Puerarin mediated miR-30b-5p targeting fibroblast activation protein against oral submucous fibrosis

Background:Puerarin(Pue)has been reported to be a natural active ingredient with multiple antifibrotic properties.This work aimed at exploring the function of Pue in oral submucousfibrosis(OSF)treatment.Methods:Human oral mucosafibroblasts(hOMF)were induced with transforming growth factor beta1(TGF-β1)and intervened with Pue.Expressions offibrosis-related markers were analyzed by Western blot and IF staining.Cell viability was characterized by the CCK-8 assay.Expressions of miR-30 family members were quantified by qRT-PCR.The correlation betweenfibroblast activation protein(FAP)and miR-30 family expression was evaluated by the Pearson correlation coefficient.Bioinformatics prediction and dual-luciferase reporter assay were employed to verify the regulation between FAP and miR-30b-5p.The specific mechanism of Pue on OSF was explored through the promotion or inhibition of miR-30b-5p.Results:After induction by TGF-β1,hOMF showed upregulated Collagen I,Collagen III,and FAP expressions,while miR-30 family expression was downregulated with miR-30b-5p being the most significant.Pue intervention inhibited the excessive proliferation of TGF-β1-induced hOMF,downregulated FAP,collagen type 3(COL3A1),collagen type 1(COL1A1),matrix metalloproteinase 1(MMP1),and matrix metalloproteinase 3(MMP3)expressions,and restored miR-30 family expression.Bioinformatics prediction and dual-luciferase reporter assay revealed that miR-30b-5p selectively inhibited FAP expression.Mechanistically,miR-30b-5p mimic suppressed the excessive proliferation of TGF-β1-induced hOMF and declinedfibrosis levels.Pue intervention significantly reversed the promotion of TGF-β1-induced OSF by miR-30b-5p inhibition.Conclusion:Pue mediated miR-30b-5p targeting FAP against OSF,which provided a theoretical basis for the pathogenesis research and Pue application in OSF.

Analysis of clinical symptoms and signs in cases of oral submucous fibrosis which have transformed into squamous cell carcinoma

To analyze the clinical symptoms and signs in cases of oral submucous fibrosis which has transformed into squamous cell carcinoma, twenty-nine patients with squamous cell carcinoma correlated with oral submucous fibrosis, leukoplakia, lichen planus were taken biopsy for positive diagnosis. The clinical and pathological diagnosis of oral submucous fibrosis depended on clinical and pathological diagnostic standards. The oral submucous fibrosis involving different portions and size in the mouth was observed and recorded. The portions of oral cancer, clinical findings and symptoms, cancerous size, the conditions of the local lymphatic nodes and of oral submucous fibrosis correlated with squamous cell carcinoma in the mouth, oral leukoplakia, lichen planus were also observed and recorded. In twenty-nine patients with oral squamous cell carcinoma, duration from oral submucous fibrosis transformed into oral cancer ranged from 2 to 15 years with an average year of 6.69. The serious degrees of oral submucous fibrosis in the patients with oral cancer were that the moderately advanced stage was found in 3 cases (10.34%) and the advanced stage was found in 26 cases (89.65%). The cancerous portion was that the buccal mucosa was found in 9 cases (31.03%);the tongue was found in 14 cases (48.27%);the other portion was found in the rest 6 cases (20.68%). Among the twenty-nine patients twenty-three patients with squamous cell carcinoma were associated with leukoplakia (79.31%), five patients were associated with lichen planus (17.24%). Twenty-seven patients (93.1%) had habits of betel quid chewing, smoked cigarette and drank alcohol, the rest two patients had habits of betel quid chewing and cigarette smoking. The present study demonstrates that oral submucous fibrosis is a real precancerous lesion and may transform into squamous cell carcinoma in the mouth.

Clinical efficacy of Danshen injection in combined with local injection of triamcinolone acetonide in the treatment of oral submucous fibrosis and the effect on serum TGF-β1 and IL-6

Objective:To explore the clinical efficacy of Danshen injection in combined with local injection of triamcinolone acetonide in the treatment of oral submucous fibrosis (OSF) and the effect on serum TGF-β1 and IL-6.Methods:A total of 70 patients with OSF who were admitted in our hospital from October, 2015 to October, 2016 were included in the study and randomized into the observation group and the control group (n=35). The patients in the control group were given multipoint injection of triamcinolone acetonide (40 mg/time) in the basal region of oral mucosal regions. On this basis, the patients in the observation group were given additional Danshen injection (4 mL/time), 1 time/week, for 12 times. VAS was used for evaluation. The oral mucosa lesion area and mouth opening were measured. The morning fasting peripheral venous blood before and after treatment in the two groups was collected. The blood rheometer was used to detect the whole blood viscosity [high shear (HS), moderate shear (MS), and low shear (LS)], plasma viscosity (PV), and fibrinogen (FIB). ELISA was used to detect the serum TGF-β1 and IL-6 levels.Results: VAS score in the observation group was significantly reduced (P<0.05), mucosal lesion area was significantly less than that in the control group (P<0.05), and mouth opening was significantly greater than that in the control group (P<0.05). LS, MS, PV, and FIB after treatment in the observation group were significantly reduced when compared with before treatment (P<0.05), while LS, MS, HS, PV, and FIB after treatment in the control group were not significantly changed (P>0.05). TGF-β1 and IL-6 levels after treatment in the observation group were significantly lower than those in the control group (P<0.05).Conclusions: Danshen injection in combined with local injection of triamcinolone acetonide in the treatment of OSF can effectively reduce the serum TGF-β1 and IL-6 levels, inhibit the connective tissue fibrosis, and improve the pain, oral mucosal lesion area, and mouth opening, with a satisfactory clinical effect.

Development of a mouse model of arecoline-induced oral mucosal fibrosis

Objective: To develop a BALB/c mouse model of oral submucous fibrosis(OSF)induced by arecoline and to exhibit an accumulation of collagen and angiogenesis changes.Methods: BALB/c mice were randomly assigned to either the control(distilled water) or experimental group(arecoline)(n = 40). Eight mice from each group were sacrificed every 4 weeks since 8 weeks post treatment. Changes in histopathologic features, levels of collagen type Ⅰ and collagen type Ⅲ, and angiogenesis were measured.Results: In the 8th week, epithelium atrophy, collagen cumulation and micrangium pathologic changes in the lamina propria were observed in the oral mucosa. In the 20th week, hyaline degeneration of the connective tissues was observed on the tongue and palate mucosa. The angiogenesis and collagen type Ⅰ changed significantly as the diseases advanced(P < 0.05); however, collagen type Ⅲ was not statistically different.Conclusions: An OSF model involving mice can be rapidly induced by drinking a highdose of arecoline. OSF angiogenic changes in mice primarily decrease and collagen accumulation is mainly collagen type Ⅰ.

Advances in molecular mechanisms of oral submucosal fibrogenic carcinogenesis

Oral submucosal fibrosis(OSF)is a chronic,progressive and insidious oral mucosal disease.Its development is highly associated with areca chewing and eventually evolves into oral squamous cell carcinoma(OSCC),which is currently recognized as a precancerous state by the International Center for Research on Cancer(IARC).Due to the extremely complex carcinogenesis process of OSF,the carcinogenesis mechanism is not clear,and most scholars believe that the disease is the result of multiple factors.Therefore,this paper mainly summarized the latest research on the molecular mechanism of OSF canceration,aiming at understanding the law of OSF canceration and providing theoretical reference for early detection,diagnosis and treatment of OSF canceration.

LncRNA H19与OSF发生及癌变的关系

目的:探讨lncRNA H19在口腔黏膜下纤维性变(OSF)发生及癌变过程中的表达变化及其意义。方法:实时荧光定量.PCR技术分别检测12例正常颊黏膜组织、33例OSF颊黏膜组织、31例伴OSF颊癌组织中的LncRNA H19的表达水平。结果:LncRNA H19在颊黏膜组织、OSF颊黏膜组织、伴OSF颊癌组织中的相对表达量分别为1.17±0.37、3.44±1.08、8.88±1.78,组间两两之间比较,P<0.01。结论:LncRNA H19可能参与OSF的发生及癌变。

曲安奈德联合丹参酮ⅡA注射液治疗OSF患者的临床疗效及对血清TGF-β1、IL-6水平的影响

目的探究曲安奈德联合丹参酮ⅡA(丹参酮单体之一)注射液治疗口腔黏膜下纤维化(OSF)的效果及对血清转化生长因子β1(TGF-β1)、白介素-6(IL-6)水平的影响。方法选择2013年4月至2016年4月间于该院就诊的92例OSF患者,按治疗方法分为对照组(46例)和试验组(46例),分别给予单纯曲安奈德、曲安奈德联合丹参酮ⅡA注射液局部注射治疗3个疗程,并进行为期12个月的随访,比较两组患者治疗前和第2、4、6、12个月随访的张口度和VAS评分情况,以及治疗前后的TGF-β1、IL-6、病损面积和口腔健康影响程度量表(OHIP-14)评分等的变化情况。结果随访6个月,对照组和试验组患者的最大张口度较治疗前均有明显增加,试验组患者的改善速度和改善程度明显优于对照组,差异有统计学意义(P<0.05);随访第2、4、6、12个月,两组患者的VAS评分较治疗前均有明显降低,试验组降低更为明显,差异有统计学意义(P<0.05),两组12个月的VAS评分较6个月出现升高,对照组更为明显,差异有统计学意义(P<0.05);治疗后,试验组TGF-β1和IL-6水平明显低于对照组和本组治疗前,差异有统计学意义(P<0.05);治疗后,试验组的病损面积显著低于对照组、OHIP-14评分明显高于对照组,与本组治疗前比较,差异有统计学意义(P<0.05)。结论在OSF患者中应用曲安奈德联合丹参酮注射液局部注射治疗具有较好的疗效,能明显降低血清中TGF-β1和IL-6的水平。

TNF-α和IFN-γ对OSF成纤维细胞的增殖影响

目的 :研究TNF α、IFN γ对正常人颊黏膜成纤维细胞 (NM FB)和口腔黏膜下纤维化 (OSF)患者颊黏膜成纤维细胞 (OSF FB)增殖活性的影响。方法 :取 6例OSF患者及 5例正常人颊黏膜组织 ,采用组织块法进行成纤维细胞原代培养并传代 ,用MTT比色法观察不同浓度TNF α及IFN γ对NM FB和OSF FB的增殖效应。结果 :浓度为 10 0～ 10 0 0 0U/ml的TNF α能明显促进NM FB和OSF FB的增殖(P <0 .0 5) ;浓度为 40 0～ 40 0 0U/ml的IFN γ则明显抑制两者的生长 (P <0 .0 5) ,且 40 0U /ml的IFN γ其抑制率接近峰值 (与 40 0 0U/ml的IFN γ组比 ,P >0 .0 5)。结论 :TNF α促进NM FB和OSF FB的增殖 ;而IFN

三七总皂苷通过激活Nrf2/GCLC信号通路抑制槟榔碱诱导的HaCaT细胞氧化应激而缓解口腔黏膜下纤维化

目的:探讨三七总皂苷(PNS)在槟榔碱(ANE)诱导的口腔黏膜下纤维化中的抗纤维化作用,并分析PNS对核因子E2相关因子2(Nrf2)∕谷氨酸-半胱氨酸连接酶催化亚基(GCLC)信号通路的影响。方法:采用CCK-8法评估不同浓度的PNS和ANE对人永生化角质形成细胞系HaCaT活力的影响,根据CCK-8结果选择75 mg/L的ANE及25、50和100 mg/L的PNS作为后续实验浓度;细胞设为空白对照组、模型组和PNS低、中、高剂量(25、50和100 mg/L)组,采用Western blot和RT-qPCR法检测各组细胞中I型胶原(COL-I)、上皮钙黏素(E-cadherin)、Nrf2、GCLC和谷胱甘肽还原酶(GR)的蛋白及mRNA表达情况;采用免疫荧光法检测Nrf2入核情况;采用生化试剂盒检测各组细胞中谷胱甘肽(GSH)、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)和丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性;采用DCFH-DA荧光探针检测细胞内活性氧(ROS)含量。结果:与空白对照组相比,模型组细胞中COL-I蛋白及mRNA表达上调,E-cadherin、Nrf2、GCLC、核Nrf2和GR的蛋白及mRNA表达下调,细胞中NADPH、MDA和ROS含量升高,GSH含量和SOD活性明显减弱;与模型组相比,PNS 50和100 mg/L组细胞中COL-I蛋白及mRNA表达下调,E-cadherin、Nrf2、GCLC、核Nrf2和GR的蛋白及mRNA表达上调,细胞中NADPH、MDA和ROS含量下降,GSH含量和SOD活性明显增强(P<0.05,P<0.01)。结论:PNS具有抗HaCaT细胞纤维化作用,其作用机制可能与激活Nrf2/GCLC信号通路,进而抗氧化应激改善口腔黏膜下纤维化有关。

活血化瘀解毒法对OSF患者GSK-3β、β-连环蛋白水平的影响及作用机制的研究

目的:探究活血化瘀解毒法对口腔黏膜下纤维化(oral submucous fibrosis,OSF)患者GSK-3β、β-连环蛋白(β-catenin)水平的影响及作用机制。方法:选取2018年5月~2019年12月本院收治的120例OSF患者,按照抽签法分为常规治疗组和活血化瘀解毒组各60例。常规治疗组使用曲安奈德注射液治疗,活血化瘀解毒组使用活血化瘀解毒法治疗。观察两组口腔黏膜病损面积、张口度、SFRP1在龈沟液及唾液中的水平、中医症候评分、TGF-β1、IL-6、GSK-3β、β-连环蛋白表达量及临床疗效。结果:治疗后活血化瘀解毒组口腔黏膜病损面积小于常规治疗组(P<0.05),活血化瘀解毒组张口度大于常规治疗组(P<0.05);治疗后活血化瘀解毒组患者SFRP1在龈沟液及唾液中的水平、临床疗效高于常规治疗组(P<0.05);治疗后活血化瘀解毒组中医症候评分、TGF-β1、IL-6、GSK-3β、β-连环蛋白表达量低于常规治疗组,差异均具有统计学意义(P<0.05)。结论:活血化瘀解毒法可通过作用于Wnt/β-catenin信号通路,调控TGF-β1、IL-6、GSK-3β、β-连环蛋白的表达,改善口腔黏膜下纤维化患者临床症状,缓解机体炎症,减轻疼痛,效果显著。

非编码小RNA在口腔黏膜下纤维性变中的研究进展

口腔黏膜下纤维性变(oral submucous fibrosis,OSF)是口腔黏膜最常见的癌前病变之一,其发病机制至今尚未完全阐明。非编码小RNA(small non-coding RNAs,SncRNAs)是一类不编码蛋白质的RNA分子,已被广泛报道参与多种人类疾病的调控。越来越多的研究表明多种SncRNAs在OSF发病过程中发挥重要作用。现有研究表明,微RNA(microRNAs,miRNAs)通过调控相关转录因子和基因表达或上皮间充质转化来调控成纤维细胞(fbroblasts,FB)活化而参与OSF疾病进展;长非编码RNA(long noncoding RNAs,lncRNAs)通过调控转化生长因子-β/母体抗瘫抑制因子(transforming growth factor-β/suppressor of mothers against decapentaplegic,TGF-β/Smad)信号通路或与miRNA相互作用参与OSF的发生发展;环状RNA(circular RNAs,circRNAs)通过与miRNA相互作用在OSF中发挥作用;tRNA衍生的小RNA(tRNA-derived small RNAs,tsRNAs)参与多种纤维化疾病的进展,但其在OSF中的具体作用机制仍需进一步探索。未来仍需重点关注SncRNAs介导OSF进展的作用靶点,探究其对OSF的作用功能及分子机制,以期为诊断和治疗OSF提供新思路。

口腔黏膜下纤维化大鼠模型造模方法的优化

目的比较三种口腔黏膜下纤维化(OSF)大鼠模型造模方法:槟榔碱涂擦、颊黏膜下注射槟榔碱、槟榔碱涂擦联合颊黏膜下注射槟榔碱,探讨建立大鼠OSF模型的更优造模方法。方法选择雄性SPF级SD大鼠40只,随机分成空白对照组、槟榔碱涂擦组、槟榔碱注射组、槟榔碱涂擦+注射组各10只。观察处理后每周各组大鼠的生存状况、张口度及体重变化;处理8周后处死大鼠,取颊黏膜进行苏木精-伊红(HE)染色观察颊黏膜组织炎症变化、免疫组化检验颊黏膜组织中转化生长因子-β1(TGF-β1)阳性程度及蛋白质印迹法(WB)检测颊黏膜组织中TGF-β1蛋白表达量。结果各组大鼠经过8周造模后,双颊部颊黏膜出现不同程度发白、呈纤维条索样改变;其中,槟榔碱涂擦组大鼠双颊黏膜下纤维化程度不一、分布不均。槟榔碱涂擦组大鼠体重与空白对照组比较,差异无统计学意义(P>0.05),槟榔碱注射组、槟榔碱涂擦+注射组大鼠体重均下降(P<0.05),其中槟榔碱涂擦+注射组大鼠体重下降最明显(P<0.05)。与空白对照组比较,三种造模方式的OSF大鼠张口度均降低(P<0.05),与槟榔碱涂擦组比较,槟榔碱注射组和槟榔碱涂擦+注射组张口度降低更明显(P<0.05);与空白对照组比较,三种造模方式的OSF大鼠TGF-β1表达量均显著上升(P<0.05),且各模型组间比较,差异有统计学意义(P<0.05)。其中TGF-β1在槟榔碱涂擦+注射组中表达量最高。结论槟榔碱涂擦联合颊黏膜下注射槟榔碱是建立大鼠口腔黏膜下纤维化的优选造模方法。

长链非编码RNA MALAT1对槟榔碱诱导的颊黏膜成纤维细胞活化能力影响的研究

目的:本研究旨在明确长链非编码RNA(long non-coding RNA,lncRNA) MALAT1对槟榔碱诱导的颊黏膜成纤维细胞(buccal mucosal fibroblasts, BMFs)活化能力影响的作用,为寻找新的口腔黏膜下纤维化(oral submucous fibrosis, OSF)治疗途径提供理论基础。方法:体外培养BMFs,采用不同浓度槟榔碱刺激BMFs,在细胞水平上构建OSF疾病模型。采用细胞计数试剂盒(cell counting kit-8,CCK-8)法检测细胞活力;Transwell法和胶原凝胶收缩法检测BMFs活化;实时荧光定量聚合酶链反应(quantitative real-time PCR, qRT-PCR)检测BMFs中平滑肌肌动蛋白-α(α-smooth muscle actin, α-SMA)和lncRNA MALAT1的表达情况;siRNA-MALAT1瞬时转染到BMFs中,评估槟榔碱刺激下BMFs迁移和收缩能力的变化。结果:10μg/mL槟榔碱是体外构建OSF疾病模型的最佳浓度,此时细胞内α-SMA和lncRNA MALAT1表达升高。Transwell实验和胶原凝胶收缩实验提示敲低lncRNA MALAT能抑制槟榔碱诱导的BMFs收缩和迁移。结论:lncRNA MALAT1对槟榔碱诱导的BMFs激活,细胞收缩和迁移有重要作用。

红花-牛膝治疗口腔黏膜下纤维化作用机制的网络药理学分析

目的联合使用网络药理学和分子对接技术探究红花-牛膝的有效成分作用于口腔黏膜下纤维化(OSF)的治疗靶点。方法通过网络药理学方法获得“红花”“牛膝”的有效成分及作用靶点,以及OSF的疾病靶点。利用STRING平台构建蛋白互作PPI网络图;对关键活性成分与核心靶点进行分子对接分析,并对交集靶点进行了GO和KEGG富集分析。结果发现红花-牛膝作用于OSF的主要活性成分为木犀草素、黄芩苷、黄连素和黄连碱等;主要核心靶点为肿瘤坏死因子(TNF)、基质金属蛋白酶9(MMP9)、丝裂原活化蛋白激酶3(MAPK3)、低氧诱导因子1α(HIF-1α)等;主要涉及癌症中的蛋白多糖、丝裂原活化蛋白激酶(MAPK)、白介素-17(IL-17)、C型凝集素受体等信号通路。分子对接结果亦表明活性分子与靶点基因均具备良好的结合力。结论本研究结果提示红花-牛膝可能通过多活性成分、多靶点以及多种信号通路来治疗OSF。

槟榔碱对口腔黏膜下纤维化NF-κB、TGF-β_(1)信号通路的影响研究

目的探讨槟榔碱对口腔黏膜下纤维化(OSF)核因子-κB(NF-κB)、转化生长因子-β_(1)(TGF-β_(1))信号通路的影响。方法选取10份上颚正常口腔黏膜样本,随机分为观察组和对照组,各5份。均经细胞培养后,观察组第6代口腔黏膜成纤维细胞(OMFb)内加入40μg/ml浓度的槟榔碱溶液,对照组加入含10%胎牛血清的DMEM培养液。比较两组样本中NF-κB、TGF-β_(1)mRNA表达、蛋白含量及12、24、48 h后细胞增殖情况。结果观察组样本NF-κB、TGF-β_(1)mRNA表达分别为(2.04±0.31)、(1.54±0.22),显著高于对照组的(1.47±0.23)、(1.17±0.18),差异具有统计学意义(P<0.05)。观察组样本NF-κB、TGF-β_(1)蛋白含量分别为(123.56±3.56)、(90.14±4.15)kDa,均显著高于对照组的(77.45±2.14)、(54.11±2.18)kDa,差异具有统计学意义(P<0.05)。观察组样本24、48 h后细胞增殖数量分别为(320.82±48.59)×10^(9)、(380.45±37.85)×10^(9),显著低于对照组的(600.76±45.37)×10^(9)、(1200.98±100.95)×10^(9),差异具有统计学意义(P<0.05)。结论高浓度槟榔碱可能通过上调NF-κB及TGF-β_(1)通路促进口腔黏膜下纤维性变,对后续的临床研究具有重要意义。

Quality of life and oral potentially malignant disorders: Critical appraisal and prospects

Quality of life(QoL) is a vital and often required health outcome measure that is relevant to patient care. A healthy oral cavity enables person to perform daily activities without any limitations. However, any disturbance may result in impaired QoL. The oral health-remains an essential element of people's health and well-being. In recent years, the tradition of clinical practice and research has been changed by incorporating QoL assessment, as it helps in assessment of patients' needs and monitoring treatment responses. Oral potentially malignant disorders(OPMDs) are a group of chronic disorders including oral leukoplakia(OL), oral lichen planus and oral submucous fibrosis(OSF). It is evident that patients with OPMDs experience significant health-related symptoms, functional limitations and psycho-social impairment, compromising their Qo L. Moreover, the worsening of Qo L has been associated with advanced stages of OPMDs. Despite of increasing number of OPMD cases in recent decades, limited literature is available regarding QoL in this population. Although, there is higher prevalence of habit-related OPMDs, particularly OSF and OL in Southern Asian countries, only a few studies have been performed in these populations. Moreover, these studies administered generic Qo L instruments, which offer less sensitivity to clinical changes. However, condition-specific instruments are more sensitive and allows better measurement of QoL. As the impacts of different conditions on OHRQoL may vary, the development and validation of a QoL instrument specific to each clinical entity of OPMDs is currently needed.

Precancerous lesions of oral mucosa

Precancerous lesions of oral mucosa, known as potentially malignant disorders in recent years, are consists of a group of diseases, which should be diagnosed in the early stage. Oral leukoplakia, oral submucous fibrosis, and oral erythroplakia are the most common oral mucosal diseases that have a very high malignant transformation rate. Oral lichen planus is one of the potentially malignant disorders that may be seen in six different subtypes including papular, reticular, plaquelike, atrophic, erosive, and bullous type, clinically. Atrophic and erosive subtypes have the greater increased malignant transformation risk compared to another subtypes. Although there are various etiological studies, the etiology of almost all these diseases is not fully understood. Geographically, etiologic factors may vary.The most frequently reported possible factors are tobacco use, alcohol drinking, chewing of betel quid containing areca nut, and solar rays. Early diagnosis is very important and can be lifesaving, because in late stages, they may be progressed to severe dysplasia and even carcinoma in situ and/or squamous cell carcinoma. For most diseases, treatment results are not satisfactory in spite of miscellaneous therapies. While at the forefront of surgical intervention, topical and systemic treatment alternatives such as corticosteroids, calcineurin inhibitors, and retinoids are widely used.

CTHRC1与TGF-1、Smad7在OSF组织中的表达及相关性分析

[目的]探讨胶原三股螺旋重复蛋白1(CTHRC1)、转化生长因子‐β1(TGF‐β1)、Smad蛋白 7 (Smad7)在口腔黏膜下纤维性变(OSF)组织中的表达及其相关性.[方法]选取33例OSF患者(早期10例,中期13 例,晚期 10 例)的新鲜颊黏膜组织及 10 例来源于外科手术的健康颊黏膜组织,免疫组化检测CT HRC1 、TGF‐β1 、smad7的表达水平, Pearson 软件分析 TGF‐β1表达与 CT HRC1和 Smad7的相关性.[结果]CT HRC1在OSF早、中、晚期病变组织中的表达均显著高于正常口腔黏膜组织( P =0 .007 ,0 .002 , 0 .006);TGF‐β1在早、中期病变组织中表达升高( P =0 .007 ,0 .031),晚期表达与对照比较差异无统计学意义( P =0 .323);Smad7在OSF各期病变组织中的表达也均高于对照组( P =0 .032 ,0 .007 ,0 .005 ).在 OSF病变组织中TGF‐β1与CT HRC1的表达呈正相关性(R=0 .678 ,P =0 .0010),TGF‐β1与Smad7的表达无明显相关性(R=0 .003 ,P =0 .9908).[结论]CT HRC1在OSF病变组织中表达增加,且与TGF‐β1的表达呈正相关,可作为抗纤维化新靶点.

Effect of Huoxue Huayu Detoxification on GSK-3β and β-catenin levels in OSF patients and its mechanism

Objective:To explore the effects of Huoxue Huayu detoxification method on the levels of GSK-3βandβ-catenin in OSF patients and its mechanism.Methods:A total of 120 patients with oral submucosal fibrosis(OSF)treated in our hospital from May 2018 to December 2019 were selected and divided into routine treatment group and Huoxue Huayu detoxification group of 60 cases according to the lottery method.The routine treatment group was treated with triamcinolone acetonide injection,and the Huoxue Huayu detoxification group was treated with Huoxue Huayu detoxification.The lesion area,mouth opening degree,SFRP1 level in gingival crevicular fluid and saliva,TCM symptom score,TGF-β1,IL-6,GSK-3β,β-catenin expression and clinical effect were observed in the two groups.Results:After treatment,the lesion area of oral mucosa in the Huoxue Huayu detoxification group was smaller than that in the routine treatment group(P<0.05),and the mouth opening degree in the Huoxue Huayu detoxification group was larger than that in the routine treatment group(P<0.05);After treatment,the level of SFRP1 in gingival crevicular fluid and saliva and clinical effect in the Huoxue Huayu detoxification group were higher than those in the routine treatment group(P<0.05);the TCM syndrome score,TGF-β1,IL-6,GSK-3β,β-catenin expression in the Huoxue Huayu detoxification group were lower than those in the routine treatment group(P<0.05).Conclusion:By acting on Wnt/β-catenin signaling pathway,Huoxue Huayu detoxification method can regulate the expression of TGF-β1,IL-6,GSK-3β,β-catenin,improve the clinical symptoms of patients with oral submucous fibrosis,alleviate the body inflammation,alleviate the pain,with remarkable effect.

三七总皂苷通过抑制槟榔碱诱导的HaCaT细胞铁死亡减轻口腔黏膜下纤维化

目的:探讨三七总皂苷(PNS)是否通过调控铁死亡减轻槟榔碱(ANE)诱导的口腔黏膜下纤维化(OSF),并初步研究其机制。方法:采用ANE作用于人永生化角质形成细胞系HaCaT构建OSF模型,设置空白组,模型组(50 mg/L ANE),低、中、高剂量(12.5、25和50 mg/L)PNS组,以及liproxstatin-1(铁死亡抑制剂)组,共计6组。光镜观察HaCaT细胞形态学变化;CCK-8法检测细胞活力;Western blot检测细胞中Ⅰ型胶原(Col-Ⅰ)、溶质载体家族7成员11(SLC7A11)和谷胱甘肽过氧化物酶4(GPX4)的蛋白表达水平;透射电镜观察细胞线粒体形态学变化;Ferro-Orange荧光探针检测细胞Fe^(2+)水平;DCFH-DA荧光探针检测细胞内活性氧(ROS)含量;比色法检测细胞内谷胱甘肽(GSH)含量。结果:与空白组比较,模型组HaCaT细胞多数呈长梭形,纤维化样外观,贴壁不牢靠,形态学改变显著,电镜下线粒体双层膜密度增加,嵴显著减少,呈典型的铁死亡表现,Col-Ⅰ蛋白表达增加,GPX4和SLC7A11蛋白表达降低,Fe^(2+)和ROS水平升高,GSH含量降低(均P<0.05);与模型组相比,低、中、高剂量PNS组细胞多数恢复圆润,呈上皮细胞样,线粒体膜密度减小,嵴数目增加,形态趋于正常,Col-Ⅰ蛋白表达减少,ROS水平下降,GSH含量升高(均P<0.05);与模型组相比,中、高剂量PNS组GPX4和SLC7A11蛋白表达显著上升,Fe^(2+)含量显著降低(均P<0.05);liproxstatin-1组细胞和线粒体形态及各指标水平与高剂量PNS组相比无显著差异(均P>0.05)。结论:PNS可通过抑制ANE诱导的HaCaT细胞铁死亡减轻OSF。