Small Molecules Accelerate Skin Wound Healing: Shikonin Efficacy and Mechanism of Action in Mice / Las Moléculas Pequeñas Aceleran la Curación de Heridas en la Piel: Eficacia de Shikonin y Mecanismo de Acción en Ratones

SUMMARY: The objective of this study was to investigate the therapeutic wound healing potential and molecular mechanisms of shikonin as small molecules in vitro. A mouse burn model was used to explore the potential therapeutic effect of shikonin; we traced proliferating cells in vivo to locate the active area of skin cell proliferation. Through the results of conventional pathological staining, we found that shikonin has a good effect on the treatment of burned skin and promoted the normal distribution of skin keratin at the damaged site. At the same time, shikonin also promoted the proliferation of skin cells at the damaged site; importantly, we found a significant increase in the number of fibroblasts at the damaged site treated with shikonin. Most importantly, shikonin promotes fibroblasts to repair skin wounds by regulating the PI3K/AKT signaling pathway. This study shows that shikonin can effectively promote the proliferation of skin cell, and local injection of fibroblasts in burned skin can play a certain therapeutic role.

El objetivo de este trabajo fue investigar el potencial terapéutico de cicatrización de heridas y los mecanismos moleculares de la shikonina como moléculas pequeñas in vitro. Se utilizó un modelo de quemaduras en ratones para explorar el posible efecto terapéutico de la shikonina; Rastreamos las células en proliferación in vivo para localizar el área activa de proliferación de células de la piel. A través de los resultados de la tinción para patología convencional, encontramos que la shikonina tiene un buen efecto en el tratamiento de la piel quemada y promueve la distribución normal de la queratina de la piel en el sitio dañado. Al mismo tiempo, la shikonina también promovió la proliferación de células de la piel en el sitio dañado. Es importante destacar que encontramos un aumento significativo en la cantidad de fibroblastos en el sitio dañado tratado con shikonina. Lo más importante es que la shikonina promueve la función reparadora de fibroblastos en las heridas de la piel regulando la vía de señalización PI3K/ AKT. Este estudio muestra que la shikonina puede promover eficazmente la proliferación de células de la piel y que la inyección local de fibroblastos en la piel quemada puede desempeñar un cierto papel terapéutico.

Fibroblasts inhibit osteogenesis by regulating nuclear-cytoplasmic shuttling of YAP in mesenchymal stem cells and secreting DKK1

Background Fibrous scars frequently form at the sites of bone nonunion when attempts to repair bone fractures have failed. However, the detailed mechanism by which fibroblasts, which are the main components of fibrous scars, impede osteogenesis remains largely unknown. Results In this study, we found that fibroblasts compete with osteogenesis in both human bone nonunion tissues and BMP2-induced ectopic osteogenesis in a mouse model. Fibroblasts could inhibit the osteoblastic differentiation of mesenchymal stem cells (MSCs) via direct and indirect cell competition. During this process, fibroblasts modulated the nuclear-cytoplasmic shuttling of YAP in MSCs. Knocking down YAP could inhibit osteoblast differentiation of MSCs, while overexpression of nuclear-localized YAP-5SA could reverse the inhibition of osteoblast differentiation of MSCs caused by fibroblasts. Furthermore, fibroblasts secreted DKK1, which further inhibited the formation of calcium nodules during the late stage of osteogenesis but did not affect the early stage of osteogenesis. Thus, fibroblasts could inhibit osteogenesis by regulating YAP localization in MSCs and secreting DKK1. Conclusions Our research revealed that fibroblasts could modulate the nuclear-cytoplasmic shuttling of YAP in MSCs, thereby inhibiting their osteoblast differentiation. Fibroblasts could also secrete DKK1, which inhibited calcium nodule formation at the late stage of osteogenesis.

Effect of electrochemically dealloying Ti6Al4V abutments on human gingival fibroblasts / 口腔疾病防治

Objective@#To investigate the effects of electrochemically dealloying of Ti6Al4V abutments on human gingival fibroblasts (HGFs) and to provide experimental evidence for surface modification of implant abutments.@*Methods@#The samples were divided into an NC group (negative control, no other treatment on a smooth surface), an NM-1 group (nanomesh-1, electrochemical dealloying treatment in 1 mol/L NaOH 1 h on 2 V voltage), and an NM-2 group (nanomesh-2, electrochemical dealloying treatment in 5 mol/L NaOH 1 h on 2 V voltage). The surface morphologies of the samples and the adhesion of HGFs on the sample surfaces were observed with scanning electron microscopy (SEM). The surface hydrophilicities of the samples were measured with a contact angle measuring instrument. The proliferation of HGFs on the different samples were evaluated with CCK-8, and the expression of adhesion-related genes, including collagen Ⅰ (COL1A1), collagen Ⅲ (COL3A1), fibronectin 1 (FN1), focal adhesion kinase (FAK), vinculin (VCL), integrin α2 (ITGA2), and integrin β1 (ITGB1), on the different samples was measured with qRT-PCR. The expression of vinculin on the surfaces of HGFs was observed via confocal laser scanning microscopy (CLSM) after immunofluorescent staining. Collagen fiber secretion and syntheses of HGFs from different samples were evaluated via Sirius red staining.@*Results@#SEM revealed the formation of ordered and uniform three-dimensional mesh structures on the surfaces of the NM-1 and NM-2 groups, with grid diameters of approximately 30 nm for the NM-1 group and approximately 150 nm for the NM-2 group. Compared with that of the NC group, the water contact angles of the NM-1 group and NM-2 groups were significantly lower (P<0.000 1). Cell proliferation in the NM-1 group was significantly greater than that in the NC group (P<0.01). Moreover, there was no significant difference in the water contact angles or cell proliferation between the NM-1 group and the NM-2 group. SEM revealed that HGFs were adhered well to the surfaces of all samples, while the HGFs in the NM-1 and NM-2 groups showed more extended areas, longer morphologies, and more developed pseudopodia than did those in the NC group after 24 h. qRT-PCR revealed that the expression levels of the adhesion-related genes COL1A1, COL3A1, FN1, FAK and VCL in the NM-1 group were significantly greater than those in the NC and NM-2 groups (P<0.01). The expression of vinculin protein in the NM-1 group was the highest, and the number of focal adhesions was greatest in the NM-1 group (P<0.01). The results of Sirius red staining showed that the NM-1 group had the highest secretion and syntheses of collagen fibers (P<0.000 1).@*Conclusion@#The three-dimensional nanomechanical structure of Ti6Al4V modified by electrochemical dealloying promoted the adhesion, proliferation, collagen fiber secretion and syntheses of HGFs, and electrochemical dealloying of Ti6Al4V with a grid diameter of approximately 30 nm obviously promoted HGF formation.

Physical-chemical influences and cell behavior of natural compounds on titanium dental surfaces

Abstract The present study evaluated the influence of carvacrol, terpinene-4-ol, and chlorhexidine on the physical-chemical properties of titanium surfaces, cell viability, proliferation, adhesion, and spreading of fibroblasts and osteoblasts in vitro. Titanium surfaces (Ti) were treated with Carvacrol (Cvc), Terpinen-4-ol (T4ol), Chlorhexidine (CHX), DMSO, and ultrapure water (Control group). Physical-chemical modifications were evaluated by surface wettability, the surface free energy (SFE) calculated from the contact angle values using the Owens-Wendt-Rabel-Kaeble (OWRK) equation, scanning electron microscopy (SEM) and energy dispersive spectrometry probe (EDS) system. Cells were seeded onto Ti-treated surfaces and incubated for 24 h and 72 h, then evaluated by Alamar blue assay and fluorescence microscopy. Surfaces treated with Cvc and T4ol showed the presence of Na, O, and Cl. All surfaces showed hydrophilic characteristics and SFE values between 5.5 mN/m and 3.4 mN/m. On the other hand, EDS peaks demonstrated the presence of O and Cl after CHX treatment. A reduction of cell viability and adhesion was noted on titanium surfaces treated with CHX after 24 and 72h. In conclusion, the results indicate that the decontamination with Cvc and T4ol on Ti surfaces does not alter the surface proprieties and allows an adequate interaction with cells involved in the re-osseointegration process such as fibroblasts and osteoblasts.

Resumo O presente estudo avaliou a influência do carvacrol, terpineno-4-ol e clorexidina nas propriedades físico-químicas de superfícies de titânio, viabilidade celular, proliferação, adesão e esplhamento de fibroblastos e osteoblastos in vitro. Superfícies de titânio (Ti) foram tratadas com Carvacrol (Cvc), Terpinen-4-ol (T4ol), Clorexidina (CHX), DMSO e água ultrapura (Grupo Controle). As modificações físico-químicas foram avaliadas pela molhabilidade da superfície, a energia livre de superfície (ELS) calculada a partir dos valores do ângulo de contato usando a equação de Owens-Wendt-Rabel-Kaeble (OWRK), microscopia eletrônica de varredura (MEV) e espectroscopia de raios X por energia dispersiva (EDS). As células foram semeadas em superfícies tratadas com Ti e incubadas por 24 h e 72 h, e avaliadas pelo ensaio Alamar blue e microscopia de fluorescência. As superfícies tratadas com Cvc e T4ol mostraram a presença de Na, O e Cl. Todas as superfícies apresentaram características hidrofílicas e valores de ELS entre 5,5 mN/m e 3,4 mN/m. Por outro lado, os picos de EDS demonstraram a presença de O e Cl após o tratamento com CHX. Uma redução da viabilidade celular e adesão foi observada em superfícies de titânio tratadas com CHX após 24 e 72h. Em conclusão, os resultados indicam que a descontaminação com Cvc e T4ol em superfícies de Ti não altera as propriedades da superfície e permite uma interação adequada com células envolvidas no processo de reosseointegração como fibroblastos e osteoblastos.

Ayudas diagnósticas y diagnóstico diferencial de la enfermedad de Pompe

Introducción: La enfermedad de Pompe (EP) o glucogenosis tipo II es una enfermedad autosómica recesiva causada por mutaciones en el gen GAA que codifica para la proteína alfa-1,4-glucosidasa. Su deficiencia lleva a un almacenamiento anormal de glucógeno en los lisosomas de varias células, a través de los diferentes tejidos, lo que causa un compromiso musculoesquelético predominante. Contenidos: Los fenotipos de la enfermedad dependen de las variantes genéticas y de los niveles de la actividad enzimática residual. La enfermedad se presenta como EP de inicio infantil, EP de inicio tardío y EP intermedio, por lo que es de suma importancia su diagnóstico temprano, por medio de estudios moleculares como la secuenciación de Sanger y la secuenciación de nueva generación. Conclusiones: Se ha demostrado, mediante diferentes estudios, que las variaciones genéticas pueden diferir entre etnias, y es importante su caracterización molecular para determinar el tratamiento más adecuado, de acuerdo con el estado del material inmunológico de reacción cruzada (CRIM).

Introduction: Pompe disease (PD) or Glycogenosis Type II is a rare autosomal recessive disease caused by mutations in the GAA gene that codes for the alpha-1,4-glucosidase protein. Its deficiency leads to abnormal glycogen storage in the lysosomes of various cells throughout the different tissues causing a predominant musculoskeletal compromise. Contents: The phenotypes of the disease depend on the genetic variants and the levels of residual enzyme activity, presenting as infantile-onset PD, late-onset PD, and intermediate PD; Therefore, early diagnosis of the disease through molecular studies such as Sanger sequencing and new generation sequencing is of utmost importance. Conclusions: It has been shown through different studies that genetic variations can vary between ethnic groups and the molecular characterization of the variants is important to determine the most appropriate treatment depending on the state of the cross-reactive immunological material (CRIM)

Tumor inflamatorio miofibroblástico gastrointestinal. Reporte de caso / Gastrointestinal myofibroblastic inflammatory tumor. Case report

Introducción. El tumor miofibroblástico inflamatorio es una enfermedad proliferativa rara, de etiología incierta, caracterizada por la proliferación de miofibroblastos epitelioides o fusionados mezclados con células inflamatorias, predominantemente mononucleares. En general se considera una lesión benigna, aunque en algunos casos esta neoplasia ha mostrado un comportamiento agresivo en cuanto a recidiva local y metástasis. El tratamiento definitivo es la resección quirúrgica completa. Caso clínico. Paciente de 67 años con dos meses de evolución de fiebre y masa abdominal, en quien se realizó una tomografía computarizada de abdomen que identificó una lesión de aspecto infiltrativo tumoral, comprometiendo la grasa retroperitoneal en la transcavidad de los epiplones. Por vía percutánea se tomó una biopsia que informó un pseudotumor inflamatorio retroperitoneal. Fue llevado a cirugía radical abdominal, con patología quirúrgica final que describió un tumor miofibroblástico inflamatorio de compromiso multifocal, adherido a la serosa del estómago e intestino delgado, sin compromiso muscular. Discusión. El tumor inflamatorio miofibroblástico es una entidad rara, de etiología por esclarecer y difícil diagnóstico. Presentamos el caso clínico de un paciente con tumor inflamatorio miofibroblástico gastrointestinal.Conclusión. Se describe el caso clínico de un paciente con un tumor inflamatorio miofibroblástico gastrointestinal, de presentación rara en nuestro medio. Es importante la comparación con casos similares para poder hacer conclusiones útiles en la práctica clínica

Introduction. Inflammatory myofibroblastic tumor is a rare proliferative disease of uncertain etiology, characterized by the proliferation of epithelioid or fused myofibroblasts mixed with predominantly mononuclear inflammatory cells. In general, it is considered a benign lesion, although in some cases this neoplasm has shown aggressive behavior in terms of local recurrence and metastasis. The definitive treatment is complete surgical resection. Clinical case. A 67-year-old patient with a two-month history of fever and an abdominal mass underwent a computed tomography scan of the abdomen that identified an infiltrative tumor, compromising the retroperitoneum fat in the lesser cavity. A biopsy was taken percutaneously, which reported a retroperitoneal inflammatory pseudotumor. He was taken to radical abdominal surgery, with final surgical pathology describing an inflammatory myofibroblastic tumor with multifocal involvement attached to the serosa of the stomach and small intestine without muscle involvement. Discussion. Inflammatory myofibroblastic tumor is a rare entity, of unknown etiology and difficult to diagnose. We present a clinical case of gastrointestinal myofibroblastic inflammatory tumor to better understand this entity.Conclusion. The clinical case of a patient with a gastrointestinal myofibroblastic inflammatory tumor, a rare presentation in our environment, is described. Comparison with similar cases is important to draw useful conclusions in clinical practice

Research progress of stroma-targeted therapies for pancreatic ductal adenocarcinoma / 中华肝胆外科杂志

Desmoplastic stroma of pancreatic ductal adenocarcinoma plays an important role in tumor progression and treatment resistance. Stroma-targeted therapies are therefore promising for clinical application and extensive related researches are undergoing. In this article, recent advances in stromal targeting strategies were reviewed from three perspectives: cancer-associated fibroblasts, extracellular matrix and angiogenesis, and an outlook for the future of this strategy was also provided.

Effects of curcumin on proliferation and apoptosis and migration of human pterygium fibroblasts / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the effects of curcumin on the proliferation and apoptosis and migration of human pterygium fibroblasts(HPF)in vitro.METHODS: A total of 7 cases of pterygium tissue removed at our hospital from November 24, 2021 to December 16, 2021 were collected. Then, primary fibroblasts were cultured in vitro and identified by immunofluorescence staining. HPF were treated with 0, 10, 20, 40, 80 and 160μmol/L curcumin containing equal amount of dimethyl sulfoxide for 24h, then the cell proliferation was detected by CCK8 assay. According to the results of CCK8, the cells were divided into control group, 20μmol/L curcumin group and 40μmol/L curcumin group, and the cells were treated with corresponding concentration of curcumin for 24h in each group. Flow cytometry was used to detect apoptosis, Transwell migration assay was used to detect cell migration, and real-time fluorescence quantitative polymerase chain reaction and Western blot were used to detect the expression of mRNA and protein of B-cell lymphoma-2 associated X protein(Bax), B-cell lymphoma-2(Bcl-2), Cyclin D1 and matrix metalloproteinase 2(MMP2).RESULTS: Compared with the control group, both 20μmol/L curcumin group and 40μmol/L curcumin group can inhibit the proliferation and migration of HPF and induce its apoptosis(all P&#x003C;0.05). Compared with the control group, 20μmol/L curcumin group can down-regulate the mRNA expression of Cyclin D1 and MMP2, up-regulate the mRNA expression of Bax, and down-regulate the protein expression of Bcl-2(all P&#x003C;0.05). Compared with the control group, 40μmol/L curcumin group can down-regulate the expression of mRNA and protein of Bcl-2, Cyclin D1 and MMP2, and up-regulate the expression of mRNA and protein of Bax(all P&#x003C;0.05). Compared with 20 μmol/L curcumin group, the 40 μmol/L curcumin group can down-regulate the mRNA expression of MMP2, down-regulate the protein expression of Bcl-2, and up-regulate the mRNA and protein expression of Bax(all P&#x003C;0.05).CONCLUSION: Curcumin can inhibit the proliferation of HPF by inhibiting the expression of Cyclin D1, induce the apoptosis of HPF by down-regulating Bcl-2 and up-regulating the expression of Bax, and inhibit the migration of HPF by down-regulating the expression of MMP2.

PAFR/Stat3 axis maintains the symbiotic ecosystem between tumor and stroma to facilitate tumor malignancy

Stroma surrounding the tumor cells plays crucial roles for tumor progression. However, little is known about the factors that maintain the symbiosis between stroma and tumor cells. In this study, we found that the transcriptional regulator-signal transducer and activator of transcription 3 (Stat3) was frequently activated in cancer-associated fibroblasts (CAFs), which was a potent facilitator of tumor malignancy, and formed forward feedback loop with platelet-activating factor receptor (PAFR) both in CAFs and tumor cells. Importantly, PAFR/Stat3 axis connected intercellular signaling crosstalk between CAFs and cancer cells and drove mutual transcriptional programming of these two types of cells. Two central Stat3-related cytokine signaling molecules-interleukin 6 (IL-6) and IL-11 played the critical role in the process of PAFR/Stat3 axis-mediated communication between tumor and CAFs. Pharmacological inhibition of PAFR and Stat3 activities effectively reduced tumor progression using CAFs/tumor co-culture xenograft model. Our study reveals that PAFR/Stat3 axis enhances the interaction between tumor and its associated stroma and suggests that targeting this axis can be an effective therapeutic strategy against tumor malignancy.

Fibroblasts overpressing WNT2b cause impairment of intestinal mucosal barrier / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which fibroblasts with high WNT2b expression causes intestinal mucosa barrier disruption and promote the progression of inflammatory bowel disease (IBD).@*METHODS@#Caco-2 cells were treated with 20% fibroblast conditioned medium or co-cultured with fibroblasts highly expressing WNT2b, with the cells without treatment with the conditioned medium and cells co-cultured with wild-type fibroblasts as the control groups. The changes in barrier permeability of Caco-2 cells were assessed by measuring transmembrane resistance and Lucifer Yellow permeability. In Caco-2 cells co-cultured with WNT2b-overexpressing or control intestinal fibroblasts, nuclear entry of β-catenin was detected with immunofluorescence assay, and the expressions of tight junction proteins ZO-1 and E-cadherin were detected with Western blotting. In a C57 mouse model of dextran sulfate sodium (DSS)-induced IBD-like enteritis, the therapeutic effect of intraperitoneal injection of salinomycin (5 mg/kg, an inhibitor of WNT/β-catenin signaling pathway) was evaluated by observing the changes in intestinal inflammation and detecting the expressions of tight junction proteins.@*RESULTS@#In the coculture system, WNT2b overexpression in the fibroblasts significantly promoted nuclear entry of β-catenin (P < 0.01) and decreased the expressions of tight junction proteins in Caco-2 cells; knockdown of FZD4 expression in Caco-2 cells obviously reversed this effect. In DSS-treated mice, salinomycin treatment significantly reduced intestinal inflammation and increased the expressions of tight junction proteins in the intestinal mucosa.@*CONCLUSION@#Intestinal fibroblasts overexpressing WNT2b causes impairment of intestinal mucosal barrier function and can be a potential target for treatment of IBD.

Arecoline induces activation of human oral fibroblasts by promoting macrophage secretion of exosomes containing miR-155-5p / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which arecoline regulates the level of miR-155-5p in macrophage-secreted exosomes to induce the transformation of human oral mucosal fibroblasts (HOMFs) into fibroblast phenotype.@*METHODS@#Exosomes were harvested from human monocytic cell line THP-1 with or without arecoline treatment. The effects of arecoline-treated THP-1 cell culture supernatant (CS), THP-1-derived exosomes (EXO), exosome-depleted THP-1 cell supernatant (NES), miR-155-5p overexpression, and miR-155-5p inhibitor on migration ability of arecoline-treated HOMF cells were examined using Transwell migration assay. The polarization of THP-1 cells was detected using flow cytometry. DCFH-DA was used to detect the level of oxidative stress in the cells with different treatments. The mRNA and protein expressions of α- SMA, type I collagen and SOCS1 in the cells were detected with qRT-PCR and Western blotting.@*RESULTS@#Flow cytometry showed that arecoline-treated THP-1 cells exhibited obvious polarization from M0 to M1. Both the supernatant and exosomes from arecoline-treated THP-1 cells significantly enhanced the migration ability of HOMF cells, increased intracellular oxidative stress, up-regulated the expressions of miR-155- 5p and the mRNA and protein levels of α-SMA and type I collagen, and lowered the mRNA and protein expressions of SOCS1. In HOMF cells treated with exosomes from arecoline- treated THP-1 cells, overexpression of miR-155-5p significantly enhanced cell migration ability and increased cellular expressions of α-SMA and type I collagen, and miR-155-5p inhibitor caused the opposite changes.@*CONCLUSION@#Arecoline can up-regulate miR-155-5p expression in THP-1 cells and inhibit the expression of SOCS1 protein in HOMF cells via the exosome pathway, thus promoting the fibrotic phenotype transformation of HOMF cells.

Inhibitory effect of phloretin on inflammation and oxidative stress in IL-1β induced orbital fibroblasts in Graves orbitopathy and its mechanism / 中华实验眼科杂志

Objective:To investigate the inhibitory effect of phloretin on inflammation and oxidative stress in interleukin (IL)-1β induced orbital fibroblasts (OFs) from Graves orbitopathy (GO) patients and its mechanism.Methods:The orbital fat and connective tissue from 6 eyes of 6 patients diagnosed as inactive GO who underwent orbital decompression in Henan Eye Hospital from January 2019 to December 2020 were collected.Primary OFs were isolated and passaged by explant culture and were identified by cell immunofluorescence assay.OFs were divided into control group, IL-1β induced group, and groups of various phloretin concentrations (25, 50, 75, 100 and 200 μmol/L). The viability of OFs after 24- and 48-hour treatment of the various phloretin concentrations was determined by cell counting kit-8 (CCK-8). OFs were induced by IL-1β to simulate an inflammatory environment of GO in vitro.Intracellular reactive oxygen species (ROS) levels of the normal control group, IL-1β induced group, 50 μmol/L phloretin group and 100 μmol/L phloretin group were detected by fluorescent probe (H 2DCF-DA). The concentrations of pro-inflammatory cytokines IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in cell culture supernatant of the normal control group, IL-1β induced group and phloretin treated groups (25, 50, 75, and 100 μmol/L) were examined by enzyme-linked immunosorbent assay (ELISA). The expressions of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor (Nrf2) proteins, as well as P38, extracelluar regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) proteins as well as their phosphorylated proteins in the MAPK signal pathway of the normal control group, IL-1β induced group and 100 μmol/L phloretin group, were detected by Western blot.The purpose and methods of the study were explained to the patients and their family members.Written informed consent was obtained.The study protocol was approved by the Ethics Committee of Henan Provincial People's Hospital (No.HNEECKY-2020[07]). Results:For cultured OFs, the mesenchymal origin was confirmed by positive expression of vimentin and fibroblasts were identified by negative expression of desmin, S-100 and cytokeratin-18.CCK-8 showed that there was no significant difference in absorbance value after 24- and 48-hour treatment between 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups and control group (all at P>0.05). The ROS levels of 50 μmol/L and 100 μmol/L phloretin groups were 21.95±1.71 and 10.01±1.03, respectively, which were significantly lower than 39.27±4.01 of IL-1β induced group (both at P<0.01). ELISA showed that IL-6 concentrations in 25 μmol/L, 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups were (4 544.25±572.98), (1 000.25±133.96), (724.25±98.63), (519.50±118.02)pg/ml, respectively, which were all significantly lower than (7 581.75±565.93)pg/ml in IL-1β induced group (all at P<0.01). IL-8 concentrations in 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups were (3 679.50±676.76), (2 143.75±616.20), (1 174.75±284.18)pg/ml, respectively, which were all significantly lower than (8 411.00±939.67)pg/ml in IL-1β induced group (all at P<0.01). The concentrations of MCP-1 in 50 μmol/L, 75 μmol/L and 100 μmol/L phloretin groups were (3 783.25±610.24), (1 565.75±457.89), (745.75±227.01)pg/ml, respectively, which were all significantly lower than (5 533.00±602.87)pg/ml in IL-1β induced group (all at P<0.01). The relative expression levels of HO-1 and Nrf2 were significantly higher and the relative expression levels of p-P38, p-ERK, and p-JNK were significantly lower in 100 μmol/L phloretin group than IL-1β induced group (all at P<0.01). Conclusions:Phloretin reduces the oxidative stress level of IL-1β induced OFs from GO patients and inhibits the production of pro-inflammatory cytokines.The mechanism is related to the activation of Nrf2/HO-1 and the inhibition of the MAPK signal pathway.

Promoting effect of AGTR1 blocker olmesartan on the apoptosis of HTF and its mechanism / 中华实验眼科杂志

Objective:To study the effect and mechanism of angiotensin type 1 receptor (AGTR1) blocker olmesartan (OMS) on the apoptosis of human Tenon capsule fibroblasts (HTF).Methods:Tenon capsule tissues were obtained from patients during strabismus surgery in the Second Affiliated Hospital of Xi'an Jiaotong University.Primary HTF were cultured by explant culture.Primary cells were identified by vimentin immunofluorescence staining and flow cytometry.The fibrosis model of HTF was established using 10 ng/ml transforming growth factor-β2 (TGF-β2). The cells were divided into normal control group cultured in culture medium, TGF-β2 group in culture medium containing TGF-β2, TGF-β2+ OMS group in culture medium containing TGF-β2 and OMS, and OMS group in culture medium containing OMS, and were cultured for 48 hours.Cell apoptosis was detected by flow cytometry with annexin V/PI staining.The early apoptosis, late apoptosis, and total apoptosis rates were analyzed.The protein expression of procaspase-9, cleaved caspase-9, bax and bcl-2 in the mitochondrial apoptosis pathway was detected by Western blot.The activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) was detected by colorimetry.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (No.2019-014).Results:Primary HTF were successfully isolated and cultured.The cultured cells were long spindle-shaped and positive for vimentin.The expression rate of vimentin in the primary cells was greater than 99%.A statistically statistical difference was found in the early apoptosis rate, late apoptosis rate, and total apoptosis rate among the four groups ( F=24.92, 3.96, 41.82; all at P<0.05). The early and total apoptosis rates were significantly higher in TGF-β2+ OMS group than normal control group and TGF-β2 group, and the late apoptosis rate in TGF-β2+ OMS group was significantly higher than that of normal control group (all at P<0.05). There were statistically significant differences in cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 among the four groups ( F=4.40, 7.98, 4.61; all at P<0.05). The bax/bcl-2 expression was significantly increased in TGF-β2+ OMS group in comparison with normal control group, and the expressions of cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 were significantly elevated in TGF-β2+ OMS group compared with TGF-β2 group (all at P<0.05). LDH activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (783.99±79.97), (913.16±196.86), (2 529.06±240.21), and (2 134.29±138.96) μmol/(min·L), respectively, showing a statistically significant difference ( F=24.95, P<0.05). Compared with normal control group and TGF-β2 group, LDH activity in TGF-β2+ OMS group was increased, and the differences were statistically significant (both at P<0.05). SOD activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (50.35±0.97), (41.61±4.56), (28.88±3.26), and (37.61±4.83) μmol/(min·L), respectively, showing a statistically significant difference ( F=5.71, P<0.05). SOD activity was reduced in TGF-β2+ OMS group compared with normal control group and TGF-β2 group, reduced in OMS group compared with normal control group, and the differences were statistically significant (all at P<0.05). Conclusions:AGTR1 blocker OMS can promote the apoptosis of HTF effectively.Mitochondrial apoptosis pathway mediated by bax/bcl-2/caspase-9 and oxidative stress pathway are the potential mechanisms that OMS regulates the apoptosis of HTF.

Effect of modified citrus pectin on synovial fibroblasts / 国际生物医学工程杂志

Objective:To study the effects of modified citrus pectin (MCP) on the viability and gene expressions of synovial fibroblasts (SF) as well as SF treated by galectin-3 (Gal-3).Methods:Rabbit SF was isolated and cultured in vitro. Then SF was treated with different concentrations of MCP (0, 250, 500, and 750 mg/L). In addition, SF was further treated with the same different concentrations of MCP after treatment with 10 μg/ml Gal-3 for 24 h. The viability of SF was detected by CCK-8 on the first, third, and fifth day after treatment. The mRNA expression of transforming growth factor-β1 (TGF-β1), type I collagen (COL1A2), and Gal-3 in SF was detected by real-time quantitative PCR. The synthesis of type I collagen in SF was investigated by immunofluorescence staining. Results:MCP, especially at a concentration of 500 mg/L can inhibit the proliferation of SF significantly (all P < 0.05) on the first, third, and fifth day after treatment. Compared with the control group, MCP at different concentrations induced different gene expression profiles. In particular, MCP at high concentrations can upregulate the expression of TGF-β1, COL1A2 and Gal-3 in SF. However, MCP shows no significant effect on the synthesis of type I collagen in SF. MCP can down-regulate the expression of TGF-β1, COL1A2, and significantly reduce the synthesis of type I collagen in SF after Gal-3 treatment. Particularly, the effect of MCP at a concentration of 500 mg/L on inhibiting the expression of TGF-β1, COL1A2, and Gal-3 in SF is significant. Conclusions:MCP can inhibit the excessive proliferation of SF and regulate gene expression in SF.

Effects of EW-7197 on the proliferation and migration of TGF-β1 induced human Tenon fibroblasts and its mechanism / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the effect of ALK5 inhibitor EW-7197 on the proliferation and migration of human Tenon fibroblasts(HTFs)induced by transforming growth factor-β1(TGF-β1)and its mechanism.METHODS: The cell proliferation rate was detected by MTS assay, and the optimal concentration and time of EW-7197 were explored. Then HTFs were divided into three groups: normal control group, TGF-β1 induced group and TGF-β1+EW-7197 group. Cell migration was observed by Transwell assay. The protein expression levels of Fibronectin, α-SMA, as well as the phosphorylated Smad2, Smad3(p-Smad2, p-Smad3)were measured by Western blot.RESULTS: MTS assay showed that the proliferation rate of cells treated with 6.0 μmol/L EW-7197 for 24h was the lowest(all P&#x003C;0.01). Transwell assay showed that the migrated number of HTFs in TGF-β1 induced group was 228.0±17.0/field, which was significantly more than that in normal control group(149.0±15.0/field)and TGF-β1+EW-7197 group(46.0±8.0/field; all P&#x003C;0.01). Western blot showed that the protein relative expression levels of Fibronectin, α-SMA and p-Smad2, p-Smad3 of HTFs in TGF-β1 induced group were significantly higher than that in normal control group and TGF-β1+EW-7197 group(all P&#x003C;0.001).CONCLUSION:EW-7197 can suppress the proliferation and migration of TGF-β1-induced HTFs through TGF-β/Smad signaling pathways.

Colorectal cancer cells induce the formation of cancer-associated fibroblasts by activating the ERK signaling pathway in fibroblasts / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which conditioned medium of colorectal cancer cells promotes the formation of cancer-associated fibroblasts (CAFs).@*METHODS@#Normal human colorectal fibroblasts (CCD-18Co cells) in logarithmic growth phase were treated with the conditioned media of colorectal cancer HCT116 cells (HCT116-CM) or Caco-2 cells (Caco-2-CM) alone or in combination with 300 nmol/L ERK inhibitor SCH772984. The expression levels of CAFs-related molecular markers were detected in the treated cells with real-time quantitative PCR (RT- qPCR) and immunofluorescence assay, and the changes in cell proliferation, colony formation and migration were assessed with RTCA, colony formation and wound healing assays; Western blotting was performed to detect the activated signaling pathways in the fibroblasts and the changes in CAFs formation after blocking of the signaling pathway.@*RESULTS@#HCT116-CM and Caco-2-CM significantly upregulated mRNA expression levels of CAFs markers (including α-SMA, FAP, FN and TGF-β) in CCD-18Co cells, and strongly promoted fibroblast transformation into CAFs (P < 0.05). The two conditioned media also promoted the proliferation, colony formation and migration of CCD-18Co cells (P < 0.05) and significantly increased the levels of α-SMA protein and ERK phosphorylation in the cells (P < 0.05). The ERK inhibitor SCH772984 obviously inhibited the expression of α-SMA and the transformation of CCD-18Co cells into CAFs induced by the conditioned medium of colorectal cancer cells (P < 0.05).@*CONCLUSION@#Colorectal cancer cells may induce the formation of colorectal CAFs by activating the ERK pathway in the fibroblasts.

Cell-derived nanovesicles from mesenchymal stem cells as extracellular vesicle-mimetics in wound healing

Wound healing is a dynamic process that involves a series of molecular and cellular events aimed at replacing devitalized and missing cellular components and/or tissue layers. Recently, extracellular vesicles (EVs), naturally cell-secreted lipid membrane-bound vesicles laden with biological cargos including proteins, lipids, and nucleic acids, have drawn wide attention due to their ability to promote wound healing and tissue regeneration. However, current exploitation of EVs as therapeutic agents is limited by their low isolation yields and tedious isolation processes. To circumvent these challenges, bioinspired cell-derived nanovesicles (CDNs) that mimic EVs were obtained by shearing mesenchymal stem cells (MSCs) through membranes with different pore sizes. Physical characterisations and high-throughput proteomics confirmed that MSC-CDNs mimicked MSC-EVs. Moreover, these MSC-CDNs were efficiently uptaken by human dermal fibroblasts and demonstrated a dose-dependent activation of MAPK signalling pathway, resulting in enhancement of cell proliferation, cell migration, secretion of growth factors and extracellular matrix proteins, which all promoted tissue regeneration. Of note, MSC-CDNs enhanced angiogenesis in human dermal microvascular endothelial cells in a 3D PEG-fibrin scaffold and animal model, accelerating wound healing in vitro and in vivo. These findings suggest that MSC-CDNs could replace both whole cells and EVs in promoting wound healing and tissue regeneration.

Preparation of tanshinone IIA self-soluble microneedles and its inhibition on proliferation of human skin fibroblasts / 中草药·英文版

OBJECTIVE@#Hypertrophic scars (HS) are a variety of skin tissue fibrosis disease that occurs in human skin, the effective therapeutic method of which is still inaccessible up to now. As a bioactive constituent of a well-known medical plant, Salvia miltiorrhiza (Danshen in Chinese), tanshinone IIA (TSA) is reported to inhibit cell proliferation in HS. Therefore, the aim of this study was to prepare TSA self-soluble microneedles to strengthen its dermal retention and break through the difficulty of significantly thickening epidermal connective tissue and stratum corneum at the HS site. The possible mechanism of action in suppressing HS was studied using human skin fibroblasts (HSF).@*METHODS@#Tanshinone IIA self-dissolving microneedles (TSA-MN) was prepared using a negative mold casting method. The prescription process of microneedle was optimized by Box-Behnken effect surface method. Different media were selected to investigate the ability of transdermal absorption and in vitro release. Furthermore, according to Cell Counting Kit-8 (CCK8) method as well as the Western blot method, the effect of TSA-MN on the biological characteristics of HSF was investigated.@*RESULTS@#With remarkable slow release effect and dermal retention, the release and transdermal properties of TSA-MN in vitro were better than both TSA and ordinary dosage forms. Its effect of HSF confirmed the essential decrease in cell motility during cell proliferation and cell migration in vitro, which plays a significant role in down-regulating the secretion of transforming growth factor-β1 (TGF-β1) in HSF and increasing the expression level of Smad7.@*CONCLUSION@#The prepared TSA self-soluble microneedles is helpful in solving the problem of hypertrophic scars, with a stable dermal retention effect after process optimization.

Effects of Foxp3 gene silencing on the expression of inflammatory cytokines and the proliferation and migration of human periodontal ligament fibroblasts in an inflammatory environment / 华西口腔医学杂志

OBJECTIVES@#This study aimed to clarify the effects of Foxp3 silencing on the expression of inflammatory cytokines in human periodontal ligament cells (hPDLFs) in an inflammatory environment and on cell proliferation and invasiveness, as well as to explore the role of Foxp3 gene in the development of periodontitis.@*METHODS@#An small interfering RNA (siRNA) construct specific for Foxp3 was transfected into hPDLFs. Foxp3 silencing efficiency was verified by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, and the siRNA with the optimum silencing effect of Foxp3 gene was screened. Using lipopolysaccharide to simulate an inflammatory environment in vitro, CCK-8 detected the effect of silencing Foxp3 on hPDLFs proliferation under inflammatory conditions. Wound-healing experiments and transwell assays were conducted to detect the effect of silencing Foxp3 on hPDLF migration under inflammatory conditions. The expression of the inflammatory cytokines interleukin (IL)-6 and IL-8 was detected by RT-PCR and Western blotting under inflammatory conditions.@*RESULTS@#After siRNA transfection, RT-PCR and Western blotting analyses showed that the expression of Foxp3 mRNA in the Foxp3-si3 group decreased significantly (t=21.03, P<0.000 1), and the protein expression of Foxp3 also decreased significantly (t=12.8, P<0.001). In the inflammatory environment, Foxp3 gene silencing had no significant effect on hPDLFs proliferation (P>0.05), and Foxp3 gene silencing promoted hPDLFs migration (P<0.05). Moreover, the expression of IL-6 and IL-8 increased (P<0.05).@*CONCLUSIONS@#In an inflammatory environment, Foxp3 gene silencing promoted hPDLFs migration but had no significant effect on hPDLFs proliferation. The expression of inflammatory factors expressed in hPDLFs increased after Foxp3 gene silencing, indicating that Foxp3 gene inhibited inflammation in periodontitis.

C5aR antagonist inhibits LPS-induced inflammation in human gingival fibroblasts via NF-κB and MAPK signaling pathways

Abstract Objective Abnormal complement activation is associated with periodontitis. W54011 is a novel non-peptide C5aR antagonist (C5aRA) that exhibits favorable anti-inflammatory effects in various inflammatory models. However, whether W54011 inhibits periodontitis has not yet been fully elucidated. To address this, we have investigated the probable anti-inflammatory mechanism of W54011 in LPS-treated inflammation in human gingival fibroblasts (HGFs). Methodology HGFs were isolated from healthy gingival tissue samples using the tissue block method and were identified with immunofluorescence staining. The CCK8 assay and reverse transcription-PCR (RT-PCR) were used to select the optimal induction conditions for Lipopolysaccharide (LPS) and C5aRA (according to supplementary data S1, S2 and S3). The levels of inflammatory cytokines, C5aR, and the activation of NF-κB/MAPK signaling pathways were determined by RT-quantitative PCR (RT-qPCR) and Western blotting. Results Immunofluorescence results showed that vimentin and FSP-1 were positive in HGFs and Keratin was negative in HGFs. Immunofluorescence staining demonstrated that C5aRA inhibited LPS-stimulated nuclear translocation of p-p65. RT-qPCR and Western blotting showed that C5aRA reduced the expression of IL-1β, IL-6, TNF-α, C5aR, p-p65, p-IκBα, p-JNK, p-c-JUN, and TLR4 in LPS-induced HGFs. Conclusion These findings suggested that C5aRA attenuated the release of inflammatory cytokines in LPS-induced HGFs by blocking the activation of the NF-κB and MAPK signaling pathways.