Mechanism of Huatan Sanjie Fang in improving goiter in Graves'disease mice based on the Hippo signaling pathway

Objective:To explore the mechanism of Huatan Sanjie Fang(HTSJ)in regulating goiter in Graves'disease(GD)mice by detecting key factors of the Hippo signaling pathway.Methods:A mouse model of GD was established by injecting Ad-TSHR289 adenovirus into the bilateral quadriceps femoris of female mice.Successful mouse models were then randomly divided into a model group,methimazole(MMI)group,and HTSJ group,and fed with deionized water,MMI(4.5 mg/kg per day),and HTSJ(35.10 g/kg per day),respectively,for 10 weeks.Histopathological changes of the thyroid gland were subsequently observed by hematoxylin-eosin staining.Radioimmunoassay was used to detect serum total thyroxine(T4)and thyrotrophin-receptor antibody(TRAb)levels.The relative expression of mRNA of Mst1,YAP,and TAZ were detected by quantitative real-time polymerase chain reaction,while the protein expression of Mst1,YAP,TAZ,pMst1,and pYAP were detected by western blot.Results:After 10 weeks of drug intervention,goiter and other pathological changes in the HTSJ group significantly improved compared with the model group,and the levels of serum T4 and TRAb significantly decreased(P=.002,P<.001,respectively).Decreased mRNA expression of Mst1,YAP,and TAZ,the key factors of the Hippo signaling transduction pathway,was also observed(P=.002,P=.022,P<.001,respectively).In contrast,protein expression of Mst1(P=.046),pMst1(P=.026),and p YAP(P=.004)increased,while protein expression of YAP and TAZ decreased(P=.041,P<.001,respectively).Conclusion:HTSJ can effectively improve goiter in GD mice through the Hippo signaling pathway.

Research progreess on relevant diseases of RhoA/ROCK signaling pathway

RhoA (Ras homolog gene family member A) belongs to the Rho subfamily of GTPases. ROCK (Rho—associated coiled—coil forming protein kinase) is downstream of the active RhoA and affects the generation and secretion of cellular element, which will result in relevant biologic effects. The RhoA/ROCK signaling pathway consists of these serious reactions. Therefore, the activation and inhibition of this pathway are closely related to the occurrence and development of many diseases. The research on the molecular mechanism of these diseases may be instructive and helpful to the clinical treatmen and prognosis of diseases. Recent studies of these typical diseases related to RhoA/ROCK signaling pathway are viewed in this article.

同种异体脂肪干细胞介导RhoA-YAP通路在慢性创面修复机制研究

目的分析同种异体脂肪干细胞(ADSC)移植促进慢性创面修复的效果及Ras同源基因家族成员A(RhoA)-Yes相关蛋白(YAP)信号通路活化的机制。方法选择2个月龄SD大鼠31只,雌雄不限,体质量112~120 g,平均体质量116.7 g。以其中1只提取ADSC并鉴定;30只大鼠随机分为5组:模型组(自身皮瓣移植),ADSC移植组,RhoA抑制剂C3干预组(简称C3干预组),慢病毒转染沉默YAP表达组(简称shYAP组),C3和慢病毒转染联合干预组(简称联合组)。分别在移植后7 d和21 d采用激光多普勒血流计测定创面血流量、皮瓣成活率,逆转录聚合酶链反应(RT-PCR)和Western blot法检测创面RhoA、YAP、结缔组织生长因子(CTGF)和血管内皮生长因子(VEGF)mRNA和蛋白的定量表达水平。结果体外成功分离并鉴定为ADSC。30只大鼠均成活至实验21 d。ADSC移植组7 d和21 d皮肤血流量[7 d(256.3±56.2) PU,21 d (421.5±82.8) PU]、皮瓣成活率[7 d (32.6±10.6)%,21 d (45.8±17.5)%]、RhoA mRNA(7 d0.36±0.07,21 d 0.58±0.12)、YAP mRNA(7 d 0.33±0.06,21 d 0.52±0.13)、CTGF mRNA(7 d 0.49±0.11,21 d 0.67±0.15)和VEGF mRNA(7 d 0.48±0.14,21 d 0.68±0.16)及蛋白定量表达水平(7 d 0.37±0.07,21 d 0.59±0.12;7 d 0.35±0.06,21 d 0.54±0.13;7 d 0.46±0.11,21 d 0.65±0.15;7 d 0.47±0.14,21 d 0.67±0.16)均显著高于其他各组,联合组显著低于C3干预组和shYAP组,差异有统计学意义(P <0.05)。结论同种异体ADSC可提高慢性创面移植皮瓣的存活率,促进创面愈合,增加创面组织CTGF和VEGF表达,可能通过介导RhoA-YAP信号通路的活化程度来实现。

溶血磷脂酸通过RhoA-YAP通路调控脂肪干细胞增殖的研究

目的探讨溶血磷脂酸(lysophosphatidic acid,LPA)调控脂肪干细胞(adipose-derived stem cells,ASCs)增殖的作用及其分子机制。方法分离SD大鼠ASCs,利用LPA对其进行干预,干预时间为1 h,采用Western Blot检测YES相关蛋白(yes associated protein,YAP)、结缔组织生长因子(connective tissue growth factor,CTGF)蛋白表达水平。利用免疫荧光检测YAP亚细胞定位,逆转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)检测CTGF和Ankrd1的m RNA表达水平。慢病毒转染ASCs,Western Blot检测不同分组YAP蛋白表达。进一步利用流式细胞术和CCK-8法检测不同分组中ASCs增殖情况。最后采用Rho A抑制剂C3干预,免疫荧光检测不同分组中YAP亚细胞定位,Western Blot检测YAP、CTGF和GTP-Rho A的表达情况。结果 LPA能显著促进YAP的表达和在细胞核内的聚集,同时LPA也能够促进YAP靶基因CTGF在蛋白水平的表达。LPA能上调YAP靶基因CTGF和锚蛋白重复域1(ankyrin repeating domain 1,Ankrd1)的m RNA表达水平。慢病毒转染敲除YAP表达后,LPA对YAP的上调作用被明显抑制。细胞周期流式细胞术和CCK-8检测结果显示LPA可显著促进ASCs的增殖,但在sh YAP慢病毒转染特异性敲除YAP后,LPA对ASCs的促增殖能力被明显削弱。Rho A抑制剂C3处理后,LPA对YAP细胞核聚集的促进作用被削弱,同时LPA对YAP、CTGF和GTP-Rho A表达的促进作用也得到了明显抑制。结论 LPA能够通过Rho A-YAP通路调控ASCs的增殖。

The roles and regulation of Yes-associated protein 1 in stem cells

Yes-associated protein 1(YAP1)is a downstream effector of the Hippo signaling pathway,and it is involved in tumorigenesis,tissue repair,growth,and development.In this review,the biological roles and the mechanisms of YAP1 in mediating stem cell fate decisions are discussed,including cell proliferation,differentiation,and apoptosis.In general,YAP1 promotes the proliferation and differentiation of stem cells,including embryonic stem cells and adult stem cells.It inhibits apoptosis by binding to the transcription factors,e.g.,transcriptional enhanced associate domain(TEAD),Smad,runt-related transcription factor 1/2,p73,p63,and Erb84,to maintain tissue homeostasis.The translocalization of YAP1 in cellular nuclei and the phosphorylation in the cytoplasm work as important and unusual events for the activation of YAP1.Moreover,YAP1 serves as the crosstalk for the Hippo pathway and other signaling pathways,including the Wnt and Notch pathways.It is highlighted in this review that YAP1 is an essential regulator for stem cells that have significant applications in regenerative medicine and reproductive medicine.

The mevalonate pathway promotes the metastasis of osteosarcoma by regulating YAP 1 activity via RhoA

Osteosarcoma is the most common malignant bone tumour,and the metastasis of osteosarcoma is an important cause of death.Evidence has shown that the mevalonate pathway is highly activated and is expected to be a new target for tumour therapy.In this study,we investigated the effect of mevalonate signalling on osteosarcoma metastasis and its molecular mechanism.First,we found that the key rate-limiting enzyme of mevalonate signalling,3-hydroxy-3-methylglutaryl-CoA reductase(HMGCR),was highly expressed in osteosarcoma cells,and inhibition of HMGCR with simvastatin significantly inhibited the motility of 143B cells.Next,we found that YAP1 activity was significantly upregulated in osteosarcoma cells and that YAP1 knockdown inhibited the motility of 143B cells.We also found that the mevalonate pathway regulated the motility of 143B cells by modulating YAP1 phosphorylation and cellular localization.Moreover,we found that the activity of YAP1 was regulated by the mevalonate pathway by modulating the cell membrane localization of RhoA.Finally,we demonstrated that inhibition of the mevalonate pathway notably reduced the lung metastasis of 143B cells,as reflected by the decreased incidence and number of metastatic nodules and the increased survival time of the nude mice.Taken together,our findings suggest that the mevalonate pathway can promote the metastasis of osteosarcoma by activating YAP1 via RhoA.Inhibition of the mevalonate pathway may be a promising therapeutic strategy for osteosarcoma metastasis.

Regulation of the Hippo signaling pathway by deubiquitinating enzymes in cancer

Regulation of the Hippo signaling pathway is essential for normal organ growth and tissue homeostasis.The proteins that act to regulate this pathway are important for ensuring proper function and cellular location.Deubiquitinases(DUBs)are a family of proteases that act upon many proteins.While ubiquitinases add ubiquitin and target proteins for degradation,DUBs act by removing ubiquitin(Ub)moieties.Changes in ubiquitin chain topology results in the stabilization of proteins,membrane trafficking,and the alteration of cellular localization.While the roles of these proteins have been well established in a cancer setting,their convergence in cancer is still under investigation.In this review,we discuss the roles that DUBs play in the regulation of the Hippo signaling pathway for homeostasis and disease.

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Effects of Bunao-Fuyuan decoction serum on proliferation and migration of vascular smooth muscle cells in atherosclerotic

Atherosclerosis(AS)is a chronic inflammatory disease,the main causes of which include abnormal lipid metabolism,endothelial injury,physical and chemical injury,hemodynamic injury,genetic factors and so on.These causes can lead to inflammatory injury of blood vessels and local dysfunction.Bunao-Fuyuan decoction(BNFY)is a traditional Chinese medicine compound that can treat cardiovascular and cerebrovascular diseases,but its effect on AS is still unknown.The aim of this study was to investigate the effect and mechanism of BNFY in proliferation and migration of vascular smooth muscle cells(VSMCs)on AS.At first,the expression ofα-SMA protein in ox-LDL-induced VSMCs,which was detected by immunofluorescence staining and western blot.CCK-8 technique and cloning technique were used to detect the cell proliferation of ox-LDL-induced VSMCs after adding BNFY.Meanwhile,the expression of proliferating protein Ki67 was detected by immunofluorescence staining.Western blot was also used to detect the expression of proliferation-related proteins CDK2,CyclinE1 and P27.Flow cytometry was used to detect the effect of BNFY on cell cycle.The effects of BNFY on proliferation and migration of cells were detected by cell scratch test and Transwell.Western blot was used to detect the expression of adhesion factors ICAM1,VCAM1,muc1,VE-cadherin and RHOA/ROCK-related proteins in cells.We found that the expression of AS markerα-SMA protein increased significantly and cells shriveled and a few floated on the medium after induction of ox-LDL on VSCMs.The proliferation rate of ox-LDL VSMCs decreased significantly after adding different doses of BNFY,and BNFY can inhibit cell cycle.Meanwhile,we also found that cell invasion and migration rate were significantly inhibited and related cell adhesion factors ICAM1,VCAM1,muc1 and VE-cadherin were inhibited too by BNFY.Finally,we found that BNFY inhibited the expression of RHOA,ROCK1,ROCK2,p-MLC proteins in the RHOA/ROCK signaling pathway.Therefore,we can summarize that BNFY may inhibit the proliferation and migration of atherosclerotic vascular smooth muscle cells by inhibiting the activity of RHOA/ROCK signaling pathway.