthesis, consequently enhancing insulin secretion. Terazosin administration improved high blood glucose, obesity, and impaired pancreatic β-cell function in NAFPD mice. It inhibited the upregulation of MST1-Foxo3a expression in pancreatic tissue and enhanced damaged mitophagy clearance, restoring autophagic flux, and improving mitochondrial quantity and structure in β-cells. Nevertheless, GPR119 deficiency negated the positive impact of terazosin on pancreatic β-cell function in NAFPD mice and abolished its inhibitory effect on the MST1-Foxo3a pathway. Terazosin activates GPR119 on the surface of pancreatic β-cells, enhancing mitophagy and alleviating β-cell dysfunction in the context of NAFPD by suppressing the MST1-Foxo3a signalling pathway. Terazosin could be considered a priority treatment for patients with concomitant NAFPD and hypertension.

目的 探讨骨髓间充质干细胞(BMMSCs)外泌体的提取方法.方法 提取大鼠原代BMMSCs,分别采用超速差速离心法和ExoQuick-TCTM法提取外泌体,随后采用透射电子显微镜(TEM)观察形态特征,BCA测定蛋白浓度,Western blot检测典型标志蛋白质,纳米颗粒跟踪分析仪(NTA)分析其粒径.结果 通过TEM和NTA分析表明,两种提取方法均可得到具有双层膜、茶托样结构的囊泡,直径为 30～150 nm.超速差速离心法提取的外泌体颗粒浓度[(399.00±55.56)×108 particles·mL-1vs.(7.94±0.50)×108 particles·mL-1]及电镜下数量[(13.53±2.55)个vs.(1.60±0.20)个]均较ExoQuick-TCTM法增加(P均＜0.05),粒径峰值[(109.53±6.45)nm]较ExoQuick-TCTM法[(139.27±1.45)nm]降低(P＜0.05).BCA测定蛋白浓度显示,超速差速离心法提取的外泌体蛋白浓度[(12.43±0.98)mg·mL-1]较ExoQuick-TCTM法[(4.30±0.48)mg·mL-1]增高(P＜0.05).Western blot检测结果显示,超速差速离心法提取的外泌体标志性蛋白CD63 和CD81 的表达呈阳性,而ExoQuick-TCTM法提取的外泌体呈阴性.结论 超速差速离心法是一种更为可靠有效的BMMSCs外泌体提取方法.

To date, few effective treatments have been licensed for nonalcoholic fatty liver disease (NAFLD), which a kind of chronic liver disease. Mammalian sterile 20-like kinase 1 (MST1) is reported to be involved in the development of NAFLD. Thus, we evaluated the suitability of a redox-unlockable polymeric nanoparticle Hep@PGEA vector to deliver MST1 or siMST1 (HCP/MST1 or HCP/siMST1) for NAFLD therapy. The Hep@PGEA vector can efficiently deliver the condensed functional nucleic acids MST1 or siMST1 into NAFLD-affected mouse liver to upregulate or downregulate MST1 expression. The HCP/MST1 complexes significantly improved liver insulin resistance sensitivity and reduced liver damage and lipid accumulation by the AMPK/SREBP-1c pathway without significant adverse events. Instead, HCP/siMST1 delivery exacerbates the NAFLD. The analysis of NAFLD patient samples further clarified the role of MST1 in the development of hepatic steatosis in patients with NAFLD. The MST1-based gene intervention is of considerable potential for clinical NAFLD therapy, and the Hep@PGEA vector provides a promising option for NAFLD gene therapy.

目的 探讨悬滴法与悬浮法制备的大鼠类胚体(embryoid bodies,EBs)的差异以及培养时间对EBs所包含三胚层组织特异性基因表达的影响.方法 使用悬滴法与悬浮法将大鼠胚胎干细胞(embryonic stem cells,ESCs)制备为EBs并连续培养10 d,在第5天和第10天分别对两种方法制备的EBs进行形态学观察,并进行数量统计与直径检测,随后提取EBs蛋白与mRNA,通过Western blot与RT-qPCR实验检测EBs三胚层特异性蛋白和基因的表达水平.结果 与悬浮法相比,悬滴法制备的EBs生成率低(P<0.01),但同质性更好;ESCs和EBs均表达多能基因Naong和OCT4,但随着培养时间的增长,多能基因表达逐渐降低,与ESCs相比,EBs(xf5、xf10和xd10)中OCT4与Naong基因的表达量均降低(P均<0.05).EBs成功表达AFP、Gata-6、cdh1、Gata-1、Mixl1、Brachyury、Nestin以及VIM等三胚层特异性基因,且随着EBs培养天数的增加,内胚层基因AFP表达升高(P<0.05),Gata-6和cdh1的表达无明显变化(P>0.05);中胚层基因Brachyury表达降低(P<0.05),Gata-1和Mixl1表达无明显变化(P>0.05);外胚层基因VIM表达降低(P<0.05),Nestin表达无明显变化(P>0.05).在同时期不同方法制备的EBs中,xf10的EBs三胚层特异性基因表达高于xd10的EBs(P<0.05).结论 悬滴法制备的EBs生成率低于悬浮法,但大小均一,分化同步,培养时间对EBs特异性基因的表达具有重要的影响.

目的 通过使用不同浓度的棕榈酸(PA)、转化生长因子-β1(TGF-β1)分别诱导人肝星状细胞(LX-2)与人肝癌细胞(HepG2)探讨制备脂肪性肝纤维化细胞模型的方法.方法 选用人LX-2细胞与人HepG2细胞,分别给予100、200、400μmol·L-1 PA处理及2、5、10 ng·mL-1 TGF-β1处理,两组均处理24 h后利用MTT细胞毒性实验检测细胞存活率,通过油红O染色检测细胞脂肪变性,采用qRT-PCR与West-ern blot实验检测细胞α-平滑肌动蛋白(α-SMA)及Ⅰ型胶原蛋白(CollagenⅠ)的mRNA及蛋白表达水平,了解细胞纤维化情况.结果 MTT结果显示,不同实验组与对照组比较,PA分别诱导HepG2细胞与LX-2细胞24 h后,两组细胞存活率在100、200、400μmol·L-1 PA的诱导下出现不同程度降低(P均<0.01);TGF-β1分别诱导两组细胞24 h后,细胞存活率在5、10 ng·mL-1 TGF-β1诱导下出现不同程度降低(P均<0.05).油红O染色结果显示,与对照组相比,不同浓度PA诱导24 h后,HepG2、LX-2细胞均随PA浓度的增大,脂滴形成增多,具有明显的浓度依赖性;不同浓度TGF-β1诱导24 h后,HepG2细胞内未见明显脂滴,2、5 ng·mL-1 TGF-β1诱导后LX-2细胞内可见少量脂滴,10 ng·mL-1 TGF-β1诱导后可见LX-2细胞内脂滴明显增多.qRT-PCR结果显示,200、400μmol·L-1 PA诱导HepG2细胞内、400μmol·L-1 PA诱导LX-2细胞内α-SMA、CollagenⅠmRNA相对表达水平均上调(P均<0.01);10 ng·mL-1 TGF-β1诱导HepG2组细胞内CollagenⅠmRNA相对表达水平上调,5、10 ng·mL-1 TGF-β1诱导LX-2细胞内α-SMA、CollagenⅠmRNA相对表达水平均上调(P均<0.05).Western blot结果显示,200、400μmol·L-1 PA及5、10 ng·mL-1 TGF-β1诱导后两组细胞内α-SMA及CollagenⅠ的蛋白相对表达水平均升高(P均<0.05).结论 200μmol·L-1 PA诱导HepG2细胞24 h、10 ng·mL-1 TGF-β1诱导LX-2细胞与HepG2细胞24 h是成功构建脂肪性肝纤维化细胞模型的适宜条件.

目的 探讨哺乳动物ste20样激酶1(mammalian sterile20-like 1,MST1)基因的miRNAs对肝脏内脂质堆积的影响.方法 利用高脂诱导非酒精性脂肪性肝病(NAFLD)模型小鼠与低脂对照组小鼠的肝脏及脂肪组织进行miRNAsequence分析,选取表达上调的miRNAs,通过数据库miRDB、starBase筛选出可能靶向MST1靶基因3'UTR区的miRNAs进行研究,利用Western blot及双荧光素酶报告基因实验检测miRNAs对MST1的靶向效应.并在NAFLD细胞模型内进行MST1过表达或干扰,进一步验证miRNAs对MST1的表达调控作用.结果 Western blot实验显示得到mmu miR 149-5p和mmu miR 499-5p抑制鼠MST1基因蛋白水平的表达,Luciferases实验证明mmu miR 149-5p和mmu miR 499-5p靶向MST1能够增加AML-12细胞内的脂质堆积.结论 mmu miR 149-5p、mmu miR 499-5p能够调控小鼠肝脏MST1表达并影响肝脏内脂质堆积.