EGR1/NOX4 pathway regulates oxidative stress and further facilitates fibrosis progression in keloids responses to TGF-ß1.

BACKGROUND: As classic benign fibroproliferative tumors, keloids remain a major therapeutic challenge due to their complex pathological mechanisms. OBJECTIVE: To determine the functional role of transforming growth factor ß1 (TGF-ß1)/early growth response factor-1 (EGR1)/NADPH oxidases 4 (NOX4) axis in the pathogenesis of keloid fibrosis. METHODS: Differentially expressed genes in keloid tissues and normal skins were analyzed by RNA sequencing. Then, the human skin fibroblast cell line was treated with TGF-ß1 at a dose of 10 ng/mL in order to stimulate the TGF-ß1/SMAD pathway and the pathway was blocked using the SB431542. Furthermore, EGR1/NOX4 was over-expressed and inhibited by transfecting overexpression plasmids and small interfering RNAs, respectively. The levels of intracellular reactive oxygen species were measured using the DCFH-DA assay, and the expression levels of fibrosis-related genes were assessed by Western blot analysis. Alternately, dual-luciferase reporter analysis verified the targeting relationship between EGR1 and NOX4. RESULTS: The TGF-ß1/SMAD signaling pathway was significantly activated in keloid tissues to promote dermal fibrosis. The level of ROS was increased in keloid fibroblasts. Moreover, TGF-ß1 could facilitate the expression of EGR1 through regulating the SMAD pathway in keloids and promoting the fibrotic phenotype of keloid fibroblasts. EGR1 could regulate the production of ROS by targeting NOX4. Furthermore, NOX4-derived ROS could promote fibrotic-like phenotype of keloid fibroblasts and play an important role in keloid fibrosis. CONCLUSION: Our findings provide new insights into the mechanisms of the TGF-ß1/EGR1/NOX4 pathway in keloid fibrosis, and the TGF-ß1/EGR1/NOX4 axis may serve as a potential therapeutic target for keloids.

Comparison of primary keloid fibroblast cultivation methods and the characteristics of fibroblasts cultured from keloids, keloid-surrounding tissues, and normal skin tissues / Comparación de los métodos de cultivo de fibroblastos queloides primarios y las características de los fibroblastos cultivados a partir de queloides, tejidos circundantes queloides y tejidos cutáneos normales

SUMMARY: The establishment of primary keloid fibroblast culture has always been a fundamental measure for studying mechanisms of keloid disease. The quality of the primary cell culture can directly affect the results of further experiments. This study was performed to investigate the optimal growth conditions, including the optimal storage time and collagenase treatment time, for in vitro cell culture models and the suitable methods for epidermis-dermis separation in different tissues. Keloid tissues, keloid-surrounding tissues, and normal skin tissues were collected from patients, for primary fibroblast culture. Two methods, tissue explant and collagenase digestion, were deployed and compared. Expression levels of the keloid-related genes α -SMA, Col1, and Col3 were assessed in cells cultured using both methods, to verify the qualities of the primary cells. A comparative analysis was conducted between the two methods and among the three different tissues used. Bacterial and lipid contamination was immediately minimized after the samples were processed. Different methods of epidermis removal and different durations of collagenase digestion were required in different tissues to generate optimal results. Real-time PCR results showed that the mRNA expression levels of keloid-related genes in cultured fibroblasts correlated to their in vivo expression profile, as previously reported in other studies. The results of this study have revealed several key points in the culture of primary keloid fibroblasts and demonstrated the correlation in gene expression between in vivo keloid fibroblasts and in vitro primary keloid fibroblasts.

RESUMEN: La identificación de un cultivo de fibroblastos queloides primarios, siempre ha sido una medida fundamental para estudiar los mecanismos de la enfermedad queloide. La calidad del cultivo de células primarias puede afectar directamente los resultados de otros experimentos. Este estudio se realizó para investigar las condiciones óptimas de crecimiento, incluido el tiempo óptimo de almacenamiento y el tiempo de tratamiento con colagenasa, para modelos de cultivo celular in vitro y los métodos adecuados para la separación epidermis-dermis en diferentes tejidos. Se recogieron de los pacientes tejidos queloides, tejidos circundantes queloides y tejidos cutáneos normales, para cultivo primario de fibroblastos. Se implementaron y compararon dos métodos, explante de tejido y digestión con colagenasa. Los niveles de expresión de los genes relacionados con queloides α -SMA, Col1 y Col3 se evaluaron en células cultivadas usando ambos métodos, para verificar las cualidades de las células primarias. Se realizó un análisis comparativo entre los dos métodos y entre los tres tejidos diferentes utilizados. La contaminación de bacterias y lípidos se minimizó inmediatamente después de que se procesaron las muestras. Se requirieron varios métodos de eliminación de la epidermis y diferentes tiempos de digestión con colagenasa en los tejidos para generar resultados óptimos. Los resultados de la PCR en tiempo real mostraron que los niveles de expresión de ARNm de genes relacionados con queloides en fibroblastos cultivados se correlacionaban con su perfil de expresión in vivo, como se informó en estudios anteriores. Los resultados de este studio indicaron varios puntos clave en el cultivo de fibroblastos queloides primarios y han demostrado la correlación en la expresión génica entre fibroblastos queloides in vivo y fibroblastos queloides primarios in vitro.

Lnc HAGLR Promotes Colon Cancer Progression Through Sponging miR-185-5p and Activating CDK4 and CDK6 in vitro and in vivo.

BACKGROUND/AIM: LncRNA plays a key role in tumor progression. HAGLR functions as an oncogene in many cancers. However, the molecular mechanism of HAGLR in colon cancer is still unclear. METHODS: qRT-PCR was used to measure the expression of HAGLR, miR-185-5p in colon cancer. The expression of CDK4 and CDK6 was detected by Western blot. CCK-8 assay, EdU staining, transwell and Annexin V-FITC/PI assay were used to analyze the effect of HAGLR and miR-185-5p on cell proliferation, invasion, migration and apoptosis. Bioinformatic analysis and luciferase were used to analyze the target genes of HAGLR and miR-185-5p. Nude mice were used to detect mouse tumor changes. RESULTS: Compared with normal colon cancer tissues and cells, the expression of HAGLR was increased in colon cancer tissues and cells. In addition, the expression of HAGLR down-regulation inhibited the growth, migration, and invasion of colon cancer cells. MiR-185-5p was reduced in colon cancer, and CDK4 and CDK6 acted as target genes of miR-185-5p to regulate the progress of colon cancer. And CDK4 and CDK6 were predicted as downstream targets of miR-185-5p. Finally, it was demonstrated that HAGLR regulated tumor progression in vivo. CONCLUSION: Lnc HAGLR promoted the development of colon cancer by miR-185-5p/CDK4/CDK6 axis, and lnc HAGLR might be potential target for colon cancer.

[Clinical observation of mild to moderate lumbodorsal fascitis treated with distal acupoints along meridian plus exercising combined with penetration needling on yang meridians of back].

OBJECTIVE: To compare the therapeutic effect between distal acupoints along meridian plus exercising combined with penetration needling on yang meridians of back and simple penetration needling on yang meridians of back for mild to moderate lumbodorsal fascitis. METHODS: A total of 60 patients with mild to moderate lumbodorsal fascitis were randomized into an observation group and a control group, 30 cases in each one. In the control group, penetration needling on yang meridians of back was applied at acupoints of the Governor vessel (T2-L5) and the first line of bladder meridian, penetration needling was performed from the top down along the governor vessel and the first line of bladder meridian of the lumbar back pain (from one acupoint down to another acupoint), until there was no pain. In the observation group, distal acupoints along meridian plus exercising were adopted on the base of treatment in the control group. The distal acupoints along meridian plus exercising was applied at Cuanzhu (BL 2) for 30 min, at the same time, lumbar back anteflexion, hypsokinesis and turning sides were used in combination for 10 min. And then penetration needling on yang meridians of back was performed. The treatments were given once a day, 5 consecutive treatments a week, 1 week as a course and 2 courses were required. The visual analogue scale (VAS) score and Oswestry disability index (ODI) before treatment, after treatment and 1-month in follow-up were observed in the two groups, and the clinical effects were compared. RESULTS: Compared before treatment, the VAS score and ODI were reduced after treatment in the two groups (P<0.01). The changes of the VAS score and ODI in the observation group were larger than those in the control group (P<0.01, P<0.05). In follow-up, the VAS score and ODI in the observation group were lower than those in the control group (P<0.05). The total effective rate in the observation group was 90.0% (27/30), which was superior to 83.3% (25/30) in the control group (P<0.05). CONCLUSION: Distal acupoints along meridian plus exercising combined with penetration needling on yang meridians of back have a better therapeutic effect than simple penetration needling on yang meridians of back in the treatment of mild to moderate lumbodorsal fascitis.

Quantitative Serum Proteomic Analysis of Essential Hypertension Using iTRAQ Technique.

Essential hypertension (EH) is a risk factor for some severe diseases. This study aimed to screen out serum special proteins and seek interaction between them, which would provide new therapeutic targets and elucidate the comprehensive pathophysiological mechanism for EH. Patients with EH (Group A, n = 47) and healthy controls (HC) (Group B, n = 47) were recruited in this study. Serums from the two groups were analyzed with isobaric tags for relative and absolute quantitation coupled two-dimensional liquid chromatography followed by electrospray ionization-tandem mass spectrometry technique, while the candidate special proteins were verified with ELISA and western blot. A total of 404 proteins were identified, of which 30 proteins were upregulated (>1.2-fold, p < 0.05) and 81 proteins were downregulated (<0.833-fold, p < 0.05) compared with HC group. With GO, KEGG analysis, and literature retrieval, 4 proteins, cathepsin G, transforming growth factor beta-1, hyaluronidase-1, and kininogen-1, were found jointly involved in the renin-angiotensin-aldosterone system and kallikrein-kinin system. The profiles of these 4 candidate proteins were confirmed with ELISA and western blot. The concentration variation of these 4 proteins could better predict the occurrence and illustrate the pathophysiological mechanism of EH. And their discovery may help pave the way for exploring new therapies of EH.