Parathyroid hormone-mitogen-activated protein kinase axis exerts fibrogenic effect of connective tissue growth factor on human renal proximal tubular cells

Background Enhanced and prolonged expression of connective tissue growth factor （CTGF） is associated with kidney fibrosis. Parathyroid hormone （PTH） is involved in the genesis of disturbed calcium/phosphate metabolism and ostitis fibrosa in renal failure. PTH activated mitogen-activated protein kinase （MAPK） signaling pathway is present in renal tubular cells. The aim of this study was to identify the mechanism how the signal is transduced to result in extracellular signal-regulated protein kinase （ERK） activation, leading to upregulation of CTGF.Methods The levels of CTGF mRNA and protein in human kidney proximal tubular cells （HK-2） treated with PTH in the presence or absence of the MAPK inhibitor PD98059 were analyzed by quantitative real-time polymerase chain reaction （RT-PCR） and immunoblotting assay. The activation of the CTGF promoter in HK-2 cells was determined by the dual-luciferase assay. The effects of the protein kinase A （PKA） activator 8-Br-cAMP and protein kinase C （PKC） activator phorbol 12-myristate 13-acetate （PMA） on MAPK phosphorylation, and the effects of the PKA inhibitor H89 and PKC inhibitor calphostin C on MAPK phosphorylation and CTGF expression were detected by immunoblotting assay.Results PD98059 inhibited the PTH stimulated expression of CTGF, which strongly suggested that the MAPK signaling pathway plays an important role in the PTH-induced CTGF upregulation in renal tubular cells. A PKA activator as well as PKC activators induced MAPK phosphorylation, and both PKA and PKC inhibitors antagonized PTH-induced MAPK phosphorylation and CTGF expression.Conclusion CTGF expression is upregulated by PTH through a PKC/PKA-ERK-dependent pathway.     

Impact of MAPK Cascade Pathway and P53 Pathway upon Liver Transplant

Summary： The change and the role of MAPK cascade pathway and P53 pathway after liver transplantation were explored. Thirty-four punctured donor liver specimens and 10 normal liver specimens were classified as group A （no rejection, n=10）, group B （mild/moderate acute rejection, n=10）, group C （serious acute rejection, n=8）, group D （chronic rejection/fibrosis, n=6） and group E （control, n= 10）. By using immunohistochemistry, the expression levels of mitogen activated protein kinase （MAPK）, Ras and P53 proteins, and by in situ hybridization, MAPK and ras mRNA expression levels were detected. The results showed that the expression levels of MAPK and Ras proteins were increased by turns in groups A, B and C, and decreased by turns in groups D and E. The protein expression of P53 was higher in the treated groups. The expression of Ras, HSP70 mRNA was identical as that of protein. It is suggested that the MAPK cascade pathway and P53 pathway can protect the hepatocytes by different mechanisms after liver transplantation. MAPKs cascade pathway repairs hepatocyte injury or accelerates hepatocytes into proliferation or differentiation. P53 pathway blocks cell cycle within G1 phase to make hepatocyte repair or apoptosis to reduce disorder differentiation.     

Cyclovirobuxinum D Alleviates Cardiac Hypertrophy in Hyperthyroid Rats by Preventing Apoptosis of Cardiac Cells and Inhibiting the p38 Mitogen-Activated Protein Kinase Signaling Pathway

Objective: To investigate the underlying mechanisms of cyclovirobuxinum D(Cvb-D) on alleviating cardiac hypertrophy in rats. Methods: Sprague-Dawley rats were randomly divided into 5 groups: control group; levothyroxine-induced cardiac hypertrophy group(model); levothyroxine-induced cardiac hypertrophy + Cvb-D group(Cvb-D); levothyroxine-induced cardiac hypertrophy + captopril group(captopril); levothyroxine-induced cardiac hypertrophy + SB203580 group(SB203580), n=10 for each group. Rats were daily administered the respective drugs continuously for14 days by gastric gavage. A rat model of cardiac hypertrophy was established by intraperitoneal injection of levothyroxine to investigate whether Cvb-D protects against cardiac hypertrophy by inhibiting the p38 mitogen-activated protein kinase(MAPK) signaling pathway and preventing apoptosis of cardiac cells. Results: Treatment with Cvb-D significantly deceased left ventricle hypertrophy, improved the histopathology, hemodynamic conditions, and cardiac function in rats with cardiac hypertrophy. Compared with the normal control group, in rats with cardiac hypertrophy, expression of bax in the heart and phospho-p38 MAPK protein levels were significantly up-regulated(P0.01 or 0.05), whereas the bcl-2 protein level was downregulated(P0.01). In contrast, Cvb-D treatment reversed the changes in bax and phospho-p38 MAPK protein levels but increased the bcl-2 protein level(P0.01 or 0.05), and these effects were similar to those of captopril and SB203580(a specific p38 MAPK inhibitor) treatment. Furthermore, both Cvb-D, captopril and SB203580 reduced m RNA expression of p38α, p38β, c-fos, and c-jun m RNA, and Cvb-D had a stronger effect(P0.01). Conclusion: These results demonstrate that Cvb-D protects against cardiac hypertrophy, which is possibly mediated by prevention of cardiac cell apoptosis and inhibition of the p38 MAPK signaling pathway.     

Thymic Stromal Lmphopoietin Pomotes Macrophage-derived Foam Cell Formation简

The effect of thymic stromal lymphopoietin（TSLP） on macrophage-derived foam cell formation and the underlying mechanism were studied. Macrophages isolated from C57BL/6 mice were co-cultured in vitro with different concentrations of TSLP or TSLPR-antibody in the presence of oxidized low density lipoprotein（ox-LDL）. The effects of TSLP on macrophage-derived foam cell formation were observed by using oil red O staining and intracellular lipid determination. The expression levels of foam cell scavenger receptors（CD36 and SRA） as well as ABCA1 and TSLPR were detected by using RT-PCR and Western blotting. As compared with the control group, TSLP treatment significantly promoted lipid accumulation in macrophages, significantly increased protein expression of CD36 and TSLPR in a dose-dependent manner, and significantly reduced the expression of ABCA1 protein in a dose-dependent manner. No significant differences were noted between the TSLPR-antibody group and the control group. TSLP may down-regulate the expression of cholesterol efflux receptor ABCA1 and up-regulate scavenger receptor expression via the TSLPR signaling pathway, thereby promoting macrophage-derived foam cell formation.     

DNA Methylation and Birth Weight:a Genome-wide Analysis

The study illustrate the inner correlation between global DNA methylation variation and different birth weights. Infant birth weight was used to identify cases and controls. Cord blood and placentas were collected. We performed DNA methylation profiling of bisulphite‐converted DNA. We have identified many differentially methylated Cp G sites in experimental groups; these sites involved in hundreds of signalings. Among these, more than ten pathways were referred to the glucose and lipid metabolism. Methylation changes in the insulin‐signaling pathway(ISP), adipocytokine signaling pathway(ASP) and MAPK signaling pathway are involved in the fetal programming of diabetes.     

Effects of Zhizi Chuanxiong Capsule (栀子川芎胶囊) on Abnormal Methylation in Rabbits with Atherosclerosis

Objective: To investigate the effects of Zhizi Chuanxiong Capsule(ZCC, 栀子川芎胶囊) on abnormal DNA methylation in a rabbit model of atherosclerosis(AS). Methods: After 1 week of adaptive feeding, 48 New Zealand white rabbits were randomly divided into 4 groups: a control group(n=12) fed with normal diet for 22 weeks; a model group(n=12) fed with high fat diet for 14 weeks followed by 8 weeks of normal diet feeding; a low-dose ZCC group(n=12) fed with high fat diet and low-dose ZCC for 14 weeks, followed by 8 weeks of normal diet and low-dose drug; a high-dose ZCC group(n=12) fed with high fat diet and high-dose drug for 14 weeks, followed by 8 weeks of normal diet and high-dose drug. After 22 weeks of feeding, blood samples were taken from the rabbit ear vein, and the genomic DNA was extracted for methylation immunoprecipitation sequencing(Medip-seq). The aorta tissues were collected for hematoxylin-eosin(HE) staining. Results: Eight rabbits died during the feeding process. HE staining showed that the size of the lipid deposition on vessel wall and atherosclerotic plaque formation were reduced in both low-and high-dose group. The Medip-seq results showed that there were 146 abnormally methylated genes(including both hypermethylated gene and hypomethylated genes) in the model group, compared with the control group. Gene Ontology(GO) and Pathway analysis showed that these abnormally methylated genes were found to be involved in multiple AS-related functions and pathways, such as protein kinase C activity, cholesterol transport, mitogen-activated protein kinase(MAPK) signaling pathway, peroxisome proliferater-activated receptor signaling pathway, vascular smooth muscle contraction, inflammation and so on. The abnormal methylated genes in AS model group were altered in both low-and high-dose groups: low-dose ZCC could change 72 of the 146 abnormally methylated genes, highdose ZCC could change 71. Through GO and Pathway analysis, these altered methylated genes were involved in protein kinase C activity, inflammatory pathway, MAPK signaling pathway, vascular endothelial growth factor signaling pathway, etc. Conclusion: ZCC could treat AS through regulating the abnormal hypermethylated and hypomethylated genes in AS rabbit model.     

壮骨健膝方通过Caveolin-p38 MAPK信号通路对白介素-1介导的软骨细胞退化产生抑制作用

Objective:To evaluate the effect of Zhuanggu Jianxi Decoction(壮骨健膝方,ZGJXD)on interleukin-1β(IL-1β)-induced degeneration of chondrocytes(CDs)as well as the activation of caveolin-p38 mitogenactivated protein kinase(MAPK)signal pathway,investigating the possible molecular mechanism that ZGJXD treats osteoarthritis.Methods:Serum pharmacology was applied in the present study,where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum(ZGJXD-S)was collected for following in vitro experiments.CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro.Upon IL-1βstimulation,the degeneration of CDs was verified by inverted microscope,toluidine blue stain and typeⅡcollagen immunocytochemistry.After IL-1β-stimulated CDs were intervened with blank control serum,ZGJXD-S,together with or without SB203580(a specific inhibitor of p38 MAPK)for 48 h,caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting,and the mRNA expression of IL-1β,tumor necrosis factorα(TNF-α),matrix metalloproteinase 3(MMP-3)and MMP-13 were examined by real-time polymerase chain reaction.Results:IL-1βstimulation induced degeneration of CDs,increased caveolin-1 expression and p38 phosphorylation,up-regulated the mRNA level of IL-1β,TNF-α,MMP-3 and MMP-13.However,the IL-1β-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs.Conclusion:ZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway,which might be one of the mechanisms that ZGJXD treats osteoarthritis.     

Blocking Ihh signaling pathway inhibits the proliferation and promotes the apoptosis of PSCs

Summary： The roles of Indian hedgehog （Ihh） signaling pathway in the proliferation and apoptosis of precartilaginous stem cells （PSCs） were investigated. PSCs, labeled with fibroblast growth factor receptor 3 （FGFR-3）, were isolated from neonatal rats by immunomagnetic separation. After identification with FGFR-3 and Col II, the cells were incubated with different concentrations of cyclopamine （cyclo）, the specific inhibitor of Ihh signaling pathway. The morphologic changes of the cells were observed under the inverted phase contrast microscope. The mRNA expression levels of Ihh, parathyroid hormonerelated peptide （PTHrP）, protein Patched （Ptch）, Bcl-2 and p21 were detected by RT-PCR. The protein expression levels of Ihh and Ptch were measured by Western blot. MTT assay was used to examine the effects of cyclo on proliferation of PSCs. Apoptosis rate of PSCs was examined by AnnexinV/PI assay of flow cytometric analyses. After PSCs were incubated with cyclo, obvious morphologic changes were observed as compared with the control group. The mRNA expression levels of PTHrP, Ptch and Bcl-2 were decreased to varying degrees in a cyclo dose-dependent manner. However, the expression levels of Ihh and p21 mRNA were increased. The protein expression of Ptch and Ihh had the same change as the mRNA expression. Meanwhile, cyclo could obvi- ously inhibit the proliferation and promote the apoptosis of PSCs. The results indicated that Ihh signaling pathway plays an important role in regulating the proliferation and apoptosis of PSCs, which is probably mediated by Bcl-2 and p21.     

Identification of drug candidate for osteoporosis by computational bioinformatics analysis of gene expression profile

Background

Osteoporosis is a condition of bones that leads to an increased susceptibility to fracture and consequent painful morbidity. It has become a major issue of life quality worldwide. However, until now, the molecular mechanism of this disease is far from being clear.

Methods

In this study, we obtained the gene expression profile of osteoporosis and controls from Gene Expression Omnibus and identified differentially expressed genes (DEGs) using classical t-test method. Then, functional enrichment analyses were performed to identify the dysregulated Gene Ontology categories and dysfunctional pathways in osteoporosis patients compared to controls. Besides, the connectivity map was used to identify compounds that induced inverse gene changes to osteoporosis.

Results

A total of 5581 DEGs were identified. We found these DEGs were enriched in 9 pathways by pathway enrichment analysis, including focal adhesion and MAPK signaling pathway. Besides, sanguinarine was identified as a potential therapeutic drug candidate capable of targeting osteoporosis.

Conclusion

Although candidate agents identified by our approach may be premature for clinical trials, it is clearly a direction that warrants additional consideration.     

葛根素通过下调P38 细胞分裂素活化蛋白激酶活性以及NF-β通道对共培养气道上皮细胞与中性粒细胞黏附分子表达的抑制作用

Objective:In this study,we aimed to investigate the expressions of adhesion molecules on human bronchial epithelial cells and neutrophils in co-culture system,assess the effects of puerarin on suppressing these adhesion molecules expressions,and explore the roles of two crucial signal-transduction elements p38mitogen-activated protein kinase(p38 MAPK)and nuclear factor kappa B(NF-k B)in modulating adhesion molecules expressions.Methods:Neutrophils and BEAS-2B cells(one human bronchial epithelial cell line)were co-cultured,and adhesion molecules expressions on cell surface were detected using flow cytometry.The mRNA levels of adhesion molecules were assessed by real-time quantitative polymerase chain reaction(real-time qPCR).Phosphorylated p38 MAPK and inhibitor k B were analyzed by Western blot.Results:In co-culture system,adhesion molecules expressions on BEAS-2B cells and neutrophils were enhanced significantly(P0.05).Correspondingly,the mRNA levels of adhesion molecules were also increased greatly.Moreover,the pretreatment of peurarin obviously suppressed adhesion molecules expressions on cell surface.Furthermore,phosphorylated p38 MAPK and inhibitor k B in BEAS-2B cells and neutrophils were elevated in co-culture system,but decreased significantly after upon the treatment of peurarin(P0.05).Conclusions:Coculture boosted the interactions between human bronchial epithelial cells and neutrophils mimicking airway inflammation,whereas peurarin decreased the expression of adhesion molecules on cell surface by suppressing the activities of d38 MAPK and NF-κB pathways,and exhibiting its anti-inflammation activity.     

Traumatic pseudoaneurysm of the internal carotid artery presenting with oculosympathetic palsy

Background

Blunt internal carotid artery (ICA) injury is rare but undiagnosed can have disastrous clinical consequences.

Aim

To report a late presentation of blunt ICA injury in a young male following a road traffic accident.

Result

A 16-year-old male presented 11 days following a head injury with a unilateral Horner’s syndrome. Imaging confirmed a pseudoaneurysm of the ICA. The patient was treated with anticoagulant therapy.

Conclusion

Diagnosis of ICA injury requires a high index of suspicion and presentation with unusual neurological signs following blunt trauma to the head and neck requires prompt investigation.

     

Velvet antler polypeptide is able to induce differentiation of neural stem cells towards neurons in vitro

Objective

To investigate the neural differentiation capacity of water extraction of velvet antler.

Effect of Acupuncture on Proliferation and Differentiation of Neural Stem Cells in Brain Tissues of Rats with Traumatic Brain Injury

Objective:To observe the effect of acupuncture on proliferation and differentiation of neural stem cells in brain tissues of rats with traumatic brain injuny.Methods:Thirty SD rats were randomly and equally allocated to the sham-operated,the model and the acupuncture groups.The traumatic brain injury model was established by the free drop method.For the rats in the acupuncture group,acupuncture was applied once a day for 7 days.Brain histotomy was carried out when treatments were completed.Immunohistochemical techniques were adopted to detect the cells that express nestin,neurofilament proteins(NF)-200 and glial fibrillary acidic proteins(GFAP),the markers of neural stem cells,neurons,astrocytes respectively.Results:Compared to the sham-operated group,the number of nestin-positive cells and NF-200-positive cells in brain tissues was decreased significantly in the model group(P<0.01),whereas the number of GFAP-positive cells was significantly increased (P<0.01).Compared to the model group,the positive cells of nestin,NF-200,GFAP in brain tissues in the acupuncture group were increased obviously(P<0.01).Conclusions:Acupuncture can significantly increase the number of nestin-positive cells,NF-200-positive cells and GFAP-positive cells,indicating the significant increase of neural stem cells,neurons and astrocytes in number.Acupuncture can improve neuranagenesis by promoting the proliferation and differentiation of neural stem cells in brain tissues.This might be one of the mechanisms for acupuncture to treat traumatic brain injury and to promote the repair of nervous function.