Mechanism of Honghua Oral Liquid in Alleviating Neuropathic Pain / 中国实验方剂学杂志

ObjectiveTo investigate the pharmacodynamic characteristics and explore the molecular mechanism of Honghua oral liquid （HOL） in relieving neuropathic pain （NP）. MethodHealthy male SD rats were randomly assigned into sham group， model group， low-， medium-， high-dose （0.5， 1.0， 2.0 mL·kg-1·d-1， respectively） HOL groups， and a positive drug （pregabalin， 25 mg·kg-1·d-1） group， with 6 rats in each group. Spinal nerve ligation （SNL） of L5 was conducted in other groups except the sham group. Drug administration was performed 3 days after the SNL surgery for 2 consecutive weeks， and samples were collected after the end of the administration. During the treatment period， the mechanical pain threshold and cold pain threshold were determined to measure the pain-relieving effect of HOL. Transcriptome sequencing was performed on hippocampal tissue samples from the sham， model， and high-dose HOL groups， and differentially expressed genes between the sham group and the model group as well as the model group and HOL high-dose group were obtained. After pathway enrichment analysis， we selected the targets which were closely related to neuroinflammation for validation， and predicted the specific binding sites of the major active components in HOL with the targets through molecular docking. In addition， the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-10 （IL-10） were determined by enzyme-linked immunosorbent assay （ELISA） to evaluate the effect of HOL on neuroinflammation in NP rats. ResultCompared with the sham group， SNL decreased the mechanical pain threshold and cold pain threshold （P<0.05）. Compared with the model group， HOL recovered the mechanical pain threshold and cold pain threshold （P<0.05）. The transcriptome data showed that 376 differentially expressed genes （DEGs） were identified between the model group and the sham group， including 124 upregulated genes and 252 downregulated genes， and 194 DEGs between the model group and the high-dose HOL group， including 33 upregulated genes and 161 downregulated genes. Among them， insulin-like growth factor 1（IGF1）， matrix metallopeptidase-2 （MMP-2）， matrix metallopeptidase-14 （MMP-14）， erb-B2 receptor tyrosine kinase 2 （ERBB2）， and integrin subunit alpha 5 （ITGA5） associated with NP were selected for further validation. The Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） results showed that compared with the sham group， the modeling up-gurelated the mRNA levels of the above five molecules in the hippocampus （P<0.01）. Compared with model group， HOL down-regulated the mRNA levels of these molecules （P<0.01）. The molecular docking results showed that the main active components of safflower， hydroxysafflor yellow A， kaempferol， and quercetin， formed stable hydrogen bonds with the amino acid residues of IGF1， MMP-2， MMP-14， ERBB2， and ITGA5. The enzyme-linked immunosorbent assay（ELISA） results showed that compared with those in the sham group， the serum levels of TNF-α and IL-10 were out of balance in the model rats （P<0.01）. Compared with the model group， HOL lowered the level of the pro-inflammatory cytokine TNF-α （P<0.01） and elevated that of the anti-inflammatory cytokine IL-10 （P<0.05）. ConclusionHOL exerts analgesic effect on SNL rats by inhibiting neuroinflammation.

The use of integrin binding domain loaded hydrogel (RGD) with minimally invasive surgical technique in treatment of periodontal intrabony defect: a randomized clinical and biochemical study

Abstract Periodontal regeneration faces multiple challenges, the most important being cellular insufficiency. In an attempt to improve defect cellularity, we aimed to demonstrate enhancing cellular attraction using arginine-glycine-aspartic acid (RGD) adhesion molecule legend blended hydrogel within the intrabony defects. Methodology Forty-five intrabony defects were selected from patients with stage III or IV - grade A or B periodontitis and divided randomly into three equal groups of 15 each: group1 (G1): received minimally invasive surgical technique (MIST) alone, group2 (G2): received MIST and placebo hydrogel injection, and group3 (G3): were treated with MIST and RGD hydrogel injection. Primary outcomes 6 months following therapy were; defect base fill (DBF) and defect width measurement (DW); secondary outcomes were clinical attachment level (CAL), pocket depth (PD), plaque index (PI), gingival index (GI), and biochemical analysis of bone morphogenetic protein (BMP-2) evaluated at 1,7,14 and 21 days following therapy. Results Significant improvements in DBF, CAL, and PD were observed in the three studied groups 6 months following therapy compared to baseline (p<0.05). A significant improvement in DBF was reported in G3 compared to G1 and 2 (p=0.005). Additionally, a significantly higher CAL gain was reported in G3 compared to that of G1 (p=0.02). Group 3 was associated with a significantly higher level of BMP-2 compared to G1 and G2 in all reported periods. Conclusion RGD peptide carried on a hydrogel delivery agent and contained with a minimally invasive flap could be a reliable option in improving the outcomes of periodontal therapy.

Bushen Jianpi Jiedu Liyan Formula Regulates Homing-associated Cell Adhesion Molecules in Rat Model of IgA Nephropathy / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Bushen Jianpi Jiedu Liyan formula on the expression of integrin alpha 4 beta 1 （α4 β1）， vascular cell adhesion molecule-1 （VCAM-1）， stromal-derived factor-1 （SDF-1）， and chemokine receptor-4 （CXCR4） in the small intestine and bone marrow of the rat model of immunoglobulin A（IgA） nephropathy. MethodA total of 120 male SD rats were used to establish the IgA nephropathy model by intragastric administration of bovine serum albumin （BSA）， subcutaneous injection of CCl4， and tail vein injection of lipopolysaccharide （LPS）. The successfully modeled rats were randomized into blank， model， lotensin （63 mg·kg-1）， and low-， medium-， and high-dose （10.4， 20.81， 41.62 g·kg-1， respectively） Bushen Jianpi Jiedu Liyan formula groups （n=16）. The rats were treated with corresponding drugs according to their body weight. After 7 weeks of administration， the rats were sacrificed for the collection of samples， and the protein and mRNA levels of α4 β1， VCAM-1， SDF-1， and CXCR4 in the small intestine and bone marrow were determined by immunohistochemistry and real-time fluorescence quantitative polymerase chain reaction， respectively. ResultCompared with the blank group， the model group showed increased red blood cell count in the urine at the 10th， 12th， 14th， 16th weeks （P<0.01）， and such increases were reduced in the drug intervention groups （P<0.05）， especially in the medium-dose Bushen Jianpi Jiedu Liyan formula group （P<0.05）. Compared with those in the blank group， the protein levels of α4 β1， VCAM-1， SDF-1， and CXCR4 in the intestinal lamina propria in the model group were up-regulated （P<0.05）， and such un-regulations were inhibited in the drug intervention groups （P<0.05）. Compared with the model group， medium-dose Bushen Jianpi Jiedu Liyan formula down-regulated the protein levels of SDF-1 and CXCR4 in the intestinal lamina propria （P<0.05）. Compared with the blank group， the model group showed down-regulated mRNA levels of α4 β1 and SDF-1 and up-regulated mRNA levels of VCAM-1 and CXCR4 （P<0.05）. Compared with the model group， the drug intervention groups showed down-regulated mRNA levels of SDF-1 and CXCR4 （P<0.05）. ConclusionBushen Jianpi Jiedu Liyan formula regulates the expression of α4 β1， VCAM-1， SDF-1， and CXCR4 in the intestinal lamina propria to inhibit the homing effect of plasma cells， which may be associated with the Toll-like receptor-mediated activation of immune response. Bushen Jianpi Jiedu Liyan formula can down-regulate the expression of adhesion molecules to inhibit the proliferation of plasmocytes in circulation， so as to reduce the renal injury of IgA nephropathy.

Relationship between DDX46 gene and invasion and metastasis behaviors of TE-1 esophageal squamous cell carcinoma cells / 中国胸心血管外科临床杂志

@#Objective    To investigate the relationship between DDX46 genes and invasion and migration of esophageal squamous cell carcinoma cells. Methods    Human esophageal squamous cell carcinoma cells TE-1 were transfected by fluorescent marker shRNA lentivirus (shDDX46 group), and an empty vector was transfected as a control (shCtrl group). The expression rate of green fluorescent protein under the microscope was used to evaluate the cell transfection efficiency. Real-time fluorescence quantitative polymerase chain reaction (RTFQ-PCR) and Western blotting (WB) detected the knockdown efficiency of the target gene at the mRNA and protein expression levels. Wound healing, invasion assay and migration assay detected the changes of invasion and metastasis ability. Classical pathway analysis was used to explore signaling pathway changes and the possible mechanism of DDX46 in the invasion and metastasis was explored by detecting fibronectin expression. Results    DDX46 gene at mRNA and protein levels was significantly inhibited after lentiviral transfection. Wound healing showed that after 8 h the cell mobility of TE-1 cells decreased significantly (P=0.001). Invasion assay showed that after 24 h the average cell metastasis rate of TE-1 cells was lower in the shDDX46 group than that in the shCtrl group (P<0.001). The cell metastasis rate in the shDDX46 group corresponding to observation points in the transwell assay was lower than that in the shCtrl group (P<0.001) after 24 h culture. The results of the classical pathway analysis showed that the integrin signaling pathway activity was inhibited, further exploration of the mechanism of action found that the expression of fibronectin associated with cell adhesion was decreased. Conclusion    DDX46 gene is related to the invasion and migration ability of esophageal squamous cell carcinoma cells. Knockdown of DDX46 genes may reduce cell adhesion by downregulating the integrin pathway signaling.

Research progress of targeted CD47 in the treatment of bladder cancer / 中华泌尿外科杂志

CD47 is a transmembrane glycoprotein widely expressed on cells and an important signal molecule for immune escape of tumor cells. CD47, which is highly expressed in bladder cancer cells, can interact with signal regulatory proteins on the surface of macrophages- α (SIRPα). It combines and transmits immunosuppressive signals to protect tumor cells from phagocytosis, thereby mediating their immune escape. CD47-SIRPα signal pathways have become the focus of tumor cell immune checkpoint research at this stage. This article reviewed the research progress in the mechanism and clinical value of CD47 in bladder cancer.

Thyroxine promotes the progression of integrin α vβ 3-positive differentiated thyroid cancer through the ERK1/2 pathway / 中华核医学与分子影像杂志

Objective:To explore whether thyroxine (T 4) could promote differentiated thyroid cancer (DTC) progression by binding to integrin α vβ 3in vitro and its downstream mechanism. Methods:Papillary thyroid cancer cell lines TPC-1, K1 and follicular thyroid cancer (FTC) cell line FTC133 were cultured in vitro, and the expressions of integrin α vβ 3 in those 3 DTC cell lines were determined with immunofluorescence and flow cytometry analysis. After the treatment of T 4, tetraiodo thyroacetic acid (Tetrac) and Arg-Gly-Asp (RGD) peptide alone or in combination, the proliferation and metastatic potential of DTC cell lines were detected by cell counting kit-8 (CCK-8), Transwell migration and invasion assays. The small interfering RNA (siRNA) transfection was used to verify whether integrin α v or β 3 subunit knockdown could reverse the effect of T 4 on DTC cells. The expression levels of downstream signaling proteins phosphorylated extracellular signal-regulated kinase (p-ERK)1/2 and total extracellular signal-regulated kinase (ERK)1/2 were detected by Western blot. The effects of mitogen-activated protein kinase kinase (MEK)1/2 inhibitor (GSK1120212) on the proliferation, migration and invasion of T 4-treated cells were detected. One-way analysis of variance and Tukey test were used for data analysis. Results:The integrin α vβ 3 expressions in TPC-1, K1 and FTC133 cells were all positive, with the relative mean fluorescence intensity (MFI) of 61.93±18.61, 16.89±2.43 and 32.36±0.83, and the percentages of positive cells of (94.38±1.30)%, (74.11±3.87)% and (50.67±1.78)%, respectively ( F values: 13.36 and 217.30, P=0.006 and P<0.001). Compared with control group, the proliferation, migration and invasion in the three DTC cell lines treated with T 4 were significantly enhanced (96 h, F values: 62.67-297.50, q values: 13.15-20.73, all P<0.001). T 4-induced cell proliferation, migration and invasion were markedly reversed by Tetrac or RGD (96 h, q values: 8.61-17.54, all P<0.001). T 4-induced cell proliferation, migration and invasion were also significantly inhibited by the knockdown of integrin α v or β 3 subunit (72 h, F values: 7.75-70.98, q values: 4.77-15.21, all P<0.05). Western blot results showed that the phosphorylation levels of ERK1/2 in DTC cells were significantly increased by T 4 treatment, and the T 4-induced activation of ERK1/2 signaling pathway could be blocked by Tetrac, RGD, integrin α v or β 3 subunit knockdown. T 4-induced cell proliferation, migration and invasion were significantly reversed by GSK1120212 (96 h, F values: 47.53-151.40, q values: 10.32-16.65, all P<0.001). Conclusion:T 4 can promote cell proliferation and metastasis of DTC cells by binding to integrin α vβ 3 and activating the ERK1/2 pathway.

Roles of focal adhesion proteins in skeleton and diseases

The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection of internal organs, production of blood cells and regulation of calcium and phosphate metabolism. The prevalence of skeletal diseases and disorders, such as osteoporosis and bone fracture, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, increases with age, causing pain and loss of mobility and creating a huge social and economic burden globally. Focal adhesions (FAs) are macromolecular assemblies that are composed of the extracellular matrix (ECM), integrins, intracellular cytoskeleton and other proteins, including kindlin, talin, vinculin, paxillin, pinch, Src, focal adhesion kinase (FAK) and integrin-linked protein kinase (ILK) and other proteins. FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell-environment communications and modulates important processes, such as cell attachment, spreading, migration, differentiation and mechanotransduction, in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways. This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.

RGD peptide inhibits tumor growth by affecting angiogenesis related signaling pathway of laryngeal cancer stem cells / El péptido RGD inhibe el crecimiento tumoral al afectar la vía de señalización relacionada con la angiogénesis de las células madre del cáncer de laringe

SUMMARY: This study is to investigate the role and mechanism of RGD peptide in laryngeal cancer stem cells (CSCs). Laryngeal cancer CD133+Hep-2 CSCs were sorted by flow cytometry. RGD peptide was co-cultured with sorted laryngeal CSCs. Cell proliferation was detected with CCK-8 assay. The mRNA levels of VEGF/VEGFR2/STAT 3/HIF-1α were detected with RT-PCR. The proteins of VEGF/ VEGFR2/STAT 3/HIF-1α were detected with Western blot. The sorted CSCs were inoculated into nude mice. Tumor volume was measured. Integrin αvβ3 expression in tumor tissues was analyzed with immunohistochemistry. The results showed that the ratio of CD133+ CSCs to the total number of cells was 1.34±0.87 %, while CD133-non-tumor stem cells accounted for 95.0±5.76 %. The sorted cancer stem cells grew well. The RGD peptide significantly inhibited the proliferation of CD133+Hep-2 laryngeal CSCs in a dose-dependent manner. The RGD peptide significantly inhibited the mRNA of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a concentration-dependent manner. Consistently, the RGD peptide significantly inhibited the protein expression of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a dose-dependent manner. At the same time, in vivo tumor experiments showed that the RGD peptide significantly inhibited tumor volume but not the body weight. Furthermore, RGD peptide significantly inhibited the expression of tumor angiogenesis-related protein integrin αvβ3. Our findings demonstrate that RGD peptide inhibits tumor cell proliferation and tumor growth. The underlying mechanism may that RGD inhibits tumor angiogenesis-related signaling pathways, thus affecting the tumor angiogenesis, and decreasing the progression of human laryngeal CSCs.

Este estudio se realizó para investigar el papel y el mecanismo del péptido RGD en las células madre del cáncer de laringe (CSC). Las CSC CD133+Hep-2 de cáncer de laringe se clasificaron mediante citometría de flujo. El péptido RGD se cocultivó con CSC laríngeas clasificadas. La proliferación celular se detectó con el ensayo CCK-8. Los niveles de ARNm de VEGF/VEGFR2/ STAT 3/HIF-1α se detectaron con RT-PCR. Las proteínas de VEGF/ VEGFR2/STAT 3/HIF-1α se detectaron con Western blot. Las CSC clasificadas se inocularon en ratones nudos. Se midió el volumen del tumor. La expresión de integrina αvβ3 en tejidos tumorales se analizó con inmunohistoquímica. Los resultados mostraron que la proporción de CSC CD133+ con respecto al número total de células fue de 1,34 ± 0,87 %, mientras que las células madre no tumorales CD133 representaron el 95,0 ± 5,76 %. Las células madre cancerosas clasificadas crecieron bien. El péptido RGD inhibió significativamente la proliferación de CSC laríngeas CD133+Hep-2 de una manera dependiente de la dosis. El péptido RGD inhibió significativamente el ARNm de VEGFR2, STAT3 y HIF-1α en CSC laríngeas de manera dependiente de la concentración. De manera consistente, el péptido RGD inhibió significativamente la expresión proteica de VEGFR2, STAT3 y HIF-1α en CSC laríngeas, de manera dependiente de la dosis. Al mismo tiempo, los experimentos con tumores in vivo mostraron que el péptido RGD inhibía significativamente el volumen del tumor pero no el peso corporal. Además, el péptido RGD inhibió significativamente la expresión de la proteína integrina αvβ3 relacionada con la angiogénesis tumoral. Nuestros hallazgos demuestran que el péptido RGD inhibe la proliferación de células tumorales y el crecimiento tumoral. El mecanismo subyacente puede ser que RGD inhiba las vías de señalización relacionadas con la angiogénesis tumoral, afectando así la angiogénesis tumoral y disminuyendo la progresión de las CSC laríngeas humanas.

Study on the correlation of integr in associated protein and disease activity and bone destruction in patients with rheumatoid arthritis / 中华风湿病学杂志

Objective:To analyze the serum levels of integrin-associated proteins (CD47) in patients with rheumatoid arthritis (RA), and to explore its association with disease activity and bone destruction in RA.Methods:Serum and clinical data were collected from 65 RA patients and 25 healthy subjects. RA patients were grouped into low, moderate, and high bone erosion groups according to 7-joint ultrasonography score (US7). The levels of serum CD47, thrombospondin-1 (TSP-1) and receptor activator of nuclear factor-κB ligand (RANKL) were measured by enzyme-linked immunosorbnent assay (ELISA) in patients with RA and healthy subjects. The statistical analysis was carried out with independent t-test, analysis of variance, nonparametric rank sum test, pearson or Spearman correlation and logistic regression. Results:① The Serum levels of CD47, TSP-1, and RANKL were higher in the RA group than in the healthy controls ( P<0.01). ② In RA patients, serum CD47 level was positively correlated with disease course ( r=0.301, P<0.05), C-reactionprotein (CRP)( r=0.316, P<0.05), number of tender joints (TJC) ( r=0.254, P<0.05), number of swollen joints (SJC) ( r=0.316, P<0.05), disease activity score in 28 joints (DAS28) ( r=0.255, P<0.05), RANKL ( r=0.252, P<0.05) and TSP-1 ( r=0.260, P<0.05). Serum TSP-1 level was positively correlated with CRP ( r=0.299, P<0.05), TJC ( r=0.335, P<0.01), DAS28 ( r=0.315, P<0.05), RANKL ( r=0.305, P<0.05). ③ The disease course [ OR(95% CI)=1.048(1.033, 1.017)] and TSP-1 [ OR(95% CI)=1.013(1.000, 1.026)] were independently relevant factors affecting bone destruction. Conclusion:CD47 levels is significantly higher in RA patients than in healthy controls, and is associated with disease activity and bone destruction. CD47 may be involved in the bone destruction process of RA by acting on TSP-1.

Effect of "patellar pushing and knee extension" manipulation on integrin β1 and p-FAK in articular cartilage of rabbit model of knee osteoarthritis / 国际生物医学工程杂志

Objective:To observe the effects of "pushing patella and extending knee" manipulation on the protein and mRNA expression levels of integrin β1 (ITGβ1) and phosphorylated adhesion plaque kinase (p-FAK) in rabbit knee osteoarthritis (KOA) model, and to investigate the mechanism of manipulations in the treatment of KOA.Methods:Twenty healthy 6-month-old New Zealand rabbits were divided into the normal group, the model group, the acupuncture group, and the manipulation group according to the random number table method. Among them, the model group, the acupuncture group, and the manipulation group were modeled using the modified Hulth method for KOA. After 7 d of successful modeling, the normal group and the model group did not receive any intervention, while the acupuncture group and the manipulation group received one acupuncture intervention and one "pushing patella and extending knee" manipulation intervention daily, respectively. After 2 weeks of treatment, the rabbit KOA model was executed by air embolization, and the protein and mRNA expression levels of ITGβ1 and p-FAK in knee cartilage were measured by Western blot and real-time polymerase chain reaction (PCR), respectively.Results:Compared with the normal group, the ITGβ1 protein expression level was decreased ( P<0.05) and p-FAK protein expression level was increased ( P<0.05), and the mRNA expression levels of ITGβ1 and p-FAK did not change significantly (all P>0.05) in the model group. Compared with the model group, the ITGβ1 protein expression level was increased ( P<0.05), the p-FAK protein expression level decreased ( P<0.05), and the mRNA expression levels of both ITGβ1 and p-FAK increased (all P<0.05) in the acupuncture group. Compared with the acupuncture group, ITGβ1 protein expression level increased ( P<0.05), p-FAK protein expression level decreased ( P<0.05), and mRNA expression levels of both ITGβ1 and p-FAK increased (all P<0.01) in the manipulation group. Conclusions:The "pushing patella and extending knee" manipulation can optimize the protein and mRNA expression levels of ITGβ1 and p-FAK in the articular cartilage of the rabbit KOA model.

Expression and Role of Integrin β-like 1 in Hepatocellular Carcinoma / 肿瘤防治研究

Objective To investigate the expression of ITGBL1 in hepatocellular carcinoma (HCC) and its role in promoting the proliferation, migration, and invasion of HCC cell lines. Methods RT-qPCR and immunohistochemistry were used in investigating ITGBL1 expression in 12 pairs of fresh HCC and adjacent normal liver tissue samples and 160 paraffin HCC specimens. The relationships of ITGBL1 expression level with clinicopathological parameters and prognosis were analyzed. HUH7 cell line with the stable overexpression of ITGBL1 and LM3 cell line with stable downregulated expression of ITGBL1 were constructed. The effects of ITGBL1 on the proliferation, migration, and invasion of HCC cells were examined by CCK8 assay, wound-healing assay, and Transwell invasion assay. Results The expression levels of ITGBL1 gene and protein in tumor tissues were higher than those in surrounding liver tissues. The high expression of ITGBL1 was correlated with serum AFP level, tumor capsular invasion, vascular invasion, tumor differentiation, and clinical stage (P < 0.05). The results of Kaplan-Meier analysis showed that patients with high expression of ITGBL1 had shorter DFS. In vitro, increased ITGBL1 expression promoted HCC cell proliferation, migration, and invasion, whereas ITGBL1 knockdown inhibited these processes. Conclusion ITGBL1 expression is highly expressed in HCC tissues, and ITGBL1 can increase the proliferation, migration, and invasion of HCC cells. However, the mechanism needs further study.

Avaliação da carga microbiana e fúngica de sistemas de canais radiculares infectados e a expressão apical de integrinas, citocinas e quimiocinas nos tecidos / Assessment of the microbial and fungal load of infected root canal systems and the apical expression of integrins, cytokines and chemokines in adjacent perirradicular tissues

Este estudo busca avaliar quantitativamente a expressão do RNA mensageiro (mRNA) das integrinas alfa1, alfa4, alfa 5, alfa L, citocinas e quimiocinas, a partir de células presentes no líquido intersticial periapical adjacente a dentes com infecção do canal radicular. Foram selecionados 22 indivíduos com necessidade de tratamento endodôntico e encaminhados à Faculdade de Odontologia da Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brasil). As amostras foram coletadas em 11 dentes necróticos e portadores de infecções endodônticas e 11 dentes hígidos que necessitavam de tratamento endodôntico por motivos protéticos. Após a cirurgia de acesso e antes dos procedimentos de limpeza e modelagem do sistema de canais radiculares (T0), imediatamente após a limpeza e formatação do sistema de canais radiculares(T1), em 7 (T2) e 14 dias (T3), um cone de papel esterilizado endodôntico # 20 foi inserido no SCR, mantido por 2 min, e posteriormente armazenado a -70°C. Real-Time PCR analisou microbiologicamente essas amostras para ler a expressão gênica do rRNA microbiano 16S e fragmentos da região ITS do gDNA fúngico da espécie Candida. Após os procedimentos de limpeza e formatação do SCR, três cones de papel absorvente esterilizados foram inseridos. Passivamente, a ponta do papel ultrapassou o ápice radicular em 2 mm e permaneceu por 2 minutos. As amostras foram coletadas imediatamente após a limpeza e modelagem do RCS, 7 e 14 dias após a primeira sessão. As pontas de papel tiveram os 4 mm finais cortados, inseridos em Eppendorf e armazenados a - 70°C. Com este procedimento, o RNA foi extraído do líquido intersticial periapical para caracterizar as expressões dos genes ITGA1, ITGA4, ITGA5, ITGAL, IL-1ß, TNF-α, IL-17A, IL-10, IFN-γ, CCL2/MCP-1, CCL5, CXCR4, e 16S usando PCR em tempo real. O DNA genômico (gDNA) foi extraído para se avaliar a abundância de Candida utilizando-se sequências ITS, por PCR em tempo real. Os resultados demonstraram que os níveis de expressão de mRNA do 16S diminuíram após os procedimentos de limpeza e modelagem e que a abundância de Candida foi insipiente na amostra analisada. As citocinas pro- inflamatórias IL-1ß e IL-17 apresentaram níveis de expressão elevados frente a infecção, reduzindo significativamente após os procedimentos de limpeza e formatação. Os níveis de expressão gênica de TNF-α significantemente aumentaram, em ambos os grupos. Não se observou diferença significativa quanto a expressão gênica das citocinas IFN-γ, IL-10, CCL-2 e CCL-5 e das integrinas ITGAL e ITGA5 nos tempos avaliados. A expressão gênica de CXCR4 reduziu significativamente do tempo T1 para o T2, no grupo experimental. As expressões gênicas de ITGA1 e ITGA4, no grupo experimental, reduziram significativamente de maneira tempo dependente. Finalmente, não houve alteração significativa na expressão de marcadores de macrófagos (CD64), enquanto expressão de marcadores de fibroblastos (S100A4) aumentou significativamente no grupo controle. Concluiu-se que a carga microbiana e a abundância de leveduras correlacionam-se positivamente com a expressão de mediadores pró-inflamatórios e que a que terapia endodôntica negativamente impacta a expressão dos mediadores pró-inflamatórios e das integrinas nos tecidos perirradiculares.

This study seeks to quantitatively evaluate the expression of messenger RNA (mRNA) of integrins alpha1, alfa4, alpha 5, alpha L, cytokines, and chemokines, from cells present in the periapical interstitial fluid adjacent to teeth with root canal infection. Twenty-two individuals needing endodontic treatment and referred to the School of Dentistry of the Federal University of Minas Gerais (Belo Horizonte, MG, Brazil) were selected. The samples were collected in 11 necrotic teeth and carriers of endodontic infections and 11 healthy teeth needing endodontic treatment for prosthetic reasons. After access surgery and before root canal system (RCS) cleaning and shaping procedures (T0), immediately after cleaning and shaping the root canal system (T1), in 7 (T2) and 14 days (T3) an endodontic sterilized paper point #20 was inserted into the RCS, maintained for 2 min, and subsequently stored at -70°C. Real-Time PCR microbiologically analyzed these samples to read the gene expression of microbial rRNA 16S and fragments of the ITS region of the Fungus Candida species gDNA. After RCS cleaning and shaping procedures, three sterilized absorbent paper cones were inserted. Passively, the paper point exceeded the root apex by 2 mm and remained for 2 minutes. Samples were collected immediately after RCS cleaning and shaping, 7 and 14 days after the first session. The paper points have the 4 mm of their tip cut, inserted in Eppendorf, and stored at - 70°C. This procedure extracted RNA from the periapical interstitial fluid to characterize the expressions of the genes ITGA1, ITGA4, ITGA5, ITGAL, IL-1ß, TNF- α, IL-17A, IL-10, IFN-γ, CCL2/MCP-1, CCL5, CXCR4, ITS using Real-Time PCR. The results showed that 16S mRNA expression levels decreased after cleaning and modeling procedures and that Candida abundance was incipient in the analyzed sample. Pro-inflammatory cytokines IL-1ß and IL-17 showed high expression levels against infection, significantly reduced after cleaning and formatting procedures. TNF-α gene expression levels significantly increased in both experimental and control groups. No significant difference was observed regarding the gene expression of the cytokines IFN-γ, IL-10, CCL-2, and CCL-5 and the integrins ITGAL and ITGA5. The gene expressions of ITGA1 and ITGA4 in the experimental group were significantly reduced time-dependent. Finally, there was no significant change in their macrophage markers (CD64) expression, while fibroblast markers (S100A4) expression significantly increased in the control group. It was concluded that microbial load and yeast abundance are positively correlated with the expression of pro-inflammatory mediators and that endodontic therapy negatively impacts the expression of pro-inflammatory mediators and integrins in periradicular tissues.

Preservation of immunoexpression of type I collagen, BSP and BMP4 in the dentin-pulp complex of head and neck cancer patients after radiotherapy

Abstract This study tested the hypothesis that head and neck radiotherapy (HNRT) impacts the immunoexpression of type I collagen, bone sialoprotein (BSP) and bone morphogenetic protein 4 (BMP4), thereby leading to micromorphological changes in the dentin-pulp complex (DPC), and promoting the onset and progression of radiation caries (RC). Twenty-two demineralized sections of carious teeth (a group of 11 irradiated teeth and a control group of 11 non-irradiated teeth) extracted from 19 head and neck cancer patients were analyzed by conventional optical microscopy and immunohistochemistry to investigate the micromorphology (cellular layer hierarchy, blood vessels, odontoblasts, fibroblasts, extracellular matrix, calcification, necrosis, reactionary dentin formation, and chronic inflammation), and the patterns of staining/immunolocalization of type I collagen, BSP and BMP4 in the dental pulp of irradiated and control samples. No significant differences attributable to the direct impact of radiotherapy were detected in DPC micromorphology between the groups. In addition, the patterns of immunohistochemical staining and immunolocalization of the proteins studied did not differ between the irradiated and the control samples for type I collagen, BSP or BMP4. This study rejected the hypothesis that HNRT directly damages dentition by changing the organic components and the microstructure of the DPC, ultimately leading to RC.

Differential Regulation of Integrin α5 and ß4 in Normal and Psoriatic Epidermal Keratinocytes

Abstract Psoriasis is a chronic skin inflammation, characterized by impaired differentiation, hyperproliferation of keratinocytes involving pro-inflammatory factors interleukin (IL)-13/17A, tumor necrosis factor (TNF)-α, interferon (IFN)-γ. Among the integrin family, α5 is important for blood vessel formation, and ß4 for proliferation, differentiation of keratinocytes. To investigate the expression and regulation of integrin α5 and ß4 in psoriatic keratinocytes. Skin biopsies were obtained from 14 psoriatic patients and 12 normal volunteers. We compared the immunolocalization and regulation of α5 and ß4 between the psoriatic and normal ones, before and after incubation with MEK/ERK pathway inhibitor U0126 by immunohistochemistry and western blot separately. Immunohistochemistry showed psoriatic keratinocytes had higher α5 than normal ones. According to western blot, IL-17A and IL-13 increased normal keratinocytes' α5 and ß4 respectively, but psoriatic keratinocytes were the exact opposite. Incubated with U0126, normal keratinocytes' α5 was enhanced by the 5 cytokines ; while IL-13/17A, IFN-γ suppressed ß4. Psoriatic keratinocytes' α5 was increased by IL-13/17A, decreased by IFN-γ; but ß4 increased by IL-17A, IFN-γ. IL-13/17A, TNF-α, IFN-γ regulate α5 and ß4 through ERK pathway whether normal or psoriasis. The normal and psoriatic keratinocytes respond to the same cytokines differently

MiR-4484 regulates the expression of integrin α 6 in gastric cancer tissues and its significance / 中华肿瘤杂志

Objective: To investigate the relationship between the expression of integrin α 6 (ITGA6), miR-4484 and the pathologic stage of gastric cancer. Methods: Gastric cancer tissues and normal gastric mucosa tissues adjacent to cancer (>5 cm from tumor margin) of 30 patients with primary gastric cancer who underwent direct surgical resection without adjuvant therapy from June to September 2017 in West China Hospital of Sichuan University were selected. Real-time quantitative polymerase chain reaction (PCR) was used to detect the expression levels of miR-4484 and ITGA6, western blot was used to detect the expression level of ITGA6 protein, dual luciferase reporter gene was used to verify the relationship between ITGA6 and miR-4484. Spearman's correlation analysis was used to determine the relationship between miR-4484 and ITGA6 expression levels in gastric cancer tissues. Results: The expression level of ITGΑ6 in gastric cancer (32.30±13.47) was higher than that in matched normal gastric tissues (24.55±10.25, P=0.015), the area under the receiver operating characteristic (ROC) curve was 0.660 and the diagnostic sensitivity and specificity were 43.3% and 96.7%, respectively. The expression level of miR-4484 in gastric cancer (4.11±2.87) was lower than that of matched normal gastric tissues (5.75±2.80, P=0.029), the area under the ROC curve was 0.690 and the diagnostic sensitivity and specificity were 30.0% and 86.7%, respectively. The expression level of miR-4484 was negatively correlated with ITGA6 in gastric cancer tissues (r=-0.621, P<0.001). The expression level of ITGA6 protein in gastric cancer tissues (0.65±0.19) was higher than that in normal adjacent tissues (0.26±0.12, P<0.001). Compared with ITGA6 3'UTR wild-type+ miR-NC group, ITGA6 3'UTR wild-type+ miRNA mimics group had lower luciferase activity (50.69±5.10, 34.00±1.19, P<0.001), while the luciferase activity of ITGA6 3'UTR wild-type+ ASO miR-4484 group was higher than that of ITGA6 3'UTR wild-type+ miR-NC group (82.44±6.37, 50.69±5.10, P<0.001), indicated that ITGA6 was the direct target gene of miR-4484. The expression levels of miR-4484 in T1, T2, T3 and T4 (4a and 4b) gastric cancer tissues were 9.98±2.24, 5.28±2.03, 2.92±2.04 and 4.11±2.87, respectively, with statistical significance (P<0.001). The expression levels of ITGA6 in N0, N1, N2 and N3 gastric cancer tissues were 29.55±8.32, 21.71±3.75, 24.60±8.79 and 40.69±15.83, respectively, with statistical significance (P=0.022). The expression levels of miR-4484 in N0, N1, N2 and N3 gastric cancer tissues were 5.01±3.52, 5.48±2.76, 5.88±1.83 and 2.30±1.56, respectively, with statistical significance (P=0.032). The expression levels of ITGA6 in M0 and M1 gastric cancer tissues were 26.28±7.66 and 52.08±8.12, respectively, with statistical significance (P<0.001). The expression levels of miR-4484 in M0 and M1 gastric cancer tissues were 4.95±2.74 and 1.34±0.80, respectively, with statistical significance (P<0.001). Conclusions: ITGA6 is upregulated in gastric cancer tissues, while miR-4484 is downregulated in the gastric cancer group, and its expression level is related to the clinicopathological features of gastric cancer. ITGA6 is the direct target gene of miR-4484, implicates that miR-4484 may inhibit the invasion and metastasis of gastric cancer by regulating the expression of ITGA6. Both miR-4484 and ITGA6 may be the new prognostic markers and potential therapeutic targets of gastric cancer.

Experimental study of vascular endothelial growth factor receptor 2 and integrin α vβ 3 dual-targeted microbubble to assess tumor angiogenesis of renal cell carcinoma in vivo / 中华超声影像学杂志

Objective:To evaluate the ability of vascular endothelial growth factor receptor 2(VEGFR2)/integrin α vβ 3 dual-targeted microubble (MBD) to target angiogenesis of renal cell carcinoma (RCC) in vivo. Methods:Non-targeted microbubble (MBN) USphere LA was employed as a template to prepare single- and dual-targeted microbubbles which could bind VEGFR2 and/or integrin α vβ 3 (MBV and MBI) by the biotin-avidin bridging method. A total of 40 RCC nude mice models were established by subcutaneously injecting 786-O cells.Twenty of the models were all injected with MBN, MBV, MBI and MBD in a random order, and the other 20 models were registered for antibody blocking assays. The results of ultrasound images were used for quantitative analyses, and the following quantitative parameters were obtained: intensity increment (a 1), peak halving speed (a 2), curve rising slope (a 3), perfusion time (t 0), time to peak (TTP), peak intensity (PI), mean transit time (MTT) and area under the curve (AUC) for the first three minutes, peak intensity at 10 s before (P 1) and after (P 2) ultrasound destruction, and the differences of tissue enhancement (dTE) between P 1 and P 2 (dTE=P 1-P 2). All the quantitative parameters of four contrast agents and the antibody blocking assays were compared.Besides, the immunohistochemical assays were performed to evaluate the expression of CD31, VEGFR2 and integrin α vβ 3 in tumor tissues. Results:The differences of parameters of a 1, a 3, t 0, TTP, PI and P 2 among four different microbubbles had no statistical significances (all P>0.05), and all parameters between the two single-targeted contrast agents were not statistically different (all P>0.05). The parameters of AUC, MTT, P 1 and dTE all showed a trend that dual-targeted bubbles > single-targeted bubbles > non-targeted bubbles (all P<0.05). On the contrary, the trend of dual-targeted bubbles < single-targeted bubbles < non-targeted bubbles (all P<0.05) was observed for a 2. In the antibody blocking experiment, a 2 was faster after the antibody injection ( P<0.001), while AUC, MTT, P 1 and dTE were all lower than those before the antibody injection ( P<0.001), and the other parameters were not statistically different before and after the antibody injection (all P>0.05). Immunohistochemical analyses confirmed the high expression of CD31, VEGFR2 and integrin β 3 in tumor tissues. Conclusions:The VEGFR2 and integrin α vβ 3 dual-targeted microbubble has a good potential to target the angiogenesis of human RCC in vivo.

Inhibitory effect of ILK-siRNA-AAV on scar formation after glaucoma filtering surgery in rats / 中华实验眼科杂志

Objective:To investigate the role of integrin-linked kinase (ILK)-small interfering RNA (siRNA)-adeno-associated virus (AAV) in scar formation after glaucoma filtering surgery in rat eyes.Methods:Forty-eight Sprague Dawley rats of SPF grade, aged 8 to 9 weeks old, were selected and divided into blank control group, ILK-siRNA-AAV group, NC-siRNA-AAV group and mitomycin C (MMC) group by random number table method, with 12 rats in each group.Left eyes of the rats were taken as experimental eyes, and no intervention was administered to fellow eyes.The bulbar conjunctival filtering bleb after glaucoma filtration surgery in rats was established by anterior chamber drainage tube implantation.One day after operation, phosphate buffer solution, ILK-siRNA-AAV, and NC-siRNA-AAV were injected into the filtering bleb of blank control group, ILK-siRNA-AAV group and NC-siRNA-AAV group, 5 μl each group, respectively.Cotton tablets containing 0.4 mg/ml MMC were placed under conjunctival flap for 5 minutes during operation in MMC group.Intraocular pressure (IOP) was measured with a handheld tonometer before surgery and at 1, 2, 3, 7, 14, 21, 28 days after surgery.Formation of filtering blebs in rats was observed with a surgical microscope at 1, 2, 3, 7, 14, 21, 28 days after operation, and the bleb survival time was calculated by Kaplan-Meier survival analysis.The mRNA and protein expression levels of ILK in conjunctival and subconjunctival tissues at the surgical sites were detected by reverse transcription PCR and Western blot, respectively, on the 28th day after operation.Silencing of ILK gene was identified.Effect of ILK gene silencing on the morphology of drainage pathway was observed by hematoxylin-eosin staining.Effect of ILK gene silencing on collagen fiber deposition in the bulbar conjunctiva at filtration area was examined by Masson staining, and the percentage of positive area of collagen fiber staining in the total tissue visual field was calculated.The use and care of the animals complied with the ARVO Statement.This research protocol was approved by an Ethics Committee of Xi'an Jiaotong University (No.2013-772). Results:There were statistically significant differences in IOP at different time points between before and after surgery among four groups ( Fgroup=76.84, P<0.001; Ftime=114.49, P<0.001). The IOP of ILK-siRNA-AAV group on the 1st, 7th, 14th and 28th day after operation and the IOP of MMC group on the 2nd, 3rd, 7th, 14th and 28th day after operation were lower than those of blank control group, and the differences were statistically significant (all at P<0.05). The IOP of ILK-siRNA-AAV group was lower on 7th, 14th, 21st and 28th day after operation than those of NC-siRNA-AAV group, with statistically significant differences (all at P<0.05). The bleb survival time of blank control group, NC-siRNA-AAV group, ILK-siRNA-AAV group and MMC group was (3.50±1.51), (5.00±3.41), (31.50±3.15) and (31.33±2.46) days, respectively, with a significant difference among them ( F=395.83, P<0.05). The bleb survival time of ILK-siRNA-AAV group and MMC group was higher than that of blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). There were statistically significant differences in the relative expression levels of ILK mRNA and protein among four groups ( F=222.32, 752.69; both at P<0.05), and the relative expression levels of ILK mRNA and protein were significantly lower in ILK-siRNA-AAV group than blank control group and NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Proliferative fibrous connective tissue and a large number of cells at surgical sites were found in blank control group and NC-siRNA-AAV group, and the fibroblasts were of a high density and grew in clumps.In ILK-siRNA-AAV group, the bulbar conjunctiva was thin, and the arrangement of fibrous connective tissue was loose, and a few proliferative fibroblasts were found.In MMC group, the conjunctival fibrous layer was loose and thin, forming cavities, and scarce cells were found.There was statistically significant difference in the percentage of collagen fiber positive staining area among four groups ( F=741.66, P<0.05). The positive staining percentage of ILK-siRNA-AAV group and MMC group was significantly lower than that of blank control group, among which there was lower positive staining percentage in ILK-siRNA-AAV group than NC-siRNA-AAV group, and the differences were statistically significant (all at P<0.05). Conclusions:Silencing of ILK can inhibit the scar formation after glaucoma filtering surgery and maintain low IOP in rats.

Clinical study of endoscopic surgery through thoraco mammary approach in treatment of thyroid papillary carcinoma / 中华内分泌外科杂志

Objective:To investigate the effect of endoscopic thyroidectomy through breast milk approach in patients with papillary thyroid carcinoma and its influence on Wnt and integrin signaling pathways.Methods:A total of 136 patients diagnosed with papillary thyroid carcinoma in our hospital from Jul. 2018 to Mar. 2020 were selected and were hospitalized for surgical treatment. According to different surgical procedures, they were divided into a study group (68 cases) and a control group (68 cases) . The control group was treated with open thyroidectomy and the study group was treated with thoracoscopic thyroidectomy. The two groups were compared in terms of immune function [CD4+, CD8+ and CD4+/CD8+], and pain index [PGE2, IL-6, Cor and VAS score]. RT-PCR method was used to detect WNT1, β-catenin, GSK3β and integrin Signal pathway before and after surgery.Results:Three days after operation, compared with the control group, the study group had significantly higher CD4+ and CD4+/CD8+ levels [ (27.62±2.52) vs (24.63±2.67) , (0.66±0.18) vs (0.52±0.13) ], while the CD8+ level was significantly lower [ (41.62±3.54) vs (45.62±3.63) ] ( P<0.001) ; PGE2, IL-6, Cor, VAS of the study group were significantly lower than the control group [ (48.54±9.86) vs (57.21±8.12) , (5.13±0.71) vs (6.99±0.95) , (511.23±67.52) vs (633.12±71.47) , (1.26±0.56) vs (3.99±2.06) ] ( P<0.001) ; WNT1, β-catenin, GSK3β, integrin β1, FAK, Ras, and MAPK mRNA expression levels in the study group were significantly lower than those of the control group[ (1.79±0.15) vs (2.85±0.25) , (1.94±0.15) vs (2.64±0.24) , (2.13±0.19) vs (2.97±0.28) , (1.95±0.17) vs (2.58± 0.23) , (2.15±0.16) vs (2.87±0.22) , (1.95±0.18) vs (2.91±0.27) , (1.89±0.12) vs (2.87±0.31) ] ( P<0.001) . Conclusion:Endoscopic thyroidectomy through thoracolumbar approach can effectively reduce postoperative pain in patients with papillary thyroid cancer, have a smaller impact on immune function, and block the expression of Wnt and integrin signaling pathways to reduce tumor metastasis risk.

Main Factors Influencing the Platelet Spreading / 中国实验血液学杂志

OBJECTIVE@#To explore the main factors of platelet spreading and provide the foundation for related research.@*METHODS@#Platelets (2×107/ml) were draw from C57BL/6J mouse and kept at 22 ℃ for 1-2 hours. Platelets (2×107/ml) were were allowed to adhere and spread on the fibrinogen-coated slides, after staining F-actin in platelets, the platelets were observed with the confocal microscopy. The effects of different concentrations of fibrinogen (10 μg/ml, 30 μg/ml, 100 μg/ml) and kinds of agonists ［thrombin(0.01,0.05,0.1 U/ml), ADP（5,10,20 μmol/L）, U46619（0.125,0.25,0.5 μmol/L）］ on platelets were analyzed. The platelet spreading was successful if the spreading rate was higher after treated with agonists.@*RESULTS@#Compared to the group which coated with 10 μg/ml and 100 μg/ml fibrinogen, the platelet density is optimal when coated with 30 μg/ml fibrinogen. In addition, under the stimulation of thrombin, ADP and U46619, the spreading rate of platelets showed a certain concentration-dependent increasing.@*CONCLUSION@#The platelet spreading is easily influenced by various factors, the platelet spreading can be induced successfully at 0.1 U/ml thrombin, 20 μmol/L ADP and 0.5 μmol/L U46619 on the slide coated with 30 μg/ml fibrinogen.

Super-sensitive bifunctional nanoprobe: Self-assembly of peptide-driven nanoparticles demonstrating tumor fluorescence imaging and therapy

The development of nanomedicine has recently achieved several breakthroughs in the field of cancer treatment; however, biocompatibility and targeted penetration of these nanomaterials remain as limitations, which lead to serious side effects and significantly narrow the scope of their application. The self-assembly of intermediate filaments with arginine-glycine-aspartate (RGD) peptide (RGD-IFP) was triggered by the hydrophobic cationic molecule 7-amino actinomycin D (7-AAD) to synthesize a bifunctional nanoparticle that could serve as a fluorescent imaging probe to visualize tumor treatment. The designed RGD-IFP peptide possessed the ability to encapsulate 7-AAD molecules through the formation of hydrogen bonds and hydrophobic interactions by a one-step method. This fluorescent nanoprobe with RGD peptide could be targeted for delivery into tumor cells and released in acidic environments such as endosomes/lysosomes, ultimately inducing cytotoxicity by arresting tumor cell cycling with inserted DNA. It is noteworthy that the RGD-IFP/7-AAD nanoprobe tail-vein injection approach demonstrated not only high tumor-targeted imaging potential, but also potent antitumor therapeutic effects in vivo. The proposed strategy may be used in peptide-driven bifunctional nanoparticles for precise imaging and cancer therapy.