Down-regulation of transforming growth factor β1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells

AIM: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-β1 (TGFβ1)/ activin receptor-like kinase 1 (ALK1, type Ⅰ receptor) signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells. METHODS: LX-2 cells were treated with TGFβ1 (5 ng/mL) Cpd861 (0.1 mg/mL), TGFβ1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGFβ1/ALK1 pathway. Real-time PCR was performed to examine the expression of α-SMA (α-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of α-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of α-SMA. RESULTS: In LX-2 cells, TGFβ1/ALK1-pathway-related gene expression could be stimulated by TGFβ1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of α-SMA mRNA and protein expression. This effect was related to inhibition of the above TGFβ1/ALK1-pathway- related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGFβ1 co-treatment for 24 h. CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGFβ1/ALK1/Smad1 pathway.

Role of activin receptor-like kinase 1 in vascular development and cerebrovascular diseases

Activin receptor-like kinase 1(ALK1)is a transmembrane serine/threonine receptor kinase of the transforming growth factor beta(TGFβ)receptor superfamily.ALK1 is specifically expressed in vascular endothelial cells,and its dynamic changes are closely related to the proliferation of endothelial cells,the recruitment of pericytes to blood vessels,and functional differentiation during embryonic vascular development.The pathophysiology of many cerebrovascular diseases is today understood as a disorder of endothelial cell function and an imbalance in the proportion of vascular cells.Indeed,mutations in ALK1 and its co-receptor endoglin are major genetic risk factors for vascular arteriovenous malformation.Many studies have shown that ALK1 is closely related to the development of cerebral aneurysms,arteriovenous malformations,and cerebral atherosclerosis.In this review,we describe the various roles of ALK1 in the regulation of angiogenesis and in the maintenance of cerebral vascular homeostasis,and we discuss its relationship to functional dysregulation in cerebrovascular diseases.This review should provide new perspectives for basic research on cerebrovascular diseases and offer more effective targets and strategies for clinical diagnosis,treatment,and prevention.

子痫前期孕妇血清PLGF、sFlt-1及激活素A的表达意义及母婴不良结局的影响因素分析

目的 分析子痫前期孕妇血清胎盘生长因子(PLGF)、可溶性FMS样酪氨酸激酶1(sFlt-1)及激活素A的表达意义及母婴不良结局的影响因素。方法 选择自2021年1月至2023年11月宣城市中心医院接诊的100例子痫前期孕妇纳入观察组,另选100例正常妊娠孕妇纳入对照组。检测所有孕妇血清PLGF、sFlt-1及激活素A的水平,使用受试者工作特征曲线(ROC)分析血清PLGF、sFlt-1及激活素A对子痫前期的诊断效能,记录观察组子痫前期孕妇的母婴不良结局,使用单因素分析及多因素Logistic回归分析子痫前期孕妇母婴不良结局的影响因素。结果 观察组血清PLGF水平为(55.26±8.34) pg/mL,低于对照组[(86.12±15.08) pg/mL],sFlt-1及激活素A水平分别为(986.59±168.73)、(398.08±86.45) pg/mL,均高于对照组[(712.42±102.36)、(201.23±45.71) pg/mL],差异均有统计学意义(P<0.05)。重度子痫前期孕妇血清PLGF水平低于轻度子痫前期孕妇,sFlt-1及激活素A的水平均高于轻度子痫前期孕妇,差异均有统计学意义(P<0.05)。经Pearson相关性分析,子痫前期孕妇血清PLGF与尿蛋白呈负相关(P<0.05),与血小板计数呈正相关(P<0.05);子痫前期孕妇血清sFlt-1及激活素A与尿蛋白均呈正相关(P<0.05),与血小板计数均呈负相关(P<0.05)。血清PLGF、sFlt-1联合激活素A诊断子痫前期的敏感度为90.47%,特异度为51.64%、AUC为0.926。经单因素及多因素Logistic回归分析,产次、分娩孕周、白蛋白、PLGF、sFlt-1及激活素A均是子痫前期孕妇发生母婴不良结局的独立影响因素(P<0.05)。结论 血清PLGF、sFlt-1及激活素A与子痫前期发生及其严重程度有关,联合应用可提高对子痫前期的诊断效能,且均是母婴不良结局的独立影响因素。

OsCERK1-Mediated Chitin Perception and Immune Signaling Requires Receptor-like Cytoplasmic Kinase 185 to Activate an MAPK Cascade in Rice

Conserved pathogen-associated molecular patterns （PAMPs）, such as chitin, are perceived by pattem recognition receptors （PRRs） located at the host cell surface and trigger rapid activation of mitogen- activated protein kinase （MAPK） cascades, which are required for plant resistance to pathogens. However, the direct links from PAMP perception to MAPK activation in plants remain largely unknown. In this study, we found that the PRR-associated receptor-like cytoplasmic kinase Oryza sativa RLCK185 transmits immune signaling from the PAMP receptor OsCERK1 to an MAPK signaling cascade through interaction with an MAPK kinase kinase, OsMAPKKKε, which is the initial kinase of the MAPK cascade. OsRLCK185 interacts with and phosphorylates the C-terminal regulatory domain of OsMAPKKKε. Coexpression of phosphomi- metic OsR LCK185 and OsMAPKKKε activates MAPK3/6 phosphorylation in Nicotiana benthamiana leaves. Moreover, OsMAPKKKε interacts with and phosphorylates OsMKK4, a key MAPK kinase that transduces the chitin signal. Overexpression of OsMAPKKKε increases chitin-induced MAPK3/6 activation, whereas OsMAPKKKε knockdown compromises chitin-induced MAPK3/6 activation and resistance to rice blast fungus. Taken together, our results suggest the existence of a phospho-signaling pathway from cell surface chitin perception to intraceilular activation of an MAPK cascade in rice.

Splicing of Receptor-Like Kinase-Encoding SNC4 and CERK1 is Regulated by Two Conserved Splicing Factors that Are Required for Plant Immunity

Plant immune receptors belonging to the receptor-like kinase （RLK） family play important roles in the recog- nition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D con- tains a gain-of-function mutation in the RLK SNC4 （SUPPRESSOR OF NPRI-1, CONSTITUTIVE4）, which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA （SUPPRESSOR OF ABI3-5） and RSN2 （REQUIRED FOR SNC4-1D 2）, that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of 5NC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 （CHITIN ELICITOR RECEPTOR KINASE1）, which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3OOOhrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1.

TGF-β1及Ⅰ型受体ALK1在人脑胶质瘤细胞的表达及意义

目的观察转化生长因子β1(TGF-β1)及活化素受体样激酶1(ALK1)在人脑胶质瘤细胞中的表达及其在胶质细胞瘤病理分级中的意义。方法采用半定量RT-PCR、Western blot及免疫组织化学染色等方法检测32例人脑胶质瘤及3例正常脑组织中TGF-β1及ALK1mRNA及蛋白的表达水平。结果人脑胶质瘤细胞中存在TGF-β1及ALK1mRNA和蛋白表达。与正常组比较,高级别胶质瘤组TGF-β1及ALK1mRNA和蛋白表达均显著升高(P<0.05),其中ALK1的高表达与胶质瘤病理级别呈明显正相关(r=0.297,P<0.05)。结论人脑胶质细胞瘤中,ALK1可能参与了胶质瘤的恶变过程。

Correlation between expression of two transforming growth factor-beta 1 receptors and microvascular density in a rat model of cerebral ischemia and reperfusion injury

The effects of transforming growth factor-β1 （TGF-β1） are currently controversial. Whether TGF-β1 promotes or inhibits revascularization under different conditions remains poorly understood. Based on previous studies, the current experiment established rat models of cerebral ischemia and reperfusion injury （IRI）, and demonstrated that pathological and functional damage was also increased after IRI. The most serious damage was observed at 3 days after reperfusion, at which time microvascular density fell to its lowest level. Soon afterwards, microvascular density increased, new collateral circulation was gradually established at 4 to 7 days after reperfusion, and pathological damage and neurological deficits were improved. TGF-β1, activin receptor-like kinase 5 （ALK5） mRNA and protein expression levels increased gradually over time. In contrast, ALK1 mRNA and protein expression decreased over the same period. A significant negative correlation was detected between microvascular density and expression of the ALK5 gene transcript. There was no correlation between microvascular density and ALK1 gene transcriptional expression following cerebral IRI in a rat model. These findings suggest that ALK5, rather than ALK1, is the critical receptor in the TGF-β1 signal pathways after cerebral IRI.

乳腺癌ALK-1表达与18F-FDG PET/CT显像的肿瘤学特点分析

目的比较乳腺癌人活化素受体样激酶1(ALK-1)表达与氟代脱氧葡萄糖(18F-FDG)PET/CT显像的肿瘤学特点。方法收集我院收治的乳腺癌患者202例作为观察对象,患者均在病理活检前、后或术前行18F-FDG PET/CT检查,且病理标本均行ALK-1检测。其中ALK-1表达阳性62例,ALK-1表达阴性140例,比较两组患者肿块的大小、形态、边缘、内部密度、钙化、淋巴结或远端转移和最大标准摄取值(SUVmax)。结果ALK-1阳性组与ALK-1阴性组比较,肿块大小、肿块边缘有无毛刺、淋巴结有无转移、SUVmax值差异有统计学意义(P<0.05),肿瘤形态、肿块内部密度和有无钙化方面,差异无统计学意义(P>0.05)。结论ALK-1表达阳性乳腺癌患者常见于肿块直径≥2 cm、肿块边缘有毛刺、有淋巴结或远端转移、SUV-max≥3.5的患者,乳腺癌病灶肿块大小、肿块边缘是否有毛刺、淋巴结或远端转移及SUVmax可能是乳腺癌患者ALK-1表达阳性的肿瘤学特点预测因素。

活化素受体样激酶1及其在血管生成中的作用

血管生成是指新的血管从已存在的毛细血管网生成的过程,受血管生成促进因子和抑制因子的严格调控。通常,血管生成发生于胚胎和出生后早期血管的发育过程中,除女性生理周期和伤口愈合等过程外,在成年阶段血管生成已处于静息状态。但是,在发生某些疾病如肿瘤、糖尿病视网膜病变、心血管疾病及类风湿性关节炎[1-2]时,血管生成过程被异常激活。

TGF-β1受体ALK1和ALK5在翼状胬肉和正常结膜组织中的差异表达

目的:研究不同种类的转化生长因子β-1(TGF-β1)激活素受体样激酶(ALK)在翼状胬肉和正常结膜组织中的表达及其意义。方法:前瞻性研究。选取2020-06/12在徐州医科大学附属医院眼科就诊的40例手术中切除的翼状胬肉标本和40例白内障手术获取的正常结膜标本纳入本次研究。使用ALK1、ALK5免疫组织化学染色观察TGF-β1受体在胬肉和正常结膜组织中的表达定位,并统计染色阳性细胞比例;使用RT-PCR检测ALK1、ALK5 mRNA在胬肉和结膜组织中的表达;并以Western Blot检测ALK1、ALK5蛋白的表达。结果:根据免疫组织化学染色结果,ALK1在翼状胬肉组中的表达水平较正常结膜组明显升高,并且在整个翼状胬肉上皮细胞中均有表达,而在正常结膜组织中仅在上皮细胞的基底层中有表达;两组上皮细胞基底层均检测到ALK5,而翼状胬肉组ALK5水平较正常结膜组下降。两组间ALK1、ALK5阳性细胞比例有显著的差异(均P<0.05)。RT-PCR结果显示,与结膜组织相比,ALK1 mRNA在翼状胬肉中的表达明显增强,ALK5的表达明显减弱,两组间有显著差异(均P<0.05),Western Blot结果显示ALK1、ALK5蛋白的表达差异与RT-PCR结果基本一致。结论:翼状胬肉和正常结膜ALK的表达谱有明显的差异。与正常结膜组织相比,翼状胬肉组织的ALK1表达增强,ALK5表达减弱,表明TGF-β信号通路处于不同的活化状态,对于进一步研究翼状胬肉的发病机制提供实验依据。

活化素受体样激酶1与心血管疾病研究进展

活化素受体样激酶1(ALK1)是转化生长因子β(TGFβ)受体超家族的一种跨膜丝氨酸/苏氨酸受体激酶。ALK1主要在内皮细胞中表达,在内皮细胞生物学和血管再生中的作用已得到广泛研究。最近研究提示,ALK1在心血管内稳态中发挥重要作用,与心血管疾病的发生发展密切相关。本文旨在描述ALK1信号传导的机制,讨论其在心血管内稳态中的作用及其与心血管疾病发生、发展间的联系,从而为心血管疾病的防治疗提供潜在的新靶点和新策略。

Identifi cation and functional analysis of phosphorylation residues of the Arabidopsis BOTRYTIS-INDUCED KINASE1

Arabidopsis BOTRYTIS-INDUCED KINASE1(BIK1)is a receptor-like cytoplasmic kinase acting early in multiple signaling pathways important for plant growth and innate immunity.It is known to form a signaling complex with a cell-surface receptor FLS2 and a co-receptor kinase BAK1 to transduce signals upon perception of pathogen-asso-ciated molecular patterns(PAMPs).Although site-specifi c phosphorylation is speculated to mediate the activation and function of BIK1,few studies have been devoted to complete profiling of BIK1 phosphorylation residues.Here,we identified nineteen in vitro autophosphoryla-tion sites of BIK1 including three phosphotyrosine sites,thereby proving BIK1 is a dual-specifi city kinase for the fi rst time.The kinase activity of BIK1 substitution mutants were explicitly assessed using quantitative mass spec-trometry(MS).Thr-237,Thr-242 and Tyr-250 were found to most signifi cantly affect BIK1 activity in autophosphoryla-tion and phosphorylation of BAK1 in vitro.A structural model of BIK1 was built to further illustrate the molecular functions of specifi c phosphorylation residues.We also mapped new sites of FLS2 phosphorylation by BIK1,which are different from those by BAK1.These in vitro results could provide new hypotheses for more in-depth in vivo studies leading to deeper understanding of how phosphorylation contributes to BIK1 activation and medi-ates downstream signaling specifi city.

Gastric angiodysplasia in a hereditary hemorrhagic telangiectasia type 2 patient

Hereditary hemorrhagic telangiectasia(HHT)is a rare autosomal-dominantly inherited disease that occurs in approximately one in 5000 to 8000 people.Clinical diagnosis of HHT is made when a person presents three of the following four criteria:family history,recurrent nosebleeds,mucocutaneous telangiectasis,and arteriovenous malformations(AVM)in the brain,lung,liver and gastrointestinal(GI)tract.Although epistaxis is themost common presenting symptom,AVMs affecting the lungs,brain and GI tract provoke a more serious outcome.Heterozygous mutations in endoglin,activin receptor-like kinase 1(ACVRL1;ALK1),and SMAD4,the genes involved in the transforming growth factor-βfamily signaling cascade,cause HHT.We report here the case of a 63 year-old male patient who presented melena and GI bleeding episodes,proven to be caused by bleeding from multiple gastric angiodysplasia.Esophagogastroduodenoscopy revealed multiple angiodysplasia throughout the stomach.Endoscopic argon plasma coagulation was performed to control bleeding from a gastric angiodysplasia.The patient has been admitted several times with episodes of hemoptysis and hematochezia.One year ago,the patient was hospitalized due to right-sided weakness,which was caused by left basal ganglia hemorrhage as the part of HHT presentation.In family history,the patient's mother and elder sister had died,due to intracranial hemorrhage,and his eldest son has been suffered from recurrent epistaxis for 20 years.A genetic study revealed a mutation in exon 3 of ALK1(c.199C>T;p.Arg67Trp)in the proband and his eldest son presenting epistaxis.

Pulmonary arterial hyper-tension in a patient with hereditary hemorrhagic telangiectasia and family gene analysis:A case report

BACKGROUND Hereditary hemorrhagic telangiectasia(HHT)is a rare autosomal dominant genetic disease.Very few patients suffering from HHT present with associated pulmonary arterial hypertension(PAH),which may result in a poor prognosis.Here,we report a case of HHT with PAH.The patient’s clinical manifestations and treatment as well as genetic analysis of family members are reviewed,in order to raise awareness of this multimorbidity.CASE SUMMARY A 45-year-old Chinese woman was admitted to the hospital to address a complaint of intermittent shortness of breath,which had lasted over the past 2 years.She also had a 30-year history of recurrent epistaxis and 5-year history of anemia.She reported that the shortness of breath had aggravated gradually over the 2 years.Physical examination discovered anemia and detected gallop rhythm in the precordium.Chest computerized tomography and cardiac ultrasound demonstrated PAH and hepatic arteriovenous malformation.The formal clinical diagnosis was HHT combined with PAH.The patient was treated with ambrisentan and her condition improved for a time.She died half a year after the diagnosis.Genetic testing revealed the patient and some family members to carry an activin A receptor-like type 1 mutation(c.1232G>A,p.Arg411Gln);the family was thus identified as an HHT family.CONCLUSION We report a novel gene mutation(c.1232G>A,p.Arg411Gln)in a Chinese HHT patient with PAH.

肝再生终止阶段的研究进展

肝脏作为体内最重要的解毒器官,具有极强的再生能力.肝再生研究一直是再生医学研究领域的热点,其再生过程可分为起始阶段、增殖阶段以及终止阶段.目前研究大都集中在肝再生的起始以及增殖阶段,对于使肝再生恰当终止的机制研究仍知之甚少.肝再生终止阶段涉及多种细胞因子与生长因子,其功能主要体现在2方面:(1)抑制有丝分裂原对于肝细胞增长的促进作用;(2)通过某种途径促进过多增殖的肝细胞凋亡.本文针对目前肝再生的终止阶段研究所涉及的主要的因子综述如下.

Clinical phenotypes,ALK1 gene mutation and level of related plasma proteins in Chinese hereditary hemorrhagic telangiectasia

Background We determined the diagnosis of hereditary hemorrhagic telangiectasis (HHT) in a suspected HHT family,identified ALK1 gene mutation and established a gene diagnosis method of HHT. The level of related plasma proteins (transforming growth factor β and thrombomodulin) were also analyzed.Methods Bleeding history and family history were collected; Dilatant nasal mucosal capillaries in proband were observed under nasal cavity endoscope; exons 3,7,8 of ALK1 gene in proband and her family members were amplified with polymerase chain reaction (PCR), and the PCR products were analyzed. Using enzyme-linked immunosorbent assay (ELISA),plasma TGF-β1 and TGF-β2 concentrations were measured. Plasma thrombomodulin (TM) level was detected by Western blotting.Results Of all family members,four had epstaxis,two had evident telangiectases on skin or mucosa. Gene screening results showed that C to T substitution at position 1231 in exon 8 of ALK1 gene (CGG→TGG) existed in proband,her affected brother and their father. The mutation did not exist in proband’s sister-in-law and nephew. Plasma TGF-β1 concentrations in the affected HHT was 20538,17194,13131 pg/ml,while that of normal control and unaffected family members was 15950,20297,12836 pg/ml,respectively. Plasma TGF-β2 in HHT patients was 14502,9550,10592 and that of normal controls 8579,20297,7680 pg/ml respectively. Level of plasma TM was in HHT subjects significantly lower than in normal subjects.Conclusions Chinese HHT individuals have mutant ALK1 gene,a C1231T variation on exon 8 of ALK1 is responsible for HHT clinical phenotypes in this family. ALK1 gene analysis,together with special clinical phenotypes and family history,provides a reliable method in diagnosing HHT. In affected HHT subjects,plasma TGFβ levels were not obviously different from those of normal subject; while plasma TM concentration was significantly lower than that in normal subjects. The significance and mechanism remain to be elucidated.

BAK1 Directly Regulates Brassinosteroid Perception and BRI1 Activation

Plants utilize plasma membrane-localized receptor-like kinases （RLKs） to sense extracellular signals to coordinate growth, development, and innate immune responses. BAK1 regulates multiple signaling pathways acting as a co-receptor of several distinct ligand-binding RLKs. It has been debated whether BAK1 serves as an essential regulatory component or only a signal amplifier without pathway specificity. This issue has been clarified recently. Genetic and structural analyses indicated that BAK1 and its homologs play indispensible roles in mediating brassinosteroid （BR） signaling pathway by directly perceiving the ligand BR and activating the receptor of BR, BRII. The mechanism revealed by these studies now serves as a paradigm for how a pair of RLKs can function together in ligand binding and subsequent initiation of signaling.

BKI1 Regulates Plant Architecture through Coordinated Inhibition of the Brassinosteroid and ERECTA Signaling Pathways in Arabidopsis

Hundreds of leucine-rich repeat receptor-like kinases （LRR-RLKs） play indispensable roles in a wide range of plant developmental and physiological processes. The mechanisms controlling LRR-RLKs at a basal and inactive status are essential but rarely studied. BKI1 is the only reported inhibitor of receptor kinases in Arabidopsis, which negatively regulates BRI1 in the brassinosteroid pathway. In this study, we found that BKI1 can also interact with another important LRR-RLK, ERECTA （ER）. Phenotypic analysis showed that BKI1 and ER together regulate plant architecture, including pedicel orientation, which is a newly reported phenotype in the BR- and ER-mediated developmental processes. Gene expression analysis revealed that BKI1 regulates a subset of ER-responsive genes. Kinase assays demonstrated that BKI1 inhibits ER kinase activity. In addition, the release of BKI1 inhibition on ER signaling relies largely on BRI1 activation. Our data provide significant insights into the regulation and activation of RLKs and suggest that BKI1 functions as a common suppressor of the BRI1 and ER signaling pathways.

Ligand Perception,Activation,and Early Signaling of Plant Steroid Receptor Brassinosteroid Insensitive 1

Leucine-rich repeat receptor-like kinases （LRR-RLKs） belong to a large group of cell surface proteins involved in many aspects of plant development and environmental responses in both monocots and dicots. Brassinosteroid insensitive 1 （BRI1）, a member of the LRR X subfamily, was first identified through several forward genetic screenings for mutants insensitive to brassinosteroids （BRs）, which are a class of plant-specific steroid hormones. Since its identification, BRI1 and its homologs had been proved as receptors perceiving BRs and initiating BR signaling. The co-receptor BRIl-associated kinase 1 and its homologs, and other BRI1 interacting proteins such as its inhibitor BRI1 kinase inhibitor I （BKI1） were identified by genetic andbiochemical approaches. The detailed mechanisms of BR perception by BRI1 and the activation of BRI1 receptor complex have also been elucidated. Moreover, several mechanisms for termination of the activated BRI1 signaling were also discovered. In this review, we will focus on the recent advances on the mechanism of BRI1 phosphorylation and activation, the regulation of its receptor complex, the structure basis of BRI1 ectodomain and BR recognition, its direct substrates, and the termination of the activated BRI1 receptor complex.

Effect of vitamin B12 on cleft palate induced by 2,3.7.8-tetrachlorodibenzo-p-dioxin and dexamethasone in mice

The purpose of this study was to investigate the effect of vitamin B12 on palatal development by co-administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin （TCDD） and dexamethasone （DEX）. We examined the morphological and histological features of the palatal shelf and expression levels of key signaling molecules （trans- forming growth factor-β3 （TGF-β3） and TGF-β3 type I receptor （activin receptor-like kinase 5, ALK5）） during pala- togenesis among a control group （Group A）, TCDD＋DEX exposed group （Group B）, and TCDD＋DEX＋vitamin B12 exposed group （Group C）. While we failed to find that vitamin B12 decreased the incidence of cleft palate induced by TCDD＋DEX treatment, the expression levels of key signaling molecules （TGF-~3 and ALK5） during palatogenesis were significantly modulated. In TCDD＋DEX exposed and TCDD＋DEX＋vitamin B12 exposed groups, palatal shelves could not contact in the midline due to their small sizes. Our results suggest that vitamin B12 may inhibit the expression of some cleft palate inducers such as TGF-β3 and ALK5 in DEX＋TCDD exposed mice, which may be beneficial against palatogenesis to some degree, even though we were unable to observe a protective role of vitamin B12 in morphological and histological alterations of palatal shelves induced by DEX and TCDD.