The Impact of NLRP3 Inflammasome on Osteoblasts and Osteogenic Differentiation: A Literature Review.

Osteoblasts (OBs), which are a crucial type of bone cells, derive from bone marrow mesenchymal stem cells (MSCs). Accumulating evidence suggests inflammatory cytokines can inhibit the differentiation and proliferation of OBs, as well as interfere with their ability to synthesize bone matrix, under inflammatory conditions. NLRP3 inflammasome is closely associated with cellular pyroptosis, which can lead to excessive release of pro-inflammatory cytokines, causing tissue damage and inflammatory responses, however, the comprehensive roles of NLRP3 inflammasome in OBs and their differentiation have not been fully elucidated, making targeting NLRP3 inflammasome approaches to treat diseases related to OBs uncertain. In this review, we provide a summary of NLRP3 inflammasome activation and its impact on OBs. We highlight the significant roles of NLRP3 inflammasome in regulating OBs differentiation and function. Furthermore, current available strategies to affect OBs function and osteogenic differentiation targeting NLRP3 inflammasome are listed and analyzed. Finally, through the prospective discussion, we seek to provide novel insights into the crucial role of NLRP3 inflammasome in diseases related to OBs and offer valuable information for devising treatment strategies.

Porcine platelet lysates exert the efficacy of chondroregeneration and SMAD2-mediated anti-chondrofibrosis on knee osteoarthritis.

BACKGROUND: The lack of self-repairability in cartilage and the formation of fibrocartilage pose significant challenges in treating knee osteoarthritis, and there is still no ideal solution. Autologous platelet lysates have been clinically applied to treat kOA and exert satisfactory cartilage-repair efficacy, but the preparation of human PL brings damage to patients and is hardly standardized. METHODS: In this study, porcine PL was developed to replace hPL, and its chondroregenerative and anti-chondrofibrosis effects were explored. Enzyme-Linked Immunosorbent Assay was applied to qualify the PL products. In vivo, partial-thickness cartilage defects were created on rats as a kOA model, and the von Frey test, histopathological observation, immunohistochemical analysis, and western blot analysis were conducted. In vitro, CCK-8 assay, real-time PCR analysis, immunofluorescence test, and WB analysis were conducted for the mechanism study of pPL. RESULTS: The in vivo data showed that pPL significantly repaired the cartilage defect by improving matrix synthesis and also ameliorated the pain response in the kOA model of rats. In addition, pPL exerted an anti-fibrosis effect on cartilage by suppressing the expressions of COL1, COL3, α-SMA, VIMENTIN, SMAD2, p-SMAD2, and CTGF in cartilage. The in vitro data verified these effects and indicated that the SMAD2 pathway mediated the anti-fibrosis mechanism of pPL. Moreover, the comparable effects between pPL and rat PL indicate that there is no immune rejection from pPL. CONCLUSIONS: This study firstly demonstrated the anti-kOA effects of pPL on both cartilage-repair and anti-chondrofibrosis. It developed pPL as a promising alternative to autologous PL for clinical applications.

Gallic acid in theabrownin suppresses cell proliferation and migration in non­small cell lung carcinoma via autophagy inhibition.

The bioactive extract of green tea, theabrownin (TB), is known to exhibit pro-apoptotic and antitumor effects on non-small cell lung cancer (NSCLC). Gallic acid (GA) is a crucial component of TB; however, its mechanism of action in NSCLC has been rarely studied. To date, little attention has been paid to the anti-NSCLC activity of GA. Therefore, the present study investigated the effects of GA in vivo and in vitro. Cell Counting Kit (CCK)-8 assay, DAPI staining and flow cytometry, wound-healing assay and western blotting were used to assess cell viability, apoptosis, migration and protein expression, respectively. In addition, a xenograft model was generated, and TUNEL assay and immunohistochemistry analysis were performed. The CCK-8 data showed that the viability of H1299 cells was significantly inhibited by GA in a dose- and time-dependent manner. DAPI staining, Annexin-V/PI staining and wound-healing data showed that GA exerted pro-apoptotic and anti-migratory effects on H1299 cells in a dose-dependent manner. Furthermore, the results of western blotting showed that GA significantly upregulated the levels of pro-apoptotic proteins [cleaved (c-)PARP, c-caspase8, c-caspase-9 and the ratio of γ-H2A.X/H2A.X]. In vivo data confirmed the antitumor effect of GA through apoptosis induction in an autophagy-dependent manner. In conclusion, the present study confirmed the anti-proliferative, pro-apoptotic and anti-migratory effects of GA against NSCLC in vitro and in vivo, providing considerable evidence for its potential as a novel candidate for the treatment of NSCLC.

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects.

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

Green tea-derived theabrownin induces cellular senescence and apoptosis of hepatocellular carcinoma through p53 signaling activation and bypassed JNK signaling suppression.

BACKGROUND: Theabrownin (TB) is a bioactive component of tea and has been reported to exert effects against many human cancers, but its efficacy and mechanism on hepatocellular carcinoma (HCC) with different p53 genotypes remains unclarified. METHODS: MTT assay, DAPI staining, flow cytometry and SA-ß-gal staining were applied to evaluate the effects of TB on HCC cells. Quantitative real time PCR (qPCR) and Western blot (WB) were conducted to explore the molecular mechanism of TB. A xenograft model of zebrafish was established to evaluate the anti-tumor effect of TB. RESULTS: MTT assays showed that TB significantly inhibited the proliferation of SK-Hep-1, HepG2, and Huh7 cells in a dose-dependent manner, of which SK-Hep-1 was the most sensitive one with the lowest IC50 values. The animal data showed that TB remarkably suppressed SK-Hep-1 tumor growth in xenograft model of zebrafish. The cellular data showed TB's pro-apoptotic and pro-senescent effect on SK-Hep-1 cells. The molecular results revealed the mechanism of TB that p53 signaling pathway (p-ATM, p-ATR, γ-H2AX, p-Chk2, and p-p53) was activated with up-regulation of downstream senescent genes (P16, P21, IL-6 and IL-8) as well as apoptotic genes (Bim, Bax and PUMA) and proteins (Bax, c-Casp9 and c-PARP). The p53-mediated mechanism was verified by using p53-siRNA. Moreover, by using JNK-siRNA, we found JNK as a bypass regulator in TB's mechanism. CONCLUSIONS: To sum up, TB exerted tumor-inhibitory, pro-senescent and pro-apoptotic effects on SK-Hep-1 cells through ATM-Chk2-p53 signaling axis in accompany with JNK bypass regulation. This is the first report on the pro-senescent effect and multi-target (p53 and JNK) mechanism of TB on HCC cells, providing new insights into the underlying mechanisms of TB's anti-HCC efficacy.

Theabrownin Induces Apoptosis and Tumor Inhibition of Hepatocellular Carcinoma Huh7 Cells Through ASK1-JNK-c-Jun Pathway.

PURPOSE: Theabrownin (TB), a main pigment and bioactive component of tea, has been shown anti-tumor activities against carcinomas, but its effects on hepatocellular carcinoma (HCC) remain unclear. METHODS: Hepatocellular carcinoma Huh7 cells were used for analyses. Cell viability assay was performed to determine TB's anti-proliferative effect, and flow cytometry with annexin V-FITC/PI double staining and DAPI staining were performed to determine its pro-apoptotic effect. Real-time PCR and Western blot assays were conducted to detect the molecular actions of TB. And a xenograft model of zebrafishes was established to evaluate the in vivo effect of TB. SP600125 (JNK inhibitor) was in vivo and in vitro used to verify the regulatory role of the JNK signaling pathway in the anti-hepatic carcinoma mechanism of TB. RESULTS: TB exerted significant anti-proliferative and pro-apoptotic effects on Huh7 cells in a dose-dependent manner. The molecular data showed that TB up-regulated the gene expressions of NOXA, PUMA, P21, Bax, and Bim and up-regulated the protein expressions of ASK-1, Bax, phosphorylated JNK, and phosphorylated c-Jun with down-regulation of Bcl-2. The in vivo data showed that TB exerted significant tumor-inhibitory effect which was even stronger than that of cis-platinum. Furthermore, the JNK inhibitor significantly weakened TB's effects both in vivo and in vitro and blocked the related molecular pathway. CONCLUSION: TB exerts anti-proliferative, pro-apoptotic, and tumor-inhibitory effects on Huh7 cells through activation of the JNK signaling pathway. For the first time, this study provides new evidence of anti-HCC effects and mechanism of TB.

Evaluation of Long-Time Decoction-Detoxicated Hei-Shun-Pian (Processed Aconitum carmichaeli Debeaux Lateral Root With Peel) for Its Acute Toxicity and Therapeutic Effect on Mono-Iodoacetate Induced Osteoarthritis.

BACKGROUND: As a degenerative joint disease with severe cartilage destruction and pain, osteoarthritis (OA) has no satisfactory therapy to date. In traditional Chinese medicine (TCM), Aconitum carmichaeli Debeaux derived Hei-shun-pian (Hsp) has been developed for joint pain treatment. However, it causes adverse events in OA patients. Long-time decoction has been traditionally applied to reduce the aconite toxicity of Hsp and other aconite herbs, but its detoxifying effect is uncertain. METHODS: Hsp was extracted with dilute decoction times (30, 60, and 120 min) and evaluated by toxicological, chemical, pharmacological assays. Acute toxicity assay and chemical analysis were employed to determine the toxicity and chemoprofile of Hsp extracts, respectively. Since the detoxified Hsp (dHsp) was defined, its therapeutic effect was evaluated by using an OA rat model induced by monosodium iodoacetate. dHsp at 14 g/kg was orally administered for 28 days, and the pain assessments (mechanical withdrawal threshold and thermal withdrawal latency) and histopathological analyses (HE and safranin-O staining) were performed. Real-time PCR (qPCR) was applied to determine the molecular actions of dHsp on cartilage tissue and on chondrocytes. MTT assay was conducted to evaluate the effect of dHsp on the cell viability of chondrocytes. The cellular and molecular assays were also conducted to analyze the functions of chemical components in dHsp. RESULTS: The chemoprofile result showed that the contents of toxic alkaloids (aconitine, mesaconitine, and hypaconitine) were decreased but that of non-toxic alkaloids (benzoylaconitine, benzoylmesaconitine, and benzoylhypaconitine) were increased with increasing decoction time. Acute toxicity assay showed that only Hsp extract with 120 min decoction was non-toxic within the therapeutic dose range. Thus, it was defined as dHsp for further experiment. In OA experiment, dHsp significantly attenuated joint pain and prevented articular degeneration from MIA attack. qPCR data showed that dHsp restored the abnormal expressions of Col10, Mmp2, Sox5, Adamts4/5/9, and up-regulated Col2 expression in rat cartilage. In vitro, dHsp-containing serum significantly proliferated rat chondrocytes and regulated the gene expressions of Col2, Mmp1, Adamts9, and Aggrecan in a similar way as the in vivo data. Moreover, aconitine, mesaconitine, and hypaconitine exerted cytotoxic effects on chondrocytes, while benzoylaconitine and benzoylhypaconitine except benzoylmesaconitine exhibited similar molecular actions to dHsp, indicating contributions of benzoylaconitine and benzoylhypaconitine to dHsp. CONCLUSIONS: This study defined dHsp and demonstrated dHsp as a potential analgesic and disease modifying agent against OA with molecular actions on the suppression of chondrocyte hypertrophy and extracellular matrix degradation, providing a promising TCM candidate for OA therapy.

Osteoarthritis Pain Model Induced by Intra-Articular Injection of Mono-Iodoacetate in Rats.

The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients' daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 ß and TNF α) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.