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.

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.

Research progress on non-coding small RNA in oral submucosal fibrosis / 口腔疾病防治

@#Oral submucous fibrosis (OSF) is one of the most common precancerous lesions of the oral mucosa, and its pathogenesis has not been fully elucidated. Small noncoding RNAs (SncRNAs), a class of RNA molecules that do not code for proteins, have been widely reported to be involved in the regulation of a variety of human diseases. An increasing number of studies have shown that a variety of SncRNAs play important roles in the pathogenesis of OSF. Current studies have shown that microRNAs (miRNAs) are involved in OSF disease progression by regulating the expression of related transcription factors and genes or epithelial mesenchymal transformation to regulate the activation of fibroblasts (FBs). Long noncoding RNAs (lncRNAs) that transform growth factor-β/suppressor of mothers against decapentaplegic (TGF-β/Smad) signaling pathways or interact with miRNAs are involved in the development of OSF. Circular RNAs (circRNAs) play a role in OSF by interacting with miRNAs. tRNA-derived small RNAs (tsRNAs) are involved in the progression of various fibrotic diseases, but their specific mechanism of action in OSF still needs to be further explored. In the future, it is still necessary to focus on the targets of SncRNAs mediating OSF progression and explore their function and molecular mechanism in OSF to provide new ideas for the diagnosis and treatment of OSF.

Effect and mechanism of reactive oxygen species-responsive nanoparticles on the regulation of human gingival fibroblast function and inflammation induced by lipopolysaccharide / 口腔疾病防治

Objective@#To investigate the effects of PssL-NAC reactive oxygen species (ROS)-responsive nanoparticles on intracellular ROS production, inflammatory factor levels, collagen production, cell function and Toll-like receptor 4 (TLR4), NF-κB nuclear factor-κB (p65) pathway protein expression in human gingival fibroblasts (HGFs) induced by Porphyromonas gingivalis-lipopolysaccharide (P.g-LPS).@*Methods@#This study was reviewed and approved by the ethics committee. PssL-NAC microspheres containing oil soluble antioxidant N-acetylcysteine (NAC) were obtained by connecting the hydrophobic end of polycaprolactone (PCL) and the hydrophilic end of polyethylene glycol (PEG) via thioketal (TK) bonds in response to ROS, and self loading in the aqueous and oil phases. After preparation of the PssL-NAC microspheres and aqueous NAC solution, successful synthesis of the nanoparticles was verified by transmission electron microscopy. Then, HGFs were exposed to P.g-LPS (0, 5, or 10 μg/mL), P.g-LPS (0, 5, or 10 μg/mL)+NAC, and P.g-LPS (0, 5, or 10 μg/mL)+PssL-NAC, and the ROS levels in the different groups were observed under confocal microscopy to determine the concentration of P.g-LPS for use in subsequent experiments. The groups were as follows: control group (no treatment), P.g-LPS group (HGFs treated with P.g-LPS), NAC group (HGFs treated with P.g-LPS and NAC), and PssL-NAC group (HGFs treated with P.g-LPS and PssL-NAC). Cell counting kit-8 (CCK-8) assays verified the biosafety of PssL-NAC. The ROS levels in the different groups were detected by DCFH-DA probes and observed via confocal microscopy. Real-time qPCR (RT-qPCR) was used to monitor the gene expression levels of the intracellular inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), collagen 1 (COL1) and collagen 3 (COL3). The effect of PssL-NAC on the migration of HGFs was observed via the scratch test. The protein expression of TLR4-NF-κB, and phosphorylated p65 (p-p65) in the TLR4-NF-κB pathway was evaluated by Western blot.@*Results@#PssL-NAC had no significant effect on HGF proliferation (P>0.05). At elevated P.g-LPS concentrations, PssL-NAC maintained intracellular ROS levels approximately twice those in the control group (P<0.001). PssL-NAC significantly decreased P.g-LPS-induced IL-6 (P<0.001) and TNF-α (P<0.001) gene expression and increased COL1 gene expression (P<0.001). After P.g-LPS stimulation, PssL-NAC restored cell migration to the control level (P>0.05) and decreased the protein expression of TLR4 (P<0.001), p65 (P = 0.006), and p-p65 (P = 0.017) in the TLR4-NF-κB pathway.@*Conclusion@#PssL-NAC maintains the appropriate intracellular ROS concentration, alleviates P.g-LPS-induced inflammation in HGFs through the TLR4-NF-κB pathway, and restores the cell functions of collagen production and migration in an inflammatory environment.

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.

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 / Diagnostic aids and differential diagnosis of Pompe disease

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

Counteractive effect of mouse dermal fibroblasts during their adipogenic differentiation against Staphylococcus aureus infection and its mechanisms / 中华皮肤科杂志

Objective:To investigate the counteractive effect of mouse dermal fibroblasts (MdFBs) during their adipogenic differentiation against Staphylococcus aureus infection, and to explore its mechanisms. Methods:MdFBs were obtained from newborn C57BL/6 mice, and their adipogenic differentiation was induced by culture in an adipogenic medium for 48 hours. Real-time fluorescence-based quantitative PCR (RT-PCR) was performed to determine the mRNA expression of cathelicidin antimicrobial peptide (CAMP) on days 0-6 during the adipogenic differentiation of MdFBs, and Western blot analysis to determine the protein expression of CAMP in the culture supernatant of MdFBs during their adipogenic differentiation. MdFBs were divided into 4 groups: co-stimulation group stimulated by S. aureus suspensions and cultured in an adipogenic medium, adipogenic control group cultured in an adipogenic medium, S. aureus-stimulation group stimulated by S. aureus suspensions and cultured in a common medium, and control group stimulated by phosphate-buffered saline and cultured in a common medium; Western blot analysis and RT-PCR were conducted to determine the protein and mRNA expression of CAMP. S. aureus (5 × 10 4 CFU/ml) was cultured with the culture supernatant of MdFBs after 5-day adipogenic differentiation (adipogenic group), and the growth activity was evaluated every 2 hours during 10 - 24 hours after the start of co-culture; S. aureus cultured with the culture supernatant of MdFBs in a common medium served as the normal control group, and that cultured with cell-free culture supernatant served as the negative control group. Differences between groups were assessed using unpaired t-test or analysis of variance. Results:Significant differences were observed in the relative mRNA expression of CAMP among different time points (days 0, 1, 2, 4, and 6) during the adipogenic differentiation of MdFBs (1.14 ± 0.74, 68.04 ± 12.72, 683.12 ± 38.06, 1 390.68 ± 226.21, 454.57 ± 204.12, F = 50.08, P < 0.001) ; the CAMP mRNA expression was significantly higher on days 1, 2, 4, and 6 than on day 0 ( t = 9.09, 31.03, 10.63, 3.85, respectively, all P < 0.05), and showed an initial rise and subsequent fall during days 0 - 6. The CAMP protein expression in the culture supernatant of MdFBs peaked on days 2-5 and subsequently decreased. Significant differences were observed in the mRNA and protein expression of CAMP among the control group, S. aureus-stimulation group, adipogenic control group and co-stimulation group (mRNA: 0.08 ± 0.02, 0.38 ± 0.10, 0.49 ± 0.11, 0.80 ± 0.03, respectively, F = 43.25, P < 0.05; protein: 0.433 ± 0.176, 0.574 ± 0.176, 1.007 ± 0.176, 1.217 ± 0.176, respectively, F = 46.79, P < 0.05), and the relative mRNA and protein expression of CAMP was significantly higher in the co-stimulation group than in the adipogenic control group, S. aureus-stimulation group and control group (all P < 0.05). At 10 hours during culture, the growth activity of S. aureus was significantly lower in the adipogenic group (0.053 ± 0.015) than in the normal control group and negative control group (0.109 ± 0.015, 0.106 ± 0.015, t = 11.30, 13.26, respectively, both P < 0.05) ; during 10 - 24 hours, the growth activity of S. aureus also showed a significant decrease in the adipogenic group compared with the normal control group and negative control group (all P < 0.05) . Conclusion:MdFBs secreted CAMP during the adipogenic differentiation, and could inhibit the proliferation of S. aureus.

Effect of transmembrane protein 45A on extracellular matrix synthesis by keloid-derived fibroblasts / 中华皮肤科杂志

Objective:To determine the expression of transmembrane protein 45A (TMEM45A) in keloid tissues and fibroblasts, and to evaluate its effect on extracellular matrix (ECM) synthesis by keloid-derived fibroblasts (KFs) .Methods:Samples of surgically excised keloid and normal foreskin tissues were collected from the Department of Dermatology and Department of Urology of Yanbian University Hospital from January 2019 to December 2020, and TMEM45A protein expression was determined in keloid tissues and KFs by Western blot analysis. KFs were divided into TMEM45A-specific small interfering RNA (siRNA) group and control siRNA group to be transfected with the TMEM45A-specific siRNA and control siRNA respectively. Then, Western blot analysis was performed to evaluate the effects of down-regulation of the TMEM45A gene on the expression of myofibroblast marker protein (α-smooth muscle actin) and ECM-related proteins.Results:Compared with normal skin tissues (1.00 ± 0.11) and fibroblasts (1.00 ± 0.20), TMEM45A expression levels significantly decreased in keloid tissues (0.26 ± 0.05) and KFs (0.41 ± 0.09), respectively ( t = 10.76, 4.75, P < 0.001, = 0.009, respectively). The expression levels of α-smooth muscle actin, ECM-related type Ⅰ collagen, type Ⅲ collagen, and fibronectin were significantly higher in the TMEM45A-specific siRNA group than in the control siRNA group ( t = -5.98, -4.57, -4.90, -7.19, P = 0.004, 0.010, 0.008, 0.002, respectively) . Conclusion:Lowly expressed TMEM45A in keloids may play an important role in the pathogenesis of keloids by promoting ECM synthesis.

Effects of the ROCK1 gene on proliferation and migration of and related molecular expression in keloid fibroblasts / 中华皮肤科杂志

Objective:To investigate effects of the ROCK1 gene on proliferation and migration of and related molecular expression in keloid fibroblasts.Methods:Immunohistochemical technique was used to detect ROCK1 protein expression in human keloids and normal skin tissues, and Western blot analysis was performed to detect the expression of ROCK1, transforming growth factor β1 (TGF-β1) and E-cadherin in keloid tissues. In vitro cultured human keloid fibroblasts (HKFs) were divided into 4 groups: ROCK1 gene overexpression control group (ROCK1 NC group) transfected with ROCK1 gene overexpression control vectors, ROCK1 gene overexpression group (ROCK1 OE group) transfected with ROCK1 gene overexpression vectors, ROCK1 gene knockdown control group (sh NC group) transfected with ROCK1 gene knockdown control vectors, and ROCK1 gene knockdown group (shROCK1 group) transfected with ROCK1 gene knockdown vectors. Cell counting kit-8 (CCK8) assay was performed to evaluate the effect of ROCK1 gene on the survival rate of HKFs, Transwell assay to evaluate the effect on the migration of HKFs, and real-time fluorescence-based quantitative PCR (qRT-PCR) and Western blot analysis were conducted to determine the mRNA and protein expression of ROCK1, TGF-β1 and E-cadherin, respectively. Results:Immunohistochemical study showed that ROCK1 protein expression decreased significantly in the human keloid tissues compared with the normal tissues ( t = 6.47, P = 0.003) ; Western blot analysis showed that the expression levels of ROCK1 and E-cadherin significantly decreased ( t = 14.02, 162.20, respectively, both P < 0.001), while TGF-β1 expression significantly increased ( t = 76.01, P < 0.001) in the keloid tissues compared with the expression levels of corresponding proteins in the normal tissues. CCK8 assay showed that the cell activity was significantly lower in the ROCK1 OE group than in the ROCK1 NC group after 24-hour transfection ( t = 3.25, 3.78, P = 0.031, 0.019, respectively), and significantly higher in the shROCK1 group than in the sh NC group ( t = 3.12, 2.79, P = 0.036, 0.049, respectively). Transwell assay showed that the number of migratory cells was significantly lower in the ROCK1 OE group than in the ROCK1 NC group ( t = 5.17, P = 0.004), and significantly higher in the shROCK1 group than in the sh NC group ( t = 9.28, P < 0.001). Compared with the ROCK1 NC group, the ROCK1 OE group showed significantly increased mRNA and protein expression levels of ROCK1 and E-cadherin ( P < 0.05 or < 0.001), but decreased mRNA and protein expression levels of TGF-β1 (both P < 0.001) ; compared with the sh NC group, the shROCK1 group showed significantly decreased mRNA and protein expression levels of ROCK1 and E-cadherin ( P < 0.05 or < 0.001), but significantly increased mRNA and protein expression levels of TGF-β1 ( P = 0.005 or < 0.001) . Conclusions:The ROCK1 gene can inhibit the proliferation and migration of HKFs. Overexpression of the ROCK1 gene can down-regulate the TGF-β1 gene expression and up-regulate the E-cadherin gene expression in HKFs.

Development of fibroblast activation protein-targeted molecular probes in non-neoplastic diseases / 中华核医学与分子影像杂志

Fibroblast activation protein (FAP) is an important molecular marker of cancer-associated fibroblasts (CAFs). FAP is selectively expressed in more than 90% of epithelial carcinomas, but barely expressed in normal tissues. In recent years, a variety of radiolabeled molecular probes based on FAP inhibitor (FAPI) have been developed and used for imaging of malignant tumors. FAP is also highly expressed in some non-neoplastic diseases related to chronic inflammation and tissue remodeling, including arthritis, atherosclerosis, fibrosis of myocardial infarction, cirrhosis, and idiopathic pulmonary fibrosis. FAPI imaging shows a potential in these diseases. This paper reviews the current status of radionuclide labeled FAPI and the application of which in non-neoplastic diseases.

Relationship between the SUV max of primary lesion on 18F-FAPI-42 PET/CT imaging and the clinicopathological characteristics of patients with gastric cancer / 中华核医学与分子影像杂志

Objective:To explore the relationship between 18F-fibroblast activation protein inhibitor (FAPI)-42 SUV max of primary gastric cancer and clinicopathological factors of patients. Methods:Fifty-one patients (31males, 20 females, age: 51(47, 65) years) with gastric cancer who underwent 18F-FAPI-42 PET/CT before surgical resection in Nanfang Hospital, Southern Medical University from February 2022 to January 2023 were analyzed retrospectively. The clinicopathological factors that might affect tumor SUV max (including gender, age, tumor location, pathological type, histological grade, Lauren classification, vascular and(or) neural invasion, programmed cell death-ligand 1 (PD-L1) expression, pathologic(p)T stage, pN stage and pTNM stage) were evaluated by the univariate analysis (Mann-Whitney U test or Kruskal-Wallis rank sum test) and multivariate analysis (multiple linear regression analysis). Results:The sensitivity of 18F-FAPI-42 PET/CT in the diagnosis of patients with primary gastric cancer was 82.35% (42/51). The diagnostic sensitivities for early gastric cancer (T1) and locally advanced gastric cancer (T2-T4) were 59.09%(13/22) and 100%(29/29), respectively. The SUV max of primary lesion was 4.90(1.71, 12.51). The univariate analysis showed that SUV max of primary gastric cancer was related to tumor location ( z=-2.00, P=0.046), pT stage ( H=36.94, P<0.001), pN stage ( z=-3.89, P<0.001), pTNM stage ( H=31.49, P<0.001) and vascular and(or) nerve invasion ( z=-5.22, P<0.001), but not related to pathological type, histological grade, Lauren typing, and PD-L1 expression ( z values: from -1.78 to -0.09, all P>0.05). pT stage was found to be a significant independent factor for SUV max in primary gastric lesion by multivariate analysis ( t=2.52, P=0.015). Conclusions:The 18F-FAPI-42 SUV max of primary tumor was related to tumor location, pT stage, pN stage, pTNM stage, and vascular and(or) nerve invasion; pT stage is an independent factor affecting tumor SUV max. The ability of 18F-FAPI-42 PET/CT to detect gastric cancer is mainly affected by pT stage.

Research progress of radionuclide labeled fibroblast activation protein inhibitors PET imaging in cardiac diseases / 中华核医学与分子影像杂志

Myocardial fibrosis is one of the important pathological mechanisms in cardiac diseases. Non-invasive evaluation of fibrosis is of great clinical significance. Fibroblast activation protein (FAP) is selectively expressed in the membrane of activated fibroblasts. Radionuclide labeled FAP inhibitors (FAPI) serve as novel imaging agents, which specifically target to the process of fibrotic remodeling. This article reviews the research progress of radionuclide labeled FAPI PET imaging in cardiac diseases.

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.

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.