A natural hydrogel complex improves intervertebral disc degeneration by correcting fatty acid metabolism and inhibiting nucleus pulposus cell pyroptosis.

The degeneration of intervertebral discs is strongly associated with the occurrence of pyroptosis in nucleus pulposus (NP) cells. This pyroptosis is characterized by abnormal metabolism of fatty acids in the degenerative pathological state, which is further exacerbated by the inflammatory microenvironment and degradation of the extracellular matrix. In order to address this issue, we have developed a fibrin hydrogel complex (FG@PEV). This intricate formulation amalgamates the beneficial attributes of platelet extravasation vesicles, contributing to tissue repair and regeneration. Furthermore, this complex showcases exceptional stability, gradual-release capabilities, and a high degree of biocompatibility. In order to substantiate the biological significance of FG@PEV in intervertebral disc degeneration (IVDD), we conducted a comprehensive investigation into its potential mechanism of action through the integration of RNA-seq sequencing and metabolomics analysis. Furthermore, these findings were subsequently validated through experimentation in both in vivo and in vitro models. The experimental results revealed that the FG@PEV intervention possesses the capability to reshape the inflammatory microenvironment within the disc. It also addresses the irregularities in fatty acid metabolism of nucleus pulposus cells, consequently hindering cellular pyroptosis and slowing down disc degeneration through the regulation of extracellular matrix synthesis and degradation. As a result, this injectable gel system represents a promising and innovative therapeutic approach for mitigating disc degeneration.

IRF1 governs the expression of SMARCC1 via the GCN5-SETD2 axis and actively engages in the advancement of osteoarthritis.

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by the breakdown of joint cartilage and underlying bone. Macrophages are a type of white blood cell that plays a critical role in the immune system and can be found in various tissues, including joints. Research on the relationship between OA and macrophages is essential to understand the mechanisms underlying the development and progression of OA. Objective: This study was performed to analyze the functions of the IRF1-GCN5-SETD2-SMARCC1 axis in osteoarthritis (OA) development. Methods: A single-cell RNA sequencing (scRNA-seq) dataset, was subjected to a comprehensive analysis aiming to identify potential regulators implicated in the progression of osteoarthritis (OA). In order to investigate the role of IRF1 and SMARCC1, knockdown experiments were conducted in both OA-induced rats and interleukin (IL)-1ß-stimulated chondrocytes, followed by the assessment of OA-like symptoms, secretion of inflammatory cytokines, and polarization of macrophages. Furthermore, the study delved into the identification of aberrant epigenetic modifications and functional enzymes responsible for the regulation of SMARCC1 by IRF1. To evaluate the clinical significance of the factors under scrutiny, a cohort comprising 13 patients diagnosed with OA and 7 fracture patients without OA was included in the analysis. Results: IRF1 was found to exert regulatory control over the expression of SMARCC1, thus playing a significant role in the development of osteoarthritis (OA). The knockdown of either IRF1 or SMARCC1 disrupted the pro-inflammatory effects induced by IL-1ß in chondrocytes, leading to a mitigation of OA-like symptoms, including inflammatory infiltration, cartilage degradation, and tissue injury, in rat models. Additionally, this intervention resulted in a reduction in the predominance of M1 macrophages both in vitro and in vivo. Significant epigenetic modifications, such as abundant H3K27ac and H3K4me3 marks, were observed near the SMARCC1 promoter and 10 kb upstream region. These modifications were attributed to the recruitment of GCN5 and SETD2, which are functional enzymes responsible for these modifications. Remarkably, the overexpression of either GCN5 or SETD2 restored SMARCC1 expression in rat cartilages or chondrocytes, consequently exacerbating the OA-like symptoms. Conclusion: This research postulates that the transcriptional activity of SMARCC1 can be influenced by IRF1 through the recruitment of GCN5 and SETD2, consequently regulating the H3K27ac and H3K4me3 modifications in close proximity to the SMARCC1 promoter and 10 kb upstream region. These modifications, in turn, facilitate the M1 skewing of macrophages and contribute to the progression of osteoarthritis (OA). The Translational Potential of this Article: The study demonstrated that the regulation of SMARCC1 by IRF1 plays a crucial role in the development of OA. Knocking down either IRF1 or SMARCC1 disrupted the pro-inflammatory effects induced by IL-1ß in chondrocytes, leading to a mitigation of OA-like symptoms in rat models. These symptoms included inflammatory infiltration, cartilage degradation, and tissue injury. These findings suggest that targeting the IRF1-SMARCC1 regulatory axis, as well as the associated epigenetic modifications, could potentially be a novel approach in the development of OA therapies, offering new opportunities for disease management and improved patient outcomes.

Injectable kaempferol-loaded fibrin glue regulates the metabolic balance and inhibits inflammation in intervertebral disc degeneration.

To construct an injectable fibrin glue system loaded with kaempferol (FG@F) to improve the bioavailability of kaempferol and observe its efficacy in the treatment of intervertebral disc degeneration (IVDD). Kaempferol-loaded fibrin glue was first synthesized in advance. Subsequently, the materials were characterized by various experimental methods. Then, nucleus pulposus cells (NPCs) were stimulated with lipopolysaccharide (LPS) to establish a degenerative cell model, and the corresponding intervention treatment was conducted to observe the effect in vitro. Finally, the tail disc of rats was punctured to establish a model of IVDD, and the therapeutic effect of the material in vivo was observed after intervertebral disc injection. The FG@F system has good injectability, sustained release and biocompatibility. This treatment reduced the inflammatory response associated with IVDD and regulated matrix synthesis and degradation. Animal experimental results showed that the FG@F system can effectively improve needle puncture-induced IVDD in rats. The FG@F system has better efficacy than kaempferol or FG alone due to its slow release and mechanical properties. The drug delivery and biotherapy platform based on this functional system might also serve as an alternative therapy for IVDD.

Systemic pharmacology reveal the mechanism by which the Qiangjin Zhuanggu Qufeng mixture inhibits LPS-induced pyroptosis of rat nucleus pulposus cells.

OBJECTIVE: Low back pain (LBP) is a worldwide health issue primarily attributed to intervertebral disc degeneration (IVDD). Qiangjin Zhuang Qufeng mixture (QJZG), an approved hospital-based formula with years of clinical application, has demonstrated notable therapeutic effects in the treatment of LBP. Nevertheless, the underlying mechanism by which it alleviates LBP remains uncertain. METHODS: The bioactive constituents of QJZG were initially identified using ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS). Subsequently, network pharmacology was employed to explore the core components and targets. In vivo and in vitro experiments were then conducted to validate the specific mechanism of action of QJZG based on the identified targets and pathways. Following that, ultra-high-performance liquid chromatography/mass spectrometry combined with 16S rRNA gene sequencing of blood and faecal samples was utilized to assess the impact of gut microbiota on faecal and serum metabolites subsequent to QJZG administration in intervertebral disc degeneration (IVDD) rats. RESULTS: The principal constituents of QJZG were identified using UPLC-Q-TOF-MS/MS, revealing a substantial enrichment of flavonoids and triterpenes. Network pharmacology analysis indicated the potential inhibitory effects of QJZG on the NLRP3 inflammasome and downstream inflammatory factors. Furthermore, investigations demonstrated that intervertebral disc degeneration may be attributed to pyroptotic cell death within the nucleus pulposus. In vitro experiments were performed utilizing LPS to induce the inflammatory response in nucleus pulposus cells (NPC), and it was observed that QJZG-containing serum significantly suppressed key pyroptosis-related genes and downstream inflammatory factors. Additionally, in vivo experiments substantiated the capacity of QJZG to preserve disc height and ameliorate the progression of disc degeneration. Concurrently, oral pharmacotherapy in animal studies prominently involved the effects of Enterobacteriaceae and Clostridium, closely intertwined with lipid metabolism. CONCLUSIONS: QJZG exhibited a delaying effect on IVDD by preserving the equilibrium between extracellular matrix (ECM) synthesis and degradation in NPCs. This effect was achieved through the suppression of NLRP3 inflammasome expression and the prevention of pyroptosis in NPCs.

Circ_0001498 contributes to lipopolysaccharide-induced lung cell apoptosis and inflammation in sepsis-related acute lung injury via upregulating SOX6 by interacting with miR-574-5p.

Circular RNAs (circRNAs) have important regulation in in sepsis-related acute lung injury (ALI). Circ_0001498 was significantly overexpressed in sepsis-induced acute respiratory distress syndrome. The aims of this study were to explore role and mechanism of circ_0001498 in lipopolysaccharide (LPS)-treated WI-38 cells. Human samples were collected from 56 sepsis patients and 46 healthy volunteers at Liyang People's Hospital. Circ_0001498, microRNA-574-5p (miR-574-5p) or sex-determining region Y-related high-mobility-group box 6 (SOX6) levels were detected via reverse transcription-quantitative polymerase chain reaction assay. Cell viability was determined through Cell Counting Kit-8 assay. Apoptosis rate was examined by flow cytometry. Western blot was used for measurement of proteins. Inflammatory cytokines were detected via enzyme-linked immunosorbent assay. Target relation was analyzed via dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Circ_0001498 was overexpressed in sepsisrelated ALI patients and LPS-treated WI-38 cells. Silencing circ_0001498 reduced LPS-induced cell apoptosis and inflammation. Circ_0001498 interacted with miR-574-5p. The regulation of circ_0001498 knockdown was abolished by miR-574-5p inhibitor. Furthermore, miR-574-5p directly targeted SOX6 and circ_0001498 upregulated SOX6 via targeting miR-574-5p. Overexpression of miR-574-5p alleviated LPS-induced cell injury by downregulating SOX6. This research identified that circ_0001498 facilitated sepsis-related ALI progression by targeting miR-574-5p to upregulate SOX6.

Cuproptosis Combined with lncRNAs Predicts the Prognosis and Immune Microenvironment of Breast Cancer.

Breast cancer (BC), the most common cancer in women, is caused by the uncontrolled proliferation of mammary epithelial cells under the action of a variety of carcinogenic factors. Cuproptosis-related targets have been found to be closely associated with breast cancer development. TCGA obtained 1226 tumor samples, 1073 clinical data, and 37 lncRNAs during univariate Cox multivariate analysis. We used nonnegative matrix factoring (NMF) agglomeration to spot thirty-three potential molecular subsets with totally different cuproptosis-related lncRNA expression patterns. The least absolute shrinkage and selection operator (LASSO) formula and variable Cox multivariate analysis were not used to construct the best prognostic model. The variations in neoplasm mutation burden and factor gene ontology (GO) and gene set enrichment analysis (GSEA) within the high- and low-risk teams were analyzed, and therefore, the potential mechanism of the development of carcinoma was analyzed. We created a prognostic profile consisting of nineteen cuproptosis-related genes (NFE2L2, LIPT1, LIPT2, DLD, etc.) and their connected targets. The correlation between tumor mutational burden (TMB) and clinical manifestations of tumors demonstrates the importance of high- and low-expression bunch data on the incidence of clinical manifestations of tumors. The area under the curve (AUC) shows moderate prophetic power for copper mortality. GO enrichment analysis showed that immunorelated responses were enriched. Correlation analysis of immune cells showed that pathology could play an important role in the prevalence and prognosis of tumors, and there were variations in immune cells between the probable and low-risk groups. Our study suggests that the prognostic characteristic genes associated with cuproptosis can be used as new biomarkers to predict the prognosis of breast cancer patients. In addition, we found that immunotherapy may play a key role in breast cancer treatment regimens. Levels of immune-associated cells and pathways vary significantly among risk groups of breast cancer patients.

[Early effectiveness of unilateral biportal endoscopic discectomy combined with annulus fibrosus suture in the treatment of lumbar disc herniation].

Objective: To analyze the early effectiveness of unilateral biportal endoscopic discectomy (UBED) combined with annulus fibrosus suture in the treatment of lumbar disc herniation (LDH). Methods: The clinical data of 19 patients with LDH treated with UBED and annulus fibrosus suture between October 2020 and October 2021 were retrospectively analyzed. There were 12 males and 7 females with an average age of 39.1 years (range, 26-59 years). The operative segment was L 4, 5 in 13 cases, and L 5, S 1 in 6 cases. The mean disease duration was 6.7 months (range, 3-15 months). Preoperative neurological examination showed that muscle strength, sensation, and tendon reflex weakened or disappeared in varying degrees. Single annulus fibrosus suture (14 cases) or anchor assisted annulus fibrosus suture (5 cases) was selected according to the location of annulus fibrosus tears. Visual analogue scale (VAS) score was used to assess the low back and leg pain before operation and at 3 days, 3 months, and 6 months after operation. Oswestry disability index (ODI) was used to evaluate the function recovery of lumbar spine before operation and at 3 days, 3 months, and 6 months after operation. At 3 days and 3 months after operation, MRI was used to examine the removal of nucleus pulposus and decompression of nerve root. MacNab criteria was used to evaluate the effectiveness at 6 months after operation and the recovery of nerve root function was recorded. Results: All operations were successfully completed with a mean operation time of 52.7 minutes (range, 40-75 minutes). There was no complication such as nerve injury, spinal cord hypertension syndrome, or dural sac tear during operation, and no complication such as infection, aggravation of nerve damage, or cerebrospinal fluid leakage after operation. All the patients were followed up 6-10 months (mean, 8.2 months). Postoperative MRI showed that the herniated disc was completely removed and nerve roots were fully decompressed. During the follow-up, there was no recurrence of disc herniation. The VAS scores of low back pain and leg pain and ODI at each time point after operation significantly improved when compared with those before operation, and those at 6 months after operation further improved than those at 3 days and 3 months after operation, all showing significant differences ( P<0.05). At 6 months after operation, MacNab standard was used to evaluate the effectiveness, and the results were excellent in 14 cases, good in 4 cases, and fair in 1 case, with an excellent and good rate of 94.7%. Neurological examination showed that the sensation and muscle strength of the affected nerve root innervated area recovered significantly when compared with those before operation ( P<0.05); the recovery of tendon reflex was not obvious, showing no significant difference when compared with that before operation ( P>0.05). Conclusion: UBED combined with annulus fibrosus suture is a safe and effective technique for LDH and early effectiveness is satisfactory.

Lumbar interbody fusion with bilateral cages using a biportal endoscopic technique with a third portal.

BACKGROUND: Unilateral biportal endoscopic lumbar interbody fusion (ULIF) with one cage results in fewer definitive fusions (Park et al. in Neurosurg Rev 42(3):753-761, 2019). We succeeded in inserting bilateral cages during ULIF. METHOD: We attempted posterior ULIF for degenerative lumbar spondylolisthesis with bilateral recess stenosis. With the help of a third portal, ULIF with bilateral cage insertion was performed under general anaesthesia. CONCLUSIONS: We successfully performed ULIF with bilateral cages with the help of a third portal. This procedure may be an alternative for treating lumbar stenosis with instability.

Electroacupuncture suppresses glucose metabolism and GLUT-3 expression in medial prefrontal cortical in rats with neuropathic pain.

BACKGROUND: Accumulating evidence has demonstrated that the electroacupuncture (EA) stimulation could effectively alleviate neuropathic pain. The medial prefrontal cortex (mPFC) is a vital part of the cortical representation of pain in the brain, and its glucose metabolism is mostly affected in the progression of pain. However, the central mechanism of EA analgesia remains unclear. METHODS: Fifty-four male SD rats were equally randomized into sham surgery (Sham) group, chronic constriction injury (CCI) group and EA stimulation (EA) group. The CCI model, involving ligature of the right sciatic nerve, was established in all animals except the Sham group. EA stimulation was applied on the right side acupoints of Huantiao (GB30) and Yanglingquan (GB34) in the EA group. Paw withdrawal threshold (PWT) and paw thermal withdrawal latency (PWL) were measured. The 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) was used to evaluate glucose metabolism changes in the mPFC. The expression of glucose transporter 3 (GLUT-3) in the mPFC was determined by immune histochemistry and ELISA. RESULTS: Comparing with CCI groups, EA treatment was obviously reversed CCI-induced mechanical allodynia (P < 0.01), thermal hyperalgesia (P < 0.01) and the increase of glucose metabolism in the left mPFC (P < 0.05). Furthermore, EA treatment significantly decreased the protein expression of GLUT-3 in the left mPFC (P < 0.01). CONCLUSIONS: Our results indicate that EA analgesia effect may be related to suppressing the glucose metabolism and GLUT-3 expression in the mPFC. This study could provide a potential insight into the central mechanisms involved in the analgesic effect of EA.

Adipose-derived stem cell sheets combined with ß-tricalcium phosphate/collagen-I fiber scaffold improve cell osteogenesis.

Transplantation of cell-based material is a promising approach for the treatment of critical bone defects. However, it is still limited by the lack of suitable scaffold material or abundant seeding cell sources. The present study aimed to establish a novel composite of an adipose-derived stem cell (ADSC) sheet and a synthetic porous ß-tricalcium phosphate/collagen-I fiber (ß-TCP/COL-I) scaffold to enhance osteogenic activity. ADSCs were isolated from 3-week-old female Sprague Dawley rats and the ADSC sheets were prepared in an osteoinductive medium. The study groups included the ADSC sheets/scaffold, scattered ADSCs/scaffold, ADSC sheet alone and scaffold alone. Scanning electron microscopy and energy-dispersive spectrometry were used to observe cell-scaffold interactions and analyze the relative calcium content on the composites' surface. Alizarin red S staining was used to examine the calcium deposition. ELISA and reverse transcription-quantitative PCR were used to detect the expression levels of alkaline phosphatase (ALP), osteocalcin (OCN) and osteopontin (OPN). The results revealed that ADSCs were able to tightly adhere to the ß-TCP/COL-I scaffold with no cytotoxicity. The calcifying nodules reaction was positive on ADSC sheets and gradually increased after osteogenic induction. In addition, the ß-TCP/COL-I scaffold combined with ADSC sheets was able to significantly enhance the expression levels of ALP, OCN and OPN and increase the superficial relative calcium content compared to scattered ADSCs/scaffold or the ADSC sheet alone (P<0.05). The results indicated that ADSCs possess a strong osteogenic potential, particularly in the cell-sheet form and when compounded with the ß-TCP/COL-I scaffold, compared to scattered ADSCs with a ß-TCP/COL-I scaffold or an ADSC sheet alone. This novel composite may be a promising candidate for bone engineering.

Electroacupuncture suppresses glucose metabolism and GLUT-3 expression in medial prefrontal cortical in rats with neuropathic pain

BACKGROUND: Accumulating evidence has demonstrated that the electroacupuncture (EA) stimulation could effectively alleviate neuropathic pain. The medial prefrontal cortex (mPFC) is a vital part of the cortical representation of pain in the brain, and its glucose metabolism is mostly affected in the progression of pain. However, the central mechanism of EA analgesia remains unclear. METHODS: Fifty-four male SD rats were equally randomized into sham surgery (Sham) group, chronic constriction injury (CCI) group and EA stimulation (EA) group. The CCI model, involving ligature of the right sciatic nerve, was established in all animals except the Sham group. EA stimulation was applied on the right side acupoints of Huantiao (GB30) and Yanglingquan (GB34) in the EA group. Paw withdrawal threshold (PWT) and paw thermal withdrawal latency (PWL) were measured. The 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET) was used to evaluate glucose metabolism changes in the mPFC. The expression of glucose transporter 3 (GLUT-3) in the mPFC was determined by immune histochemistry and ELISA. RESULTS: Comparing with CCI groups, EA treatment was obviously reversed CCI-induced mechanical allodynia (P < 0.01), thermal hyperalgesia (P < 0.01) and the increase of glucose metabolism in the left mPFC (P < 0.05). Furthermore, EA treatment significantly decreased the protein expression of GLUT-3 in the left mPFC (P < 0.01). CONCLUSIONS: Our results indicate that EA analgesia effect may be related to suppressing the glucose metabolism and GLUT-3 expression in the mPFC. This study could provide a potential insight into the central mechanisms involved in the analgesic effect of EA.

[Effect of wrist-ankle acupuncture on the expression of glutamate and NMDA receptor of the spinal dorsal horn in rats with neuropathic pain].

OBJECTIVE: To observe the effect of wrist-ankle acupuncture (WA) stimulation at "R4"- "R5" - "R6" on the expression of glutamate (Glu) and phosphorylated protein NMDAR1(p-NMDAR1) of the spinal dorsal horn in spared nerve injury (SNI) rats, so as to explore its mechanism underlying improvement of SNI. METHODS: A total of 36 SD rats were randomly divi-ded into sham operation, model and WA groups, with 12 rats in each group. The SNI procedure comprised an axotomy and ligation of the tibial and common peroneal nerves leaving the sural nerve intact. Rats of the WA group were treated by acupuncture at "R4"-"R5"-"R6" points from the 5th day to the 14th day after modeling. The mechanical pain thresholds were measured before and 5, 10 and 14 d after SNI, respectively. The cold allodynia was dectected by Acetone solution dropped onto the lateral plantar surface of the paw. Glu content and p-NMDAR1 expression of spinal dorsal horn were detected by 1H-MRS, ELISA and immunohistochemistry Methods. RESULTS: Compared with the sham operation group, the mechanical pain threshold of the model group was significantly decreased (P<0.01), the duration of cold stimulation foot contraction was increased (P<0.01), and the Glu content and p-NMDAR1 expression in the spinal dorsal horn were significantly increased (P<0.05, P<0.01). After WA intervention, the mechanical pain threshold was significantly increased (P<0.01), the duration of cold stimulation was significantly shortened (P<0.01), and Glu content and p-NMDAR1 protein expression of spinal dorsal horn were decreased significantly (P<0.05, P<0.01) in the WA group compared with the model group. CONCLUSION: WA can reduce pain sensitivity in rats with neuropathic pain, possibly by inhibiting the expression of Glu and p-NMDAR1 in the spinal dorsal horn.

[Current Researches and Ideas Regarding Correlation Between Meridians and Viscera].

The correlation between meridians and viscera is the key content of the meridian theory in acupuncture medicine. This paper introduces the close relationship between the meridians running on the body surface and viscera from ancient lite-rature, modern experimental studies (such as acupoint sensitization, neuro-endocrine-immune networks, etc.), and clinical application in the treatment of various conditions with acupuncture, moxibustion, Guasha (scraping) therapies, etc. We also presented some related research ideas (i.e., taking the clinical treatment as the forerunner, paying more attention to multi-disciplinary integration, combining clinical and basic research together, doing research from tridimensional point of review, and making full use of advanced technology, etc.) in order to provide a possibly favorable help for researchers to further reveal the scientific mechanisms underlying correlation between the meridian and viscera in the future.

[Effects of blocking two sites of transforming growth factor-ß/Smads signaling on the formation of scar-related proteins in human skin fibroblasts].

OBJECTIVE: To explore the effects of blocking two sites of TGF-ß/Smads signaling on the formation of scar-related proteins in human skin fibroblasts. METHODS: Two lentivirus vectors encoding soluble TGF-ß receptor II (sTßRII) and mutant Smad 4-Smad 4ΔM4 were respectively transfected into human skin fibroblast cell line human foreskin fibroblast 1 (HFF-1) cells with the optimum multiplicity of infection (MOI) of 50. The protein expressions of sTßRII and Smad 4ΔM4 of the two types of transfected cells were determined by Western blotting so as to compare with those of the untransfected cells. The HFF-1 cells were divided into 6 groups as named below according to the random number table, with 6 dishes in each group, 1×10(4) cells per dish. Co-transfection group, transfected with the two previous lentivirus vectors, mixed with the ratio of 1:1 and MOI of 50, and then stimulated with 5 ng/mL TGF-ß1 for 72 h; sTßRII group, transfected with lenti-sTßRII with MOI of 50, with the other treatment as above; Smad 4ΔM4 group, transfected with lenti-Smad 4ΔM4 with MOI of 50, with the other treatment as above; negative virus group, transfected with empty lentivirus vector, with the other treatment as above; positive control group, stimulated with 5 ng/mL TGF-ß1 for 72 h; and blank control group, conventionally cultured without any other treatment. After stimulation, Western blotting and real-time fluorescent quantitative RT-PCR were respectively used to determine the protein and mRNA expressions of fibronectin in cells of each group. ELISA and Sircol collagen assay were respectively used to determine the protein expressions of connective tissue growth factor (CTGF) and total collagen in the cell culture supernate of each group. Data were processed with one-way analysis of variance and SNK-(q test). RESULTS: (1) HFF-1 cells transfected with lenti-sTßRII and HFF-1 cells transfected with lenti-Smad 4ΔM4 respectively expressed higher levels of sTßRII protein and Smad 4ΔM4 protein compared with those of untransfected cells, confirming that HFF-1 cells transfected with the two lentivirus vectors can efficiently express the target proteins. (2) There were statistically significant differences in the protein and mRNA expressions of fibronectin in cells of the 6 groups (with F values respectively 53.536 and 24.365, P values below 0.001). The protein and mRNA expressions of fibronectin in cells of positive control group (respectively 1.60 ± 0.18 and 1.99 ± 0.40) were similar with those of negative virus group (respectively 1.60 ± 0.15 and 1.94 ± 0.28, with q values respectively 0.091 and 0.419, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 5.245 to 18.228, P values below 0.05). The protein and mRNA expressions of fibronectin in cells of co-transfection group (respectively 0.60 ± 0.05 and 0.70 ± 0.11) were significantly lower than those of sTßRII group (respectively 0.89 ± 0.13 and 1.24 ± 0.17) and Smad 4ΔM4 group (respectively 0.91 ± 0.14 and 1.28 ± 0.19, with q values from 3.964 to 4.294, P values below 0.05). (3) There were statistically significant differences in the protein expressions of CTGF and total collagen in the cell culture supernate of the 6 groups (with F values respectively 107.680 and 38.347, P values below 0.001). The protein expressions of CTGF and total collagen in the cell culture supernate of positive control group were similar with those of negative virus group (with q values respectively 1.106 and 0.491, P values above 0.05), and they were significantly higher than those of the rest 4 groups (with q values from 6.414 to 26.420, P values below 0.05). The protein expressions of CTGF and total collagen in the cell culture supernate of co-transfection group were significantly lower than those of sTßRII group and Smad 4ΔM4 group (with q values from 3.424 to 7.143, P values below 0.05). CONCLUSIONS: In human skin fibroblasts, blockage of two sites of TGF-ß/Smad signaling can reduce the expression of scar related proteins which are up-regulated by TGF-ß1 to a greater extent than that of blocking one single site.

[Nuclear factor I-C inhibits platelet-derived growth factor-induced enhancement of dermal fibroblast sensitivity to TGF-ß].

OBJECTIVE: To investigate the effect of nuclear factor I-C (NFI-C) on platelet-derived growth factor (PDGF)-induced up-regulation of TGF-ß receptor II (TßRII) in dermal fibroblasts. METHODS: A lentiviral vector containing NFI-C sequence (Lenti-GFP-NFI-C) was transfected into a human foreskin fibroblast cell line (HFF-1). Cultured HFF-1 cells, cells transfected with Lenti-GFP-NFI-C, and cells transfected with a negative virus were stimulated with PDGF-BB, and Western blotting and RT-qPCR were used to detect the expression levels of TßRII in the treated cells. RESULTS: PDGF treatment significantly increased the expression level of TßRII in HFF-1 cells (P<0.05). The cells transfected with Lenti-GFP-NFI-C expressed a significantly lower level of TßRII than non-transfected cells in response to PDGF stimulation (P<0.05), but the negative virus showed no such inhibitory effect (P>0.05). No significant difference was found in the expression level of TßRII protein between cells transfected with Lenti-GFP-NFI-C-transfection before PDGF stimulation and the blank control cells. CONCLUSION: NFI-C can inhibit PDGF-induced up-regulation of TßRII and thus reduce the sensitivity of the dermal fibroblasts to TGF-ß.