Incense-burning smoke ingredient Auramine enhances lincRNA-p21 expression for chemosensitization in p53-mutated non-small cell lung cancer.

Incense-burning smoke is a deleterious air pollutant that initiates cytotoxic effects by inducing apoptosis in lung epithelial cells and also acts as a risk factor for lung cancers. Auramine, an ingredient of incense smoke, has been implicated in tumor progression and cellular sensitivity in non-small cell lung cancer (NSCLC) towards anti-cancer agents through unclear mechanisms. Tumor protein p53 (TP53)-activated long intergenic non-coding RNA-p21 (lincRNA-p21) undertakes a pivotal role in regulating cell apoptosis and chemosensitivity. TP53 mutations prevalent in 50% of NSCLC, contribute to diminished therapeutic efficacy. However, the influence of auramine on chemotherapy-induced lincRNA-p21 expression and apoptosis in NSCLC with different TP53 genetic statuses remains unexplored. This study disclosed that both wild-type p53 (wtp53) and mutant p53 (mutp53) mediate lincRNA-p21 expression, albeit through distinct promoter enhancers, p53-response element (p53RE) and non-B DNA structure G-quadruplex (GQ), respectively. Intriguingly, auramine functions as an effective stabilizer of the GQ structure, augmenting mutp53-mediated lincRNA-p21 expression and enhancing apoptosis and cellular sensitivity to chemotherapy in mutp53-expressing NSCLC cells. These findings suggest a mechanism by which mutp53, in the presence of auramine, is endowed with tumor-suppressing function akin to wtp53, thereby aiding in combating chemoresistance in NSCLC cells harboring TP53 mutations.

Glucose Transporter 1-Mediated Transcytosis of Glucosamine-Labeled Liposomal Ceramide Targets Hypoxia Niches and Cancer Stem Cells to Enhance Therapeutic Efficacy.

Tumour hypoxia plays an important role in modulating tumorigenesis, angiogenesis, invasion, immunosuppression, resistance to treatment, and even maintenance of the stemness of cancer stem cells (CSCs). Moreover, the targeting and treatment of hypoxic cancer cells and CSCs to reduce the influence of tumor hypoxia on cancer therapy remains an imperative clinical problem that needs to be addressed. Since cancer cells upregulate the expression of glucose transporter 1 (GLUT1) through the Warburg effect, we considered the possibility of GLUT1-mediated transcytosis in cancer cells and developed a tumor hypoxia-targeting nanomedicine. Our experimental results indicate that glucosamine-labeled liposomal ceramide can be efficiently transported between cancer cells by GLUT1 transporters and substantially accumulated in the hypoxic area in in vitro CSC spheroids and in vivo tumor xenografts. We also verified the effects of exogenous ceramide on tumor hypoxia, including important bioactivities such as upregulation of p53 and retinoblastoma protein (RB), downregulation of hypoxia-inducible factor-1 alpha (HIF-1α) expression, disruption of the OCT4-SOX2 network of stemness, and inhibition of CD47 and PD-L1 expression. To achieve an ideal therapeutic outcome, we combined treatment of glucosamine-labeled liposomal ceramide with paclitaxel and carboplatin, and we found an excellent synergistic effect, with tumor clearance being noted in three-fourths of the mice. Overall, our findings provide a potential therapeutic strategy for the treatment of cancer.

Pim1 Kinase Inhibitors Exert Anti-Cancer Activity Against HER2-Positive Breast Cancer Cells Through Downregulation of HER2.

The proviral integration site for moloney murine leukemia virus 1 (Pim1) is a serine/threonine kinase and able to promote cell proliferation, survival and drug resistance. Overexpression of Pim1 has been observed in many cancer types and is associated with the poor prognosis of breast cancer. However, it remains unclear whether Pim1 kinase is a potential therapeutic target for breast cancer patients. In this study, we found that Pim1 expression was strongly associated with HER2 expression and that HER2-overexpressing breast cancer cells were more sensitive to Pim1 inhibitor-induced inhibitions of cell viability and metastatic ability. Mechanistically, Pim1 inhibitor suppressed the expression of HER2 at least in part through transcriptional level. More importantly, Pim1 inhibitor overcame the resistance of breast cancer cells to HER2 tyrosine kinase inhibitor lapatinib. In summary, downregulation of HER2 by targeting Pim1 may be a promising and effective therapeutic approach not only for anti-cancer growth but also for circumventing lapatinib resistance in HER2-positive breast cancer patients.

PKCδ-mediated SGLT1 upregulation confers the acquired resistance of NSCLC to EGFR TKIs.

The tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) have been widely used for non-small cell lung cancer (NSCLC) patients, but the development of acquired resistance remains a therapeutic hurdle. The reduction of glucose uptake has been implicated in the anti-tumor activity of EGFR TKIs. In this study, the upregulation of the active sodium/glucose co-transporter 1 (SGLT1) was found to confer the development of acquired EGFR TKI resistance and was correlated with the poorer clinical outcome of the NSCLC patients who received EGFR TKI treatment. Blockade of SGLT1 overcame this resistance in vitro and in vivo by reducing glucose uptake in NSCLC cells. Mechanistically, SGLT1 protein was stabilized through the interaction with PKCδ-phosphorylated (Thr678) EGFR in the TKI-resistant cells. Our findings revealed that PKCδ/EGFR axis-dependent SGLT1 upregulation was a critical mechanism underlying the acquired resistance to EGFR TKIs. We suggest co-targeting PKCδ/SGLT1 as a potential strategy to improve the therapeutic efficacy of EGFR TKIs in NSCLC patients.

Cigarette smoke-induced LKB1/AMPK pathway deficiency reduces EGFR TKI sensitivity in NSCLC.

Smoker patients with non-small cell lung cancer (NSCLC) have poorer prognosis and survival than those without smoking history. However, the mechanisms underlying the low response rate of those patients to EGFR tyrosine kinase inhibitors (TKIs) are not well understood. Here we report that exposure to cigarette smoke extract enhances glycolysis and attenuates AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR; this in turn reduces the sensitivity of NSCLC cells with wild-type EGFR (EGFRWT) to EGFR TKI by repressing expression of liver kinase B1 (LKB1), a master kinase of the AMPK subfamily, via CpG island methylation. In addition, LKB1 expression is correlated positively with sensitivity to TKI in patients with NSCLC. Moreover, combined treatment of EGFR TKI with AMPK activators synergistically increases EGFR TKI sensitivity. Collectively, the current study suggests that LKB1 may serve as a marker to predict EGFR TKI sensitivity in smokers with NSCLC carrying EGFRWT and that the combination of EGFR TKI and AMPK activator may be a potentially effective therapeutic strategy against NSCLC with EGFRWT.

Galectin-3 and TRIM16 coregulate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells at least partly via enhancing autophagy.

BACKGROUND: The osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is critical for bone homeostasis. Here, we investigated the regulation of Galectin-3 and tripartite motif protein 16 (TRIM16) on osteogenic differentiation of hBMSCs through autophagy. METHODS: Quantitative PCR (qPCR) and western blot were performed to determine the expression of osteogenic markers, autophagic markers, Galectin-3 and TRIM16. Short-hairpin RNAs (shRNAs) and overexpression plasmids were used to manipulate the expression of Galectin-3, TRIM16 and Unc-51 like autophagy activating kinase 1 (ULK1). Alkaline phosphatase (ALP) activity was measured by ALP staining assay. Calcium deposition in differentiated hBMSCs was assessed by Alizarin Red S staining. LC3 puncta formation was monitored by immunofluorescence staining. The interaction between indicated proteins was confirmed by co-immunoprecipitation (Co-IP) assay. RESULTS: Either Galectin-3 or TRIM16 knockdown led to impaired ALP activity, reduced calcium deposition, down-regulation of pro-osteogenic markers as well as restrained autophagy in osteogenic-induced hBMSCs. However, overexpression of Galectin-3 or TRIM16 promoted osteogenic differentiation of hBMSCs, which was then compromised by autophagy inhibition. Co-IP experiment demonstrated that TRIM16 associated with Galectin-3 through ULK1. Meanwhile, osteogenic induction enhanced the association between TRIM16 and ULK1 or coiled-coil myosin-like BCL2-interacting protein (Beclin1), and TRIM16 increased the stability of ULK1 and Beclin1. Moreover, either TRIM16 or ULK1 knockdown dampened the pro-osteogenic effect of Galectin-3, which elucidated that Galectin-3 mediated osteogenic differentiation was at least partly dependent on TRIM16 and ULK1. CONCLUSION: In summary, the present study revealed Galectin-3 and TRIM16 co-regulated osteogenic differentiation of hBMSCs at least partly via enhancing autophagy, which might provide a promising approach for osteoporosis treatment in future.

Aucubin Protects Chondrocytes Against IL-1ß-Induced Apoptosis In Vitro And Inhibits Osteoarthritis In Mice Model.

OBJECTIVE: Chondrocyte apoptosis has also been strongly correlated with the severity of cartilage damage and matrix depletion in an osteoarthritis (OA) joint. Therefore, pharmacological inhibitors of apoptosis may provide a novel treatment option for patients with OA. Aucubin, a natural compound isolated from Eucommia ulmoides, has been proved to possess antioxidative and anti-apoptotic properties. However, anti-osteoarthritis effect of aucubin in animal model and anti-apoptotic response of aucubin in OA chondrocytes remain unclear. This study aimed to determine whether aucubin could slow progression of OA in a mouse model and inhibit the IL-1ß-induced chondrocyte apoptosis. METHODS: OA severity and articular cartilage degradation were evaluated by Safranin-O staining, Hematoxylin-eosin (H&E) staining, and Osteoarthritis Research Society International (OARSI) standards. Chondrocyte viability was observed by Cell Counting Kit-8 (CCK8) and live/dead cells assay; the apoptotic rate of chondrocytes was evaluated by flow cytometry (FCM) with Annexin V-FITC/PI kit. Mediators of apoptosis were tested by Western blot of Bax, caspase-3, caspase-9, and Bcl-2 expression. The intracellular levels of Reactive oxygen species (ROS) were assessed by the probe of 2,7-Dichlorofluorescin diacetate (DCFH-DA). RESULTS: The articular cartilage in the limb with destabilization of the medial meniscus (DMM) exhibited early OA-like manifestations characterized by proteoglycan loss, cartilage fibrillation, and erosion, with lower OARSI score. Oral administration of aucubin remarkably attenuated the loss of proteoglycan and the articular cartilage erosion and decreased the OARSI scores underwent DMM surgery. Aucubin treatment significantly reverses IL-1ß-induced cytotoxicity and attenuated the IL-1ß-induced chondrocyte apoptosis. In addition, aucubin can significantly inhibit mediators of apoptosis in rat primary chondrocytes. Furthermore, aucubin remarkably attenuated the IL-1ß-induced intracellular ROS production. CONCLUSION: Our findings suggest that aucubin has a protective effect on articular cartilage and slowing progression of OA in a mouse model. This protective effect may result from inhibiting chondrocyte apoptosis and excessive ROS production.

Chinese expert consensus on diagnosis and treatment of infection after fracture fixation.

Currently, accurate diagnosis and successful treatment of infection after fracture fixation (IAFF) still impose great challenges. According to the onset of infection symptoms after implantation, IAFF is classified as early infection (<2 weeks), delayed infection (2∼10 weeks) and late infection (>10 weeks). Confirmation of IAFF should be supported by histopathological tests of intraoperative specimens which confirm infection, cultures from at least two suspected infection sites which reveal the same pathogen, a definite sinus or fistula which connects directly the bone or the implant, and purulent drainage from the wound or presence of pus during surgery. Diagnosis of IAFF is built on comprehensive assessment of medical history, clinical signs and symptoms of the patient, and imaging and laboratory tests. The gold standard of diagnosis is histopathological tests. Treatment of IAFF consists of radical debridement, adequate irrigation, implant handling, systematic and local antibiotics, reconstruction of osseous and/or soft tissue defects, and functional rehabilitation of an affected limb. Early accurate diagnosis and appropriate treatment of IAFF play a key role in increasing the cure rate, reducing infection recurrence and disability risk, restoring limb function and improving quality of life of the patient.

miR-193a inhibits osteogenic differentiation of bone marrow-derived stroma cell via targeting HMGB1.

BACKGROUND: miR-193a has been shown to be involved in a variety of biological processes, including cell proliferation, differentiation, and apoptosis. However, little is known about how miR-193a regulates osteogenic differentiation. METHODS: We employed RT-qPCR to determine the level of miR-193a and mRNA level of HMGB1 and osteoblast-specific markers (Runx2, ALP, OSX, OCN). Besides, westernblot was used to probe protein level of phosphorylated MAPK family members and ß-catenin. Bioinformatic analysis was used to predict the putative binding sequence of miR-193a to the 3'-UTR of HMGB1 and we confirmed this result by dual luciferase reporter assay. Alizarin red staining assay (ARS) and alkaline phosphatase activity (ALP) were performed to detect osteogenic differentiation. RESULTS: miR-193a was downregulated in OM (osteogenic medium)induced hBMSC. More interestingly, we found that miR-193a mimic attenuated matrix mineralization and alkaline phosphatase activity, whereas miR-193a inhibitor exerted the opposite phenotypes. Mechanistically, we observed that miR-193a played an inhibitory role in expression of osteoblast-specific markers and activation of MAPK and Wnt signaling pathways. Bioinformatic analysis and dual luciferase assay demonstrated that miR-193a directly targeted 3'-UTR of HMGB1. Furthermore, we overexpressed HMGB1 in miR-193a overexpressed hBMSC to establish that HMGB1 acted as downstream target of miR-193a-inhibited osteogenic differentiation. CONCLUSIONS: Here, we reveal miR-193a plays a suppressive role in osteogenic differentiation of hBMSC via targeting HMGB1. These findings provide a novel mechanism underlying osteogenic differentiation and offer therapeutical strategy for bone formation.

Cigarette smoke enhances oncogene addiction to c-MET and desensitizes EGFR-expressing non-small cell lung cancer to EGFR TKIs.

Cigarette smoking is one of the leading risks for lung cancer and is associated with the insensitivity of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, it remains undetermined whether and how cigarette smoke affects the therapeutic efficacy of EGFR TKIs. In this study, our data showed that chronic exposure to cigarette smoke extract (CSE) or tobacco smoke-derived carcinogen benzo[α]pyrene, B[α]P, but not nicotine-derived nitrosamine ketone (NNK), reduced the sensitivity of wild-type EGFR-expressing NSCLC cells to EGFR TKIs. Treatment with TKIs almost abolished EGFR tyrosine kinase activity but did not show an inhibitory effect on downstream Akt and ERK pathways in B[α]P-treated NSCLC cells. CSE and B[α]P transcriptionally upregulate c-MET and activate its downstream Akt pathway, which is not inhibited by EGFR TKIs. Silencing of c-MET reduces B[α]P-induced Akt activation. The CSE-treated NSCLC cells are sensitive to the c-MET inhibitor crizotinib. These findings suggest that cigarette smoke augments oncogene addiction to c-MET in NSCLC cells and that MET inhibitors may show clinical benefits for lung cancer patients with a smoking history.

Calcium sulfate induced versus PMMA-induced membrane in a critical-sized femoral defect in a rat model.

Aimed to investigate the characteristics of CS-induced membrane in comparison with the PMMA-induced membrane. Cellular components, histological changes, growth factor expressions of IL-6, VEGF, BMP-2, and TGF-ß1 in the two induced membranes were compared at 2, 4, 6 and 8 weeks, respectively. We also compared the histological changes at the bone defects between CS and PMMA groups. The structural characteristics of induced membrane were similar between CS and PMMA. Endochondral ossification took place in the CS-induced membrane at 8 week. Levels of VEGF, BMP-2 and TGF-ß1 in CS-induced membrane were insignificantly higher than those in PMMA-induced membrane at different time points. The expression of IL-6 was significantly higher in PMMA-induced membranes at 2nd week. In addition, osteogenic and neovascular activities of induced membranes increased with time and peaked at 6 weeks. CS promoted endochondral ossification at the broken ends of the bone defect than PMMA did. CS-induced membrane has a better capacity of generating VEGF, BMP-2 and TGF-ß1.osteogenic and neovascular activities achieve highest level at 6 week. CS may have the potential to replace PMMA as a novel spacer in Masquelet technique.

Incense burning smoke sensitizes lung cancer cells to EGFR TKI by inducing AREG expression.

Incense burning is common in Asian countries due to the religious beliefs. Environmental exposure to incense burning smoke is a potential risk factor for tumor development and progression of non-small cell lung cancer (NSCLC). Eastern Asia ethnic origin is strongly associated the clinical benefits of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in NSCLC patients. However, the impact of the oriental custom of incense burning on the cancer progression and the EGFR TKI-sensitivity of NSCLC remains unclear. Our results showed that long-term exposure to incense burning extract (IBE) increases the cellular proliferation with S phase accumulation and the motility activity of NSCLCs. Interestingly, IBE enhances EGFR signaling activity without affecting its genetic status, and increases the cellular sensitivity of NSCLC cell lines to EGFR TKIs. Auramine, a yellow dye for making incense sticks, was identified as a residual composition in the burning incense smoke, and showed similar EGFR TKI-sensitizing effects. Furthermore, IBE or auramine transcriptionally induce EGFR ligand amphiregulin (AREG) expression for the enhancement of EGFR activity. Neutralization of AREG reduced the viability of IBE-treated cells. These results indicated that exposure to incent smoke may enhance NSCLC progression and their sensitivity to EGFR TKIs through increasing their oncogenic addiction to AREG-induced EGFR signaling.

Is surgical treatment better than conservative treatment for primary patellar dislocations? A meta-analysis of randomized controlled trials.

BACKGROUND: Despite several randomized controlled trials comparing operative to nonoperative management of primary patellar dislocation, the optimal management of this condition remains a subject of controversy. The aim of this study was to compare surgical to conservative treatment of outcomes for primary patellar dislocation by meta-analysis all the relative randomized controlled trials. STUDY DESIGN: Meta-analysis. METHODS: After searching multiple online databases (MEDILINE, EMBASE, CLINICAL, OVID, BISOS and Cochrane registry of controlled clinical trials), eight randomized controlled trials including 430 patients were meta-analyzed in which operative treatment was compared with non-operative treatment for primary patellar dislocation. Outcomes evaluated were redislocation rate, Kujala score, episode of instability, Tegner activity score, Hughston visual analog score (VAS) and patient satisfaction. RESULTS: Outcomes on recurrent patellar dislocation (P = 0.004) and Hughston VAS (P = 0.03) were statistically significant in favor of operative management. Tegner activity score (P < 0.00001) was significantly higher in favor of conservative treatment, though only a few studies were identified. There was no significant difference between the two treatments regarding episode of instability (P = 0.41), Kujala score (P = 0.32) or patient satisfaction (P = 0.49). CONCLUSION: Surgical treatment may be better than conservative treatment for patients with primary patellar dislocation on incidence of redislocation. However, since these findings are built on a limited number of studies available, well-designed, multicenter clinical trials with long-term follow-up are required to provide more solid evidence concerning optimal strategies.

Association between micronucleus frequency and cervical intraepithelial neoplasia grade in Thinprep cytological test and its significance.

A micronucleus is an additional small nucleus formed due to chromosomes or chromosomal fragments fail to be incorporated into the nucleus during cell division. In this study, we assessed the utility of micronucleus counting as a screening tool in cervical precancerous lesions in Thinprep cytological test smears under oil immersion. High risk HPV was also detected by hybrid capture-2 in Thinprep cytological test smears. Our results showed that micronucleus counting was significantly higher in high-grade squamous intraepithelial lesion (HSIL) and invasive carcinoma cases compared to low-grade squamous intraepithelial lesion (LSIL) and non-neoplastic cases. Receiver operating characteristic (ROC) curve analysis revealed that micronucleus counting possessed a high degree of sensitivity and specificity for identifying HSIL and invasive carcinoma. Cut-off of 7.5 for MN counting gave a sensitivity of 89.6% and a specificity of 66.7% (P = 0.024 and AUC = 0.892) for detecting HSIL and invasive carcinoma lesions. Multiple linear regression analysis showed that only HSIL and invasive cancer lesions not age, duration of marital life and number of pregnancy are significantly associated with MN counting. The positive rate of high risk HPV was distinctly higher in LSIL, HSIL and invasive cancer than that in non-neoplstic categories. In conclusions, MN evaluation may be viewed as an effective biomarker for cervical cancer screening. The combination of MN count with HPV DNA detection and TCT may serve as an effective means to screen precancerous cervical lesions in most developing nations.

Transduction of mesenchymal stem cells with multidrug resistance gene provides protection for bone marrow toxicity after being transplanted into a nude mice model.

BACKGROUND: Myelosuppression is the main dose-related toxicity of many chemotherapeutic drugs. The human multidrug resistance (mdr1) gene is well-known for its ability to confering drug resistance. In this study, we meant to transplant the placenta mesenchymal stem cells (P-MSCs) moderated by mdr1 gene into a nude mice model radiated by γ-Co(60) and to explore the chemoprotection for bone marrow (BM) toxicity. METHODS: Human P-MSCs were isolated from trypsin-digested term placentas and then transduced by with reconstructed retroviral vector containing mdr1 gene and green fluorescent protein (GFP) reporter gene. The integration and expression of mdr1 gene was observed indirectedly by the expression of GFP. A nude mice model was constructed after irradiation with a sublethal dosage of γ-Co(60). These irradiated mice were transplanted with mdr1-MSCs through the caudal vein and then received paclitaxel (PAC) intraperitoneal chemotherapy. The Peripheral peripheral blood (PB) of the nude mice was collected, and the PB cells counts and values were determined using an automatic analyzer. RESULTS: After PAC treatment, mdr1-MSCs transplanted mice showed markedly improved survival upon compared to MSCs transplanted mice (85.7% vs. 57.1%). White blood cell (WBC) and red blood cell (RBC) counts as well as the hemoglobin (Hb) values were significantly increased in PAC treated mdr1-MSCs mice compared to PAC treated control mice when PAC chemotherapy had been finished (all P < 0.05), but the difference was not found in the plateltes (PLT) count (P > 0.05). CONCLUSION: Human P-MSCs moderated by mdr1 gene when transplanted into nude mice may provide chemoprotection for hematopoietic toxicity.

[Transfer and identification of multidrug resistance gene in mesenchymal stem cells derived from human placenta].

OBJECTIVE: To explore the feasibility and safety of mdr1 gene transferred into placenta derived mesenchymal stem cells (P-MSCs) by reconstructed retroviral vector. METHODS: Human P-MSCs were isolated and expanded by Percoll density gradient and then transduced repeatedly by reconstructed retroviral vector containing mdr1 gene. The transfection and expression of mdr1 gene was detected by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS). Meanwhile, the biological features of mdr1-MSCs were identified and analyzed. RESULTS: The expression of mdr1mRNA was found in transfected cells. The expression of P-glycoprotein (P-gp) encoded by mdr1 gene was (27.6 ± 5.1)% in the transfected P-MSCs cells versus (0.4 ± 0.1)% in the non-transfected P-MSCs cells (t = 14.291, P < 0.01). The percent of P-MSCs at quiescent phase (G0/G1 phase) was around 95.40% and it was in accord with the characterization of stem cells. The mdr1-MSCs exhibited typical ultrastructures of low-differentiated stem cells. Moreover, they still retained the potency of adipogenic and osteogenic differentiation in the presence of appropriate conditioned media. CONCLUSION: A stable expression of P-gp may be obtained by reconstructed retroviral-mediated transfection in vitro. And transfected MSCs retain the characteristics of stem cells.

Microwave-assisted extraction of oleanolic acid and ursolic acid from Ligustrum lucidum Ait.

Oleanolic acid and ursolic acid are the main active components in fruit of Ligustrum lucidum Ait, and possess anticancer, antimutagenic, anti-inflammatory, antioxidative and antiprotozoal activities. In this study, microwave-assisted extraction of oleanolic acid and ursolic acid from Ligustrum lucidum was investigated with HPLC-photodiode array detection. Effects of several experimental parameters, such as type and concentration of extraction solvent, ratio of liquid to material, microwave power, extraction temperature and microwave time, on the extraction efficiencies of oleanolic acid and ursolic acid from Ligustrum lucidum were evaluated. The influence of experimental parameters on the extraction efficiency of ursolic acid was more significant than that of oleanolic acid (p < 0.05). The optimal extraction conditions were 80% ethanol aqueous solution, the ratio of material to liquid was 1:15, and extraction for 30 min at 70 °C under microwave irradiation of 500 W. Under optimal conditions, the yields of oleanolic acid and ursolic acid were 4.4 ± 0.20 mg/g and 5.8 ± 0.15 mg/g, respectively. The results obtained are helpful for the full utilization of Ligustrum lucidum, which also indicated that microwave-assisted extraction is a very useful method for extraction of oleanolic acid and ursolic acid from plant materials.

[Effect of intra-articular ozone injection on serum and synovial TNF-α, TNFR I, and TNFR II contents in rats with rheumatoid arthritis].

OBJECTIVE: To observe the effects of intra-articular ozone injection at different concentrations on the contents of tumor necrosis factor-α (TNF-α), TNF receptor I (TNFR I), and TNFR II in the serum and synovium of rats with rheumatoid arthritis (RA) and explore the therapeutic mechanism of ozone in RA treatment. METHODS: Forty-eight Wistar rats were randomized into 8 groups, including 5 ozone groups receiving intra-articular injection of 10, 20, 30, 40 or 50 µg/ml ozone, a blank control group, an oxygen group and a RA model group. All the rats, except for those in the blank control group, were subjected to hypodermic injection of bovine collagen II and complete Freunds adjuvant to induce RA. Ozone treatment was administered once weekly for 3 weeks starting at 21 days after the modeling. The swelling and thickness of the hind paws were observed, and the serum and synovial contents of TNF-α, TNFR I, and TNFR II were detected. RESULTS: At the end of treatment, the paw thickness was reduced significantly in rats with 40 µg/ml ozone injection compared with that in the model RA group (P<0.01). The serum contents of TNF-α, TNFR I and TNFR II showed no significant difference between the RA model group, oxygen group and the ozone groups, but their synovial contents showed significant reductions in rats with 40 and 50 µg/ml ozone injection (P<0.01); the synovial TNFR I was significantly higher in 40 µg/ml ozone group than in the model group (P<0.05). CONCLUSION: Intra-articular injection of 40 µg/ml ozone can attenuate synovitis in rats with RA, the mechanism of which may involve the inhibition of TNF-α and TNFR II activity and enhancement of TNFR I activity in the synovium.