Combining Porous Se@SiO2 Nanocomposites and dECM Enhances the Myogenic Differentiation of Adipose-Derived Stem Cells.

Background: Volumetric Muscle Loss (VML) denotes the traumatic loss of skeletal muscle, a condition that can result in chronic functional impairment and even disability. While the body can naturally repair injured skeletal muscle within a limited scope, patients experiencing local and severe muscle loss due to VML surpass the compensatory capacity of the muscle itself. Currently, clinical treatments for VML are constrained and demonstrate minimal efficacy. Selenium, a recognized antioxidant, plays a crucial role in regulating cell differentiation, anti-inflammatory responses, and various other physiological functions. Methods: We engineered a porous Se@SiO2 nanocomposite (SeNPs) with the purpose of releasing selenium continuously and gradually. This nanocomposite was subsequently combined with a decellularized extracellular matrix (dECM) to explore their collaborative protective and stimulatory effects on the myogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs). The influence of dECM and NPs on the myogenic level, reactive oxygen species (ROS) production, and mitochondrial respiratory chain (MRC) activity of ADSCs was evaluated using Western Blot, ELISA, and Immunofluorescence assay. Results: Our findings demonstrate that the concurrent application of SeNPs and dECM effectively mitigates the apoptosis and intracellular ROS levels in ADSCs. Furthermore, the combination of dECM with SeNPs significantly upregulated the expression of key myogenic markers, including MYOD, MYOG, Desmin, and myosin heavy chain in ADSCs. Notably, this combination also led to an increase in both the number of mitochondria and the respiratory chain activity in ADSCs. Conclusion: The concurrent application of SeNPs and dECM effectively diminishes ROS production, boosts mitochondrial function, and stimulates the myogenic differentiation of ADSCs. This study lays the groundwork for future treatments of VML utilizing the combination of SeNPs and dECM.

Ginkgolide K delays the progression of osteoarthritis by regulating YAP to promote the formation of cartilage extracellular matrix.

Osteoarthritis (OA) is a degenerative disease characterized by cartilage wear and degradation. Ginkgolide K (GK) is a natural compound extracted from Ginkgo biloba leaves and possesses anti-inflammatory and anti-apoptotic effects. We found that the biological characteristics of GK were highly consistent with those of OA medications. This study aimed to determine and verify the therapeutic effect of GK on OA and mechanism of its therapeutic effect. For the in vivo experiment, OA rats were regularly injected in the articular cavity with GK, and the curative effects were observed after 4 and 8 weeks. For the in vitro experiment, we treated OA chondrocytes with different concentrations of GK and then detected the related indices of OA. Through the in vivo and in vitro experiments, we found that GK could promote the production of major components of the cartilage extracellular matrix. Transcriptome sequencing revealed that GK may activate hypoxia-inducible factor 1 alpha via the hypoxia signaling pathway, which, in turn, activates yes-associated protein and inhibits apoptosis of OA chondrocytes. GK has a therapeutic effect on OA and, therefore, has the potential to be developed into a new drug for OA treatment.

Robotic-assisted systems for the safe and reliable treatment of femoral neck fractures: retrospective cohort study.

BACKGROUND: Robots are being used in a wide range of surgical procedures. However, in clinical practice, the efficacy of orthopedic robotic-assisted treatment of femoral neck fractures is still poorly reported, particularly in terms of screw placement accuracy, femoral neck fracture healing rates and postoperative functional recovery. Moreover, there is a lack of comparative analysis between robot-assisted surgery and traditional surgical approaches. PURPOSE: The purpose of this study was to compare the clinical outcomes of patients with femoral neck fractures treated with TiRobot-assisted hollow screw fixation with those of patients with femoral neck fractures treated with traditional surgical approaches. METHODS: This study included 112 patients with femoral neck fracture who were treated from March 2017 to October 2021 with percutaneous hollow screw internal fixation. These included 56 cases in the TiRobot-assisted surgery group and 56 cases in the standard surgery group. After at least 1 year of follow-up, the treatment outcomes of the two groups were compared, including the amount of intraoperative bleeding, the duration of intraoperative fluoroscopy, the number of guide pin positioning adjustments, the length of hospital stay, the accuracy rate of screw placement, the final Harris Hip Score, the fracture healing rate, and the rate of femoral head necrosis. Statistical analysis software was used to process and analyze the result. RESULTS: The TiRobot-assisted group had a statistically significant improvement over the control group in terms of intraoperative bleeding, the duration of intraoperative fluoroscopy, the number of guide pin positioning adjustments, length of hospital stay, accuracy of screw placement and incidence of femoral head necrosis (P < 0.05). There was no statistically significant difference in time to surgery, final Harris hip score and fracture healing rate (P > 0.05). CONCLUSION: This study shows that TiRobot-assisted surgery has the advantages of short hospital stay, high safety, minimally invasive, high success rate of nail placement, and can reduce the amount of intraoperative radiation and the incidence of femoral head necrosis, thus achieving satisfactory clinical outcomes, and is worthy of clinical promotion.

Human Infrapatellar Fat Pad Mesenchymal Stem Cell-Derived Extracellular Vesicles Inhibit Fibroblast Proliferation by Regulating MT2A to Reduce Knee Arthrofibrosis.

Knee arthrofibrosis is one of the most serious complications of knee surgery; however, its pathogenesis is unclear, and current treatment methods have not achieved satisfactory results. Mesenchymal stem cells (MSCs) have good anti-inflammatory and antifibrotic properties, and studies have reported that human infrapatellar fat pad-derived MSCs (IPFSCs) have the advantages of strong proliferative and differentiating ability, ease of acquisition, and minimal harm to the donor. Increasing evidence has shown that MSCs function through their paracrine extracellular vesicles (EVs). Our study is aimed at exploring the effects of human IPFSC-derived EVs (IPFSC-EVs) on knee arthrofibrosis and the underlying mechanisms in vivo and in vitro. In the in vivo study, injecting IPFSC-EVs into the knee joint cavity effectively reduced surgery-induced knee arthrofibrosis in rats. In the in vitro study, IPFSC-EVs were found to inhibit the proliferation of fibroblasts in the inflammatory environment. Additionally, we screened a potential IPFSC-EV molecular target, metallothionein 2A (MT2A), using RNA sequencing. We found that silencing MT2A partially reversed the inhibitory effect of IPFSC-EVs on fibroblast proliferation in the inflammatory environment. In conclusion, IPFSC-EVs inhibit the progression of knee arthrofibrosis by regulating MT2A, which inhibits fibroblast proliferation in the inflammatory environment.

Synergistic Effect of Hydrogen and 5-Aza on Myogenic Differentiation through the p38 MAPK Signaling Pathway in Adipose-Derived Mesenchymal Stem Cells.

Background and Objectives: This study aims to clarify the systems underlying regulation and regulatory roles of hydrogen combined with 5-Aza in the myogenic differentiation of adipose mesenchymal stem cells (ADSCs). Methods and Results: In this study, ADSCs acted as an in vitro myogenic differentiating mode. First, the Alamar blue Staining and mitochondrial tracer technique were used to verify whether hydrogen combined with 5-Aza could promote cell proliferation. In addition, this study assessed myogenic differentiating markers (e.g., Myogenin, Mhc and Myod protein expressions) based on the Western blotting assay, analysis on cellular morphological characteristics (e.g., Myotube number, length, diameter and maturation index), RT-PCR (Myod, Myogenin and Mhc mRNA expression) and Immunofluorescence analysis (Desmin, Myosin and ß-actin protein expression). Finally, to verify the mechanism of myogenic differentiation of hydrogen-bound 5-Aza, we performed bioinformatics analysis and Western blot to detect the expression of p-P38 protein. Hydrogen combined with 5-Aza significantly enhanced the proliferation and myogenic differentiation of ADSCs in vitro by increasing the number of single-cell mitochondria and upregulating the expression of myogenic biomarkers such as Myod, Mhc and myotube formation. The expressions of p-P38 was up-regulated by hydrogen combined with 5-Aza. The differentiating ability was suppressed when the cells were cultivated in combination with SB203580 (p38 MAPK signal pathway inhibitor). Conclusions: Hydrogen alleviates the cytotoxicity of 5-Aza and synergistically promotes the myogenic differentiation capacity of adipose stem cells via the p38 MAPK pathway. Thus, the mentioned results present insights into myogenic differentiation and are likely to generate one potential alternative strategy for skeletal muscle related diseases.

Galangin ameliorates osteoarthritis progression by attenuating extracellular matrix degradation in chondrocytes via the activation of PRELP expression.

Osteoarthritis (OA) is primarily characterized by progressive degeneration and destruction of articular cartilage. Currently, there is no effective method to treat OA. The metabolic disturbance of cartilage extracellular matrix (ECM) and oxidative stress are critical to promote OA progression. Galangin (Gal) is a small molecule compound that pertains to flavonoids. To determine the protective effect and mechanism of Gal against OA progression, various experiments were performed in vitro and in vivo. In vitro, Gal promoted ECM production and attenuated ECM degradation in human OA chondrocytes. The expression of ECM components from human OA cartilage explants was also stimulated by Gal treatment. As demonstrated from the in vivo study, the intra-articular injection of Gal delayed OA progression in rat models. Moreover, RNA sequencing analysis showed that proline/arginine-rich end leucine repeat protein (PRELP) was a molecular target of Gal activity. Gal inhibited oxidative stress and attenuated ECM degradation by activating PRELP expression. The study demonstrated that Gal could attenuate ECM degradation and ameliorate OA progression, and PRELP may be a potential candidate drug of Gal for treating OA.

Sinensetin attenuates IL-1ß-induced cartilage damage and ameliorates osteoarthritis by regulating SERPINA3.

Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degeneration, subchondral bone sclerosis, synovial hyperplasia and osteophyte formation as the main pathological manifestations. Age, mechanical stress and inflammation are the main factors that induce joint degeneration in the pathogenesis of OA. Sinensetin (SIN) is a natural flavonoid with anti-inflammatory and antioxidant properties. This study aims to investigate the effect of SIN on OA. We have investigated the anti-inflammatory and chondroprotective effects of SIN on IL-1ß-induced human OA chondrocytes and a rat OA model. In vitro, human chondrocytes were induced by 5 ng mL-1 IL-1ß and treated with different concentrations of SIN. The results suggest that SIN can inhibit IL-1ß-induced overproduction of pro-inflammatory mediators in human OA chondrocytes, including COX2, iNOS, TNF-α and IL-6, and also reduce the production of MMP13 and MMP9, thus protecting the degradation of the extracellular matrix. In addition, SIN can inhibit the activation of NF-κB by regulating the expression of SERPINA3. In an in vivo experiment, rats were randomly divided into 3 groups, namely the sham operation group, OA model group and SIN group, and were given normal saline or 20 mg kg-1 SIN, respectively. The knee cartilage tissue was removed 6 weeks after surgery for analysis and detection, and our studies have shown that SIN can effectively delay the progression of OA in rats and protect cartilage. In conclusion, our study shows that SIN has good application potential in the treatment of OA.

DL-3-N-Butylphthalide Promotes Cartilage Extracellular Matrix Synthesis and Inhibits Osteoarthritis Development by Regulating FoxO3a.

Osteoarthritis (OA) has been reported as a progressive disease in the elderly, primarily characterized by degenerated articular cartilage. There has been no satisfactory drug for the treatment of OA. DL-3-n-butylphthalide (NBP), a small molecule compound extracted from celery seeds, may have antiapoptotic, antioxidant, and anti-inflammatory activities in numerous studies. However, the effects of NBP on OA and its mechanisms have been rarely reported. In this study, the effect of NBP on OA in vitro and in vivo and its possible mechanism were investigated. The results showed that NBP injection into the knee joint inhibited osteoarthritis development in a rat model of osteoarthritis induced by DMM+ACLT. NBP could increase the expressions of extracellular matrix-related components (such as type II collagen, aggrecan, proteoglycan 4, and SRY-box 9) in human osteoarthritic chondrocytes and cartilage explants. Moreover, NBP promoted the expressions of SOD and CAT. NBP upregulated the expression of FoxO3a by inhibiting the PI3K/AKT pathway, which subsequently inhibited the apoptosis of human OA chondrocytes. In conclusion, NBP promotes cartilage extracellular matrix synthesis and inhibits osteoarthritis development and the underlying mechanism related to the activation of FoxO3a.

The effect of cartilage decellularized extracellular matrix-chitosan compound on treating knee osteoarthritis in rats.

Knee osteoarthritis (KOA) refers to a common disease in orthopaedics, whereas effective treatments have been rarely developed. As indicated from existing studies, chondrocyte death, extracellular matrix degradation and subchondral bone injury are recognized as the pathological basis of KOA. The present study aimed to determine the therapeutic effect of decellularized extracellular matrix-chitosan (dECM-CS) compound on KOA. In this study, rat knee cartilage was decellularized, and a satisfactory decellularized extracellular matrix was developed. As suggested from the in vitro experiments, the rat chondrocytes co-cultured with allogeneic dECM grew effectively. According to the results of the alamar blue detection, dECM did not adversely affect the viability of rat chondrocytes, and dECM could up-regulate the genes related to the cartilage synthesis and metabolism. As reported from the animal experiments, dECM-CS compound could protect cartilage, alleviate knee joint pain in rats, significantly delay the progress of KOA in rats, and achieve high drug safety. In brief, dECM-CS compound shows a good therapeutic effect on KOA.

IFN-α-2b Inhibits the Proliferation and Migration of Fibroblasts via the TGFß/Smad Signaling Pathway to Reduce Postoperative Epidural Fibrosis.

Epidural fibrosis after lumbar laminectomy refers to a serious complication, and excessive proliferation of fibroblasts is considered the major factor. Interferon-alpha-2b (IFN-α-2b) can exert antiviral and antiproliferative effects, which has been suggested to effectively prevent several fibrotic diseases. However, the effect of IFN-α-2b on the prevention of epidural fibrosis (EF) and its possible mechanism remain unclear. In this study, in vitro and in vivo experiments were performed to examine the possible mechanism of IFN-α-2b for preventing EF. Cell counting kit-8 (CCK-8), cell cycle test, Edu incorporation, wound healing assay, transwell test, and Western blotting assay were performed to investigate the inhibitory effect of IFN-α-2b on the proliferation and migration of fibroblasts in vitro. As indicated from the results, IFN-α-2b was capable of inhibiting proliferation and migration of fibroblasts and inhibiting the activity of the transforming growth factor ß (TGFß)/Smad signaling pathway. In vivo, the effect of IFN-α-2b on the reduction of EF was determined by performing histological macroscopic evaluation and histological and immunohistochemical staining. As suggested from the results, IFN-α-2b significantly inhibited EF after laminectomy. As revealed from the mentioned results, IFN-α-2b may have a promising application for preventing EF in the future.

Risk factors for femoral neck fracture in elderly population. / è€å¹´è‚¡éª¨é¢ˆéª¨æŠ˜çš„å±é™©å› ç´ .

OBJECTIVES: To explore the risk factors for femoral neck fracture in elderly population. METHODS: A total of 124 elderly patients (≥60 years old) in hospital for trauma were enrolled, including 71 patients (57%) with femoral neck fracture and 53 non-femoral neck fracture patients (43%). All patients' age, gender, body mass index (BMI), bone mineral density (BMD), thigh length and average circumference were collected. Single factor analysis and multivariate logistic regression analysis were performed to explore whether the above factors were risk factors for femoral neck fracture. RESULTS: Single factor analysis showed that the age, gender, BMI, BMD, thigh length, and average thigh circumference between the 2 groups were statistically different (all P<0.05), and multi-factor logistic regression analysis showed that age, gender, BMI, BMD, thigh length, and average thigh circumference were influencing factors for femoral neck fracture in elderly population (all P<0.05). CONCLUSIONS: Older age, female, lower BMI index (low body weight), lower BMD (osteoporosis), longer thigh length, and lower average circumference are risk factors for femoral neck fracture in the elderly population.

Homoharringtonine inhibits fibroblasts proliferation, extracellular matrix production and reduces surgery-induced knee arthrofibrosis via PI3K/AKT/mTOR pathway-mediated apoptosis.

BACKGROUND: The prevention of surgery-induced intraarticular fibrosis remains a challenge following orthopedic surgery. Homoharringtonine (HHT) has been reported to have positive effects in preventing various kinds of fibrosis. However, little is known regarding its effect as well as the potential mechanism of HHT in preventing surgery-induced intraarticular fibrosis. METHODS: Various concentrations of HHTs were locally applied in vivo to reduce knee intraarticular fibrosis in rabbits. Histological macroscopic assessments such as hematoxylin and eosin (HE) staining, Masson's trichrome staining, and Picric-sirius red polarized light were used to evaluate the effect of HHT in reducing intraarticular fibrosis. CCK-8, cell cycle assay, and EdU incorporation assay were used in vitro to detect HHT's effect on inhibiting fibroblast viability and proliferation. The effect of HHT on fibroblast differentiation, extracellular matrix production, and apoptosis were evaluated by western blot, flow cytometry, immunofluorescent staining, and TUNEL analysis. Moreover, the expressions of PI3K/AKT/mTOR signaling pathway were detected. RESULTS: The results demonstrated that HHT could reduce the formation of intraarticular fibrosis. HHT was also found to induce fibroblast apoptotic cell death in a dose- and time-dependent manner in vitro. Moreover, HHT could effectively inhibit the production of the extracellular matrix secreted by fibroblasts and inhibited the expression of p-PI3K, p-AKT, and p-mTOR in a dose-dependent manner. After treating with insulin-like growth factor-1 (IGF-1), an activator of the PI3K/AKT axis, the expressions of pro-apoptosis-related proteins were decreased, and the fibroblast apoptosis rate was also inhibited. CONCLUSIONS: In conclusion, this study demonstrated that HHT could reduce the formation of intraarticular fibrosis through the inhibition of fibroblast proliferation, extracellular matrix production, and the induction of fibroblast apoptotic cell death. Furthermore, its potential mechanism may be through the suppression of the PI3K/AKT/mTOR signaling pathway.

Glabridin inhibits osteoarthritis development by protecting chondrocytes against oxidative stress, apoptosis and promoting mTOR mediated autophagy.

Osteoarthritis (OA) is a common chronic degenerative disease that affects the elderly. Thus far, no pharmacological therapy approved by regulators has shown a convincing effect on OA. Glabridin, a small molecule, is a well-known and powerful natural antioxidant, which has a strong scavenging effect on free radicals. This study attempted to explore the role and underlying mechanisms of Glabridin on OA both in vitro and in vivo. In the in vitro study, Glabridin was found to increase the expression levels of extracellular matrix (ECM) related genes, Collagen II, Aggrecan (ACAN), SRY-box 9 (SOX9) and proteoglycan 4 (PRG4). Moreover, Glabridin was observed to significantly reduce the level of oxidative stress in OA chondrocytes while effectively reducing the apoptosis of chondrocytes. Glabridin was also found to significantly increase the autophagy of human OA chondrocytes. During the in vivo study, intraarticular injection of Glabridin was observed to alleviate OA progression and protect chondrocytes against apoptosis following anterior cruciate ligament transection (ACLT) in rats. Furthermore, the mammalian target of rapamycin (mTOR) mediated autophagy was identified as one of the potential mediators of Glabridin activity. Overall, Glabridin protects articular cartilage from damage in rats with OA by protecting chondrocytes against oxidative stress, apoptosis and promoting mTOR mediated autophagy.

N-Acetyl-Cysteine-Loaded Biomimetic Nanofibrous Scaffold for Osteogenesis of Induced-Pluripotent-Stem-Cell-Derived Mesenchymal Stem Cells and Bone Regeneration.

To regenerate bone tissues, we investigated the osteogenic differentiation of induced-pluripotent-stem-cell-derived mesenchymal stem cells (iPSC-MSCs) and bone regeneration capacities using N-acetyl cysteine (NAC)-loaded biomimetic nanofibers of hydroxyapatite/silk fibroin (HAp/SF). The addition of HAp and NAC decreased the diameters of the electrospun fibers and enhanced the mechanical properties of the silk scaffold. The release kinetic curve indicated that NAC was released from NAC/HAp/SF nanofibers in a biphasic pattern, with an initial burst release stage and a later sustained release stage. This pattern of release of NAC encapsulated on the NAC/HAp/SF scaffolds prolonged the release of high concentrations of NAC, thereby largely affecting the osteogenic differentiation of iPSC-MSCs and bone regeneration. Thus, a new silk electrospun scaffold was developed. HAp was used as a separate nanocarrier for recharging the NAC concentration, which demonstrated the promising potential for the use of NAC/HAp/SF for bone tissue engineering.

Intra-articular injection of decellularized extracellular matrices in the treatment of osteoarthritis in rabbits.

BACKGROUND: We investigated the role of decellularized cartilage matrix in osteoarthritis to seek a new treatment for this disease. METHODS: Knee cartilage from rabbits was decellularized and the degree of decellularization was assessed. A grinder was used to turn acellular cartilage into particles, which were then used in a suspension. Thirty New Zealand white rabbits were subjected to an operation on their anterior cruciate ligament for the osteoarthritis model. The success of the animal model of osteoarthritis was evaluated using results from six rabbits. The remaining 24 rabbits were randomly divided into four groups (groups A, B, C, and D). Rabbits in groups A, B, C, and D were injected with 200 µl of normal saline, 200 µl of 10% (w/v) cartilage decellularized suspension, 200 µl of 20% (w/v) cartilage decellularized suspension, and 200 µl of 40% (w/v) cartilage decellularized suspension into the knee joints, respectively. Macroscopic and microscopic assessments were performed three months after surgery to assess the degree of osteoarthritic changes. RESULTS: Histological and biochemical analysis revealed that the cartilage decellularized matrix removed cells after decellularization but retained components of collagen and glycosaminoglycan. Group A exhibited the most significant changes from osteophyte and cartilage erosion, which was macroscopically observable on the surface of the femoral cartilage. HE staining in group A revealed damage to the cartilage surface, disorganized chondrocytes, and spontaneous fibrocartilage formation. Safranin O-fast green staining revealed a cavity formed at the osteochondral junction in group A that did not appear in other groups. CONCLUSION: Our study shows that decellularized cartilage matrix has a certain therapeutic effect on osteoarthritis and provides new insights in the treatment of osteoarthritis.

Emodin promotes fibroblast apoptosis and prevents epidural fibrosis through PERK pathway in rats.

BACKGROUND: Laminectomy is usually classed as a common orthopedic surgery, but postoperative epidural fibrosis often leads to less-than-desirable clinical outcomes. As demonstrated by prior studies, emodin (EMO) exerts an anti-fibrotic effect. Here, we carried out investigation into the inhibitory effect created by EMO application on epidural fibrosis after laminectomy in rats. METHODS: The paper conducts a series of experiment. In vitro, we observed the effect of EMO on fibroblasts by Cell Counting Kit-8 (CCK-8) assay. Apoptosis of fibroblasts induced by EMO was detected by western blot, TUNEL assay, and flow cytometry. The results revealed that EMO was capable of inducing fibroblast apoptosis, and the proteins of PERK pathway also changed accordingly. In vivo, the effect of EMO on epidural fibrosis in 12 male Sprague-Dawley rats was observed by histological staining. RESULTS: CCK-8 assay indicated that EMO was effective in reducing fibroblast viability in a time- and a dose-dependent manner. TUNEL assay and flow cytometry analysis have demonstrated that the apoptotic rate of fibroblasts increased as the EMO concentration rose. Western blot analysis proved that EMO promoted the relative expression of p-perk and p-eIF2α and that the expression of its downstream proteins CHOP and GRP78 was also enhanced. The expression of apoptotic protein Bax and cleaved PARP was upregulated, whereas the expression of anti-apoptotic protein Bcl-2 was downregulated. In addition, histological and immunohistochemical analysis demonstrated that EMO functioned to inhibit epidural fibrosis and increase GRP78 expression in fibrous tissue by promoting apoptosis of fibroblasts. CONCLUSIONS: EMO could have inhibitory effect on epidural fibrosis in a concentration-dependent manner. The potential mechanism might be through PERK signaling pathway to promote fibroblast apoptosis. It has a possibility to be taken as a novel method for the treatment of epidural fibrosis.

Negative regulation of PI3K/AKT/mTOR axis regulates fibroblast proliferation, apoptosis and autophagy play a vital role in triptolide-induced epidural fibrosis reduction.

Fibroblasts excessive proliferation was considered as a decisive reason for epidural fibrosis, which was known as a serious complication of lumbar laminectomy. As a traditional Chinese medicine, triptolide (TP) was used to be proved effective in preventing several fibrosis scar formation diseases. However, little is known about the effect of TP on preventing epidural fibrosis and its possible mechanism. Here, we performed in vitro and in vivo experiments to detect the possible mechanism of TP in preventing epidural fibrosis. In vitro, the effect of TP on impacting fibroblasts proliferation activities was detected by CCK-8, cell cycle assay and EdU incorporation assay. Also, the expressions of cell proliferation protein markers and the expressions of p-PI3K, p-AKT, p-mTOR were detect by Western blot. Besides, the effect of TP on inducing fibroblast apoptosis and autophagy was tested by Western blots, flow cytometry, TUNEL staining, Transmission electron microscope (TEM) analysis and LC3 immunofluorescent staining. The results suggested that TP could suppress the activation of PI3K/AKT/mTOR signaling pathway. Meanwhile, TP could inhibit fibroblast proliferation and induce fibroblast apoptosis as well as autophagy, which was known as two cellular self-destructions. Furthermore, we speculated the possible molecular pathway, through which that TP could inhibit fibroblast proliferation, induce fibroblast apoptosis and autophagy. We used PI3-kinase activator (740Y-P) to activate the PI3K/AKT/mTOR signaling. Activation of PI3K/AKT/mTOR signaling pathway increase the proliferation of fibroblasts and suppressed the autophagy and apoptosis induced by TP. In vivo, we built epidural fibrosis models in rats and locally applied TP of various concentrations. Hematoxylin-eosin (HE) and Masson's trichrome were used to detect the effect of TP on reducing epidural fibrosis. And the results showed that TP could significantly reduce the surgery-induced epidural fibrosis. In conclusion, the results above shown that TP could reduce epidural fibrosis formation, and the potential mechanism might through inhibiting fibroblast proliferation and stimulating apoptosis and autophagy via suppressing PI3K/AKT/mTOR signaling pathway. It might provide a novel thought for reducing surgery-induced epidural fibrosis.

Apigenin inhibits fibroblast proliferation and reduces epidural fibrosis by regulating Wnt3a/ß-catenin signaling pathway.

BACKGROUND: Failed back surgery syndrome (FBSS) is a common complication after the laminectomy. Epidural fibrosis is the major cause of lower back pain and other complications. Numerous studies have shown that apigenin (API) could treat various fibrotic diseases by regulating various signaling pathways, whereas no study has discussed whether API can inhibit fibroblast proliferation and reduce epidural fibrosis after the laminectomy by regulating Wnt3a/ß-catenin signaling pathway. METHODS: Human fibroblasts were cultured and treated with API in different concentrations for 24 h. CCK-8 detection and EdU incorporation assay were performed to detect cell viability and cell proliferation. Western blotting analysis was applied to detect expressions of proliferative proteins, Wnt3a, and its downstream proteins. Moreover, the Wnt3a gene was overexpressed in fibroblasts to define the relationship between Wnt3a/ß-catenin signaling pathway and fibroblast proliferation. Wnt3a overexpressed fibroblasts were treated with API to verify if it could reverse the effects of API treatment. Twenty-four Sprague-Dawley rats were randomly divided into four groups. Laminectomy was performed and the rats were gavaged with different doses of API or 5% sodium carboxyl methyl cellulose (CMC-Na) solution for 1 month. The abilities of API to inhibit fibroblast proliferation and to reduce epidural fibrosis were evaluated using histological and immunohistochemical analysis. RESULTS: CCK-8 detection and EdU incorporation assay demonstrated that API could inhibit the viability and proliferation rate of fibroblasts in a concentration-dependent manner. The Western blotting analysis revealed that API could inhibit the expressions of PCNA, cyclinD1, Wnt3a, and its downstream proteins. The overexpression of Wnt3a in fibroblasts could upregulate the expressions of proliferative proteins such as PCNA and cyclinD1. The inhibitory effect of API on PCNA, Wnt3a, and its downstream proteins was partially reversed by overexpression of Wnt3a. Moreover, the results of the histological and immunohistochemical analysis revealed that API could reduce the epidural fibrosis in rats by inhibiting fibroblast proliferation in a dose-dependent manner. CONCLUSIONS: API can inhibit fibroblast proliferation and reduce epidural fibrosis by suppressing Wnt3a/ß-catenin signaling pathway, which can be adopted as a new option to prevent epidural fibrosis after the laminectomy.

Tamoxifen inhibits fibroblast proliferation and prevents epidural fibrosis by regulating the AKT pathway in rats.

Many factors contribute to epidural fibrosis after lumbar laminectomy, particularly the excessive proliferation of fibroblasts. Many studies have shown that tamoxifen (TAM) inhibits fibroblast proliferation and reduces fibrosis, but the detailed effect and mechanism of TAM on preventing epidural fibrosis are unknown. To investigate the effect of TAM on fibroblast proliferation and epidural fibrosis, fibroblasts were cultured and treated with different concentrations of TAM. Cell Counting Kit-8(CCK-8) detection, cell cycle analysis and western blot analysis were used to detect the roles of TAM in regulating fibroblast proliferation. Lumbar laminectomies were performed in rats, and various concentrations of TAM were administered by gavage. Histological and immunohistochemical analyses were used to evaluate the effects of TAM on preventing epidural fibrosis. CCK-8 detection showed that TAM could inhibit fibroblast viability; western blot analysis showed that TAM could decrease the expression of proliferative proteins p-AKT and cyclinD1 and increase the expression of antiproliferative proteins P21 and P27. Histological analysis showed that TAM could reduce epidural fibrosis. Immunohistochemical analysis showed that the p-ATK expression in epidural scar tissue was decreased after TAM treatment. The present study demonstrated that TAM could inhibit fibroblast proliferation and prevent epidural fibrosis, potentially through the regulation of the AKT pathway.