[Rapid extraction and detection of five alkaloids in dried khat by solvent extraction-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].

Khat is a common plant that grows primarily in Eastern Africa and the Arabian Peninsula. Cathinone, norpseudoephedrine, and norephedrine are the main psychoactive components of khat. Experimental studies have shown that red and green khat have similar cathinone contents, but green khat contains more norpseudoephedrine and norephedrine than red khat. Research indicates that Ethiopians believe that red khat has stronger psychoactive effects than green khat. Therefore, we speculated that other substances in red khat may enhance its psychoactive effects. Using the sampling method, we identified two other psychoactive components in khat: methcathinone and ethcathinone. At present, only a few studies on the extraction and detection of alkaloids from khat have been published in China, and no reports on the extraction and detection of methcathinone and ethcathinone from khat are available. In this study, we established an extraction and detection method for five alkaloids in dried khat using high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF MS). To establish the extraction method, we optimized the extraction solvent and process. The amounts of dichloromethane and sodium hydroxide added during the purification step were also optimized. To establish the detection method, we optimized the chromatographic and MS conditions. The final extraction and detection method was as follows: Dried khat powder (0.1 g) was loaded into a polypropylene centrifuge tube, added with 1 mL of 0.05 mol/L hydrochloride aqueous solution, and vortex-oscillated for 3 min for extraction. The sample was centrifuged at 10000 r/min for 3 min. Next, 600 µL of the supernatant was placed in a centrifuge tube, added with 1 mL of dichloromethane, shaken for 1 min, and centrifuged at 10000 r/min for 3 min. Subsequently, 300 µL of the supernatant was placed in a centrifuge tube, added with 80 µL of 1 mol/L sodium hydroxide aqueous solution, shaken for 1 min, and added with 1 mL of acetonitrile. Vortex oscillation was performed for 2 min to extract the sample, after which solid sodium chloride (0.4 g) was added to the mixture, followed by shaking for 1 min to separate the acetonitrile and aqueous phases. The mixture was then centrifuged at 10000 r/min for 3 min. Finally, the supernatant was collected and diluted for further testing. The five target analytes were separated on a ZORBAX Eclipse Plus Phenyl-Hexyl column (100 mm×3.0 mm, 1.8 µm) via gradient elution using 0.1% acetic acid aqueous solution and acetonitrile as mobile phases with a flow rate of 0.3 mL/min and column temperature of 30 ℃. The analytes were identified using the targeted MS/MS method under positive electrospray ionization mode and quantified using the external standard method. The five alkaloids showed good correlations (all correlation coefficients (r2)≥0.9976) with their respective linear ranges. The limits of detection were between 0.08 and 0.75 µg/L, and the limits of quantification were between 0.25 and 2.50 µg/L. The average recoveries of the five alkaloids from two plants with different alkaloid contents were between 90.7% and 105.2%. The intra-sample precision ranged from 0.5% to 2.3%, the intra-day precision was between 1.0% and 2.5%, and the inter-day precision was between 1.3% and 3.3%. Using the developed method, we extracted and analyzed 15 khat samples, and detected five alkaloids. This method enables rapid sample pretreatment and has high sensitivity, good stability, and suitable accuracy. Based on the above results, we conclude that the proposed method meets the inspection and identification requirements for khat. Thus, it can provide a valuable reference for the physical and chemical identification of khat and support for further studies on its psychoactive components.

Epidemiological updates of post-traumatic related limb osteomyelitis in china: a 10 years multicentre cohort study.

BACKGROUND: Post-traumatic related limb osteomyelitis (PTRLO) is a complex bone infection. Currently, there are no available microbial data on a national scale that can guide appropriate antibiotic selection, and explore the dynamic changes in dominant pathogens over time. This study aimed to conduct a comprehensive epidemiological analysis of PTRLO in China. METHODS: The study was approved by the Institutional Research Board (IRB), and 3526 PTRLO patients were identified from 212 394 traumatic limb fracture patients at 21 hospitals between 1 January 2008 and 31 December 2017. A retrospective analysis was conducted to investigate the epidemiology of PTRLO, including changes in infection rate (IR), pathogens, infection risk factors and antibiotic resistance and sensitivity. RESULTS: The IR of PTRLO increased gradually from 0.93 to 2.16% (Z=14.392, P <0.001). Monomicrobial infection (82.6%) was significantly higher than polymicrobial infection (17.4%) ( P <0.001). The IR of Gram-positive (GP) and Gram-negative (GN) pathogens showed a significant increase from the lowest 0.41% to the highest 1.15% (GP) or 1.62% (GN), respectively. However, the longitudinal trend of GP vs. GN's composition did not show any significance (Z=±1.1918, P >0.05). The most prevalent GP strains were Methicillin-sensitive Staphylococcus aureus (MSSA) (17.03%), Methicillin-resistant Staphylococcus aureus (MRSA) (10.46%), E. faecalis (5.19%) and S. epidermidis (4.87%). In contrast, the dominant strains GN strains were Pseudomonas Aeruginosa (10.92%), E. cloacae (10.34%), E. coli (9.47%), Acinetobacter Baumannii (7.92%) and Klebsiella Pneumoniae (3.33%). In general, the high-risk factors for polymicrobial infection include opened-fracture (odds ratio, 2.223), hypoproteinemia (odds ratio, 2.328), and multiple fractures (odds ratio, 1.465). It is important to note that the antibiotics resistance and sensitivity analysis of the pathogens may be influenced by complications or comorbidities. CONCLUSIONS: This study provides the latest data of PTRLO in China and offers trustworthy guidelines for clinical practice. (China Clinical Trials.gov number, ChiCTR1800017597).

Association Between Sleep Quality and Hypertension in Chinese Adults: A Cross-Sectional Analysis in the Tianning Cohort.

Background: Poor sleep quality is becoming very common in a developed society and relates to many health disorders. However, the association between sleep quality and hypertension has not been well studied in Chinese adults. Methods: Blood pressure was measured and sleep quality was assessed by the Pittsburgh Sleep Quality Index (PSQI) for 5167 participants (mean aged 51±15 years, 41.5% males) in the Tianning Cohort. A logistic regression model was constructed to examine the association between sleep quality, as well as its components, and hypertension, adjusting for age, sex, education level, current smoking, current drinking, physical activity, obesity, glucose, and blood lipids. Results: After multivariate adjustment, a higher score Of the PSQI was significantly associated with an increased risk of prevalent hypertension (OR=1.03, P=0.018). Compared to participants with normal sleep (the PSQI score <5), those with a poor sleep quality (the PSQI score ≥5) had a 17% increased risk of prevalent hypertension (OR=1.17, P=0.042). Three of the seven components of sleep quality, such as subjective sleep quality (OR=1.17, P=0.001), sleep latency (OR=1.11, P=0.010), and sleep disturbances (OR=1.19, P=0.004), were also significantly associated with prevalent hypertension. Conclusion: Poor sleep quality is increasingly prevalent in developed societies and may be related to an increased risk of hypertension in Chinese adults. The underlying causality is waiting to be studied.

Incidence and risk factors of preoperative deep vein thrombosis in patients with intertrochanteric fractures: a retrospective study.

BACKGROUND: The risk of perioperative complications remains high in patients with intertrochanteric fractures. Immobilization after injury may predispose these patients to deep vein thrombosis (DVT) while waiting for surgery. The aims of this study were to determine the incidence of preoperative DVT in patients with intertrochanteric fractures and identify independent risk factors. METHODS: This retrospective study included patients with intertrochanteric fractures waiting for surgical interventions at our institution from June 2018 to December 2020. All patients received pharmacologic thromboprophylaxis after admission and ultrasound screening for DVT in both lower limbs before surgery. Demographic, clinical and laboratory data of these patients were collected to perform univariate analysis first. Subsequently, factors with a significant difference in univariate analysis were introduced into the multivariate logistic regression analysis to determine the independent risk factors for preoperative DVT. RESULTS: A total of 266 patients were enrolled in this study. Seventy-one patients (26.7%) developed DVTs before surgery. The majority of DVTs were distal types (91.5%). There were 40 patients with DVTs only in the affected limb, 7 patients with DVTs only in the unaffected limb, and 24 patients with DVTs in both lower limbs. Advanced age, female, prolonged period from injury to admission, combined cranial trauma, shorter thrombin time, increased level of D-dimer and lower level of albumin proved to be the independent risk factors for preoperative DVT. CONCLUSIONS: We observed a high incidence of preoperative DVT in patients with intertrochanteric fractures. Identification of patients at high risk may improve the prevention and treatment of preoperative DVT in this population.

The radiographic soft tissue thickness is associated with wound complications after open reduction and internal fixation of patella fractures.

BACKGROUND: Displaced patella fracture is commonly treated with open reduction and internal fixation (ORIF). Wound complications after surgery often lead to prolonged hospitalization and dissatisfaction of patients. Periarticular adiposity and swelling may be associated with wound complications. The purpose of this study is to determine the relationship between periarticular soft tissue thickness and wound complications after ORIF of patella fractures. METHODS: We retrospectively studied 237 patients undergoing ORIF for patella fractures from June 2017 to February 2021 at our institution. We established periarticular soft tissue thickness ratio (PSTTR) to evaluate soft tissue status on lateral knee X-ray radiographs. Univariate analysis was performed to identify the association between PSTTR and postoperative wound complication. A receiver-operating characteristic (ROC) curve analysis was performed to evaluate the predictive value of PSTTR. RESULTS: The incidence of postoperative wound complication was 10.5%. Minor wound complication occurred in 24 patients, and major wound complication occurred in one patient. The average femoral PSTTR (fPSTTR) was 0.94 ± 0.17 and the average tibial PSTTR (tPSTTR) was 0.66 ± 0.16. fPSTTR proved to be associated with postoperative wound complication. In the ROC analysis of fPSTTR predicting postoperative wound complication, the area under curve (AUC) was 0.676, which indicated a moderate predictive value. CONCLUSIONS: PSTTR was a feasible method to assess periarticular soft tissue. The increased fPSTTR was associated with wound complications after ORIF of patella fractures.

Single volar locking plating for the intra- and extra-articular distal radius fractures with dorsal metaphyseal comminution.

BACKGROUND: Volar locking plating remains a popular method for the surgical management of distal radius fractures. Dorsal metaphyseal comminution (DMC) is a common fracture pattern which weakens the stability during fracture fixation. In this study, we aimed to compare the radiographic and functional outcome of the intra- and extra-articular distal radius fractures with DMC following single volar locking plate fixation. MATERIALS AND METHODS: Patients suffered from a distal radius fracture with DMC were reviewed in the clinical database of the authors' institution between Jan 2016 and Jan 2020. The included patients were classified into the extra-articular (A3) group or the intra-articular (C2 and C3) group according to the AO/OTA system. The radiological parameters, wrist range of motion, and functional outcomes were evaluated following open reduction and volar locking plate fixation. RESULTS: A total of 130 patients were included in this study with a mean follow-up length of 17.2 months. Compared with the A3 fracture group, no significant fracture re-displacement or reduced wrist ROMs was observed in the C2 fractures after 12-month's follow-up. However, significantly decreased volar tilt (P = 0.003) as well as the extension/flexion ROMs were observed in the C3 fractures comparing to the A3 fractures. Most of the patients achieved an excellent (n = 75) or good (n = 51) Gartland and Werley wrist score. Four patients with C3 fractures resulted in a fair functional outcome due to a significant loss of volar tilt during follow-up. CONCLUSIONS: The single volar locking plate fixation provided sufficient stability for distal radius fractures with DMC, and resulted in similar radiological and functional outcomes in the intra-articular distal radius fractures with a simple articular component (C2 fractures) as those in the extra-articular fractures. Considering the intra-articular fractures with multifragmentary articular component (C3 fracture), despite of the subsequent loss of volar tilt, the majority of the patients achieved good to excellent wrist function following single volar locking plating. TRIAL REGISTRATION: This study has been registered on the ClinicalTrials.gov.

Quinone oxidoreductase 1 is overexpressed in gastric cancer and associated with outcome of adjuvant chemotherapy and survival.

BACKGROUND: Quinine oxidoreductase 1 (NQO1) plays a vital role in protecting normal cells against oxidative damage and electrophilic attack. It is highly expressed in many solid tumors, suggesting a role in cancer development and progression. However, the role of NQO1 in gastric cancer and its effect on cancer development and prognosis have not been fully investigated. AIM: To investigate the clinical relevance of NQO1 protein expression in gastric cancer and to explore the potential of NQO1 to serve as a prognostic biomarker and therapeutic target. METHODS: In this retrospective study, gastric cancer specimens of 175 patients who were treated between 1995 and 2011 were subjected to immunohistochemistry analyses for NQO1. The correlation of NQO1 expression with gastric cancer prognosis and clinical and pathological parameters was investigated. RESULTS: NQO1 protein was overexpressed in 59.43% (104/175) of the analyzed samples. Overexpression of NQO1 was associated with a significantly inferior prognosis. In addition, multivariate analysis suggested that NQO1 overexpression, along with tumor stage and patient age, are prominent prognostic biomarkers for gastric cancer. Moreover, NQO1 overexpression was correlated to a better response to 5-fluorouracil (5-FU)-based adjuvant chemotherapy. CONCLUSION: NQO1 overexpression is associated with a significantly poor prognosis and better response to 5-FU in patients with gastric cancer. These findings are relevant for improving therapeutic approaches for gastric cancer patients.

Outcomes of Femoral Neck Fractures Treated with Cannulated Internal Fixation in Elderly Patients: A Long-Term Follow-Up Study.

OBJECTIVES: To evaluate the long-term efficacy of cannulated internal fixation in patients who sustain femoral neck fracture (FNF) and risk factors influencing the outcomes. METHODS: This retrospective study analyzed data from 73 elderly patients aged ≥60 years old, hospitalized for FNF and treated with cannulated internal fixation between August 2008 and July 2016. The inclusion criteria were: (i) patients aged ≥60 years; (ii) patients with recent femoral neck fracture, with times of injury ranging from 12-72 h; and (iii) patients who underwent Garden II-IV closed reduction and internal fixation. Patients were classified based on the Garden fracture type and Pauwels angle. Clinical data and radiographs before and after the surgery were collected. Subsequently, surgery was performed with the patient in supine position, under general or lumbar epidural anesthesia. Closed reduction was performed until satisfactory fracture reduction quality was achieved. The weight of all included patients were <75 kg, and thus, minimal internal fixation was performed. The ischemic necrosis of the femoral head was diagnosed by considering the symptoms, signs, and radiological findings. Harris hip scores were used to evaluate postoperative recovery of hip function. Furthermore, relationships between Garden fracture type and necrosis rate of the femoral head, Pauwels angle and necrosis rate of the femoral head, Garden fracture type and Harris hip score, and age and Harris hip score were analyzed. RESULTS: The mean duration of surgery was 1 ± 0.17 h and blood loss for all the patients was approximately 15 mL. The included patients were followed up for 13-128 months, with an average follow-up of 61 months. Among the included 73 patients, 65 (89.0%) exhibited satisfactory union, seven (9.6%) had femoral head necrosis, and one (1.4%) had nonunion. For seven patients with femoral head necrosis, there were two, two, and three patients classified as Garden IV, Garden III, and Garden II, respectively, and two and five patients classified as Pauwels II and Pauwels III, respectively. Among the seven cases, four underwent hip replacement. Garden fracture type was not significantly associated with femoral head necrosis (χ2 = 0.44, P > 0.05) or Harris score (χ2 = 1.43, P > 0.05). Patients with Pauwels I (0%) and II (4.3%) fractures exhibited a significantly lower necrosis rate than those with Pauwels III fractures (41.7%) (P < 0.05). CONCLUSIONS: Cannulated internal fixation was more suitable for older Chinese patients with Pauwels I/II fractures than those with Pauwels III fractures.

Capsaicin suppressed activity of prostate cancer stem cells by inhibition of Wnt/ß-catenin pathway.

Cancer stem cells (CSCs) are considered as the origin and driving cells of cancer, and play a key role in the progress of cancer. Studies have shown that capsaicin exerted inhibitory effect on prostate cancer cells, however, the effects of capsaicin on prostate CSCs remain undefined. In the present study, we showed that capsaicin could downregulate prostate CSCs markers and inhibit the growth of PC-3 and DU145 prostate cancer stem cells. Further, we found capsaicin suppressed the expression of Wnt-2, p-GSK3ß and ß-catenin, along with downregulation of Wnt/ß-catenin pathway target genes c-myc and cyclinD1. Using LiCl, a activator of Wnt/ß-catenin pathway, we found activation of Wnt/ß-catenin pathway could ameliorate the downregulation of prostate CSCs markers and the growth inhibition induced by capsaicin in prostate cancer stem cells. Those data suggested that the inhibition effect of capsaicin on prostate cancer stem cells and the anti-cancer effect of capsaicin on prostate cancer stem cell may be mediated by Wnt/ß-catenin pathway. Findings from this study reveals for the first time the potential role and mechanisms of capsaicin on prostate cancer stem cells.

Identification of novel Nrf2 target genes as prognostic biomarkers in colitis-associated colorectal cancer in Nrf2-deficient mice.

AIMS: Colorectal cancer (CRC) is the third most common cancer worldwide. Nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of many cytoprotective genes, plays a protective role in carcinogenesis. Recent studies have identified a specific gene-expression signature regulated by the Nrf2 pathway in lung adenocarcinoma and head-and-neck squamous cell cancer. However, the roles of Nrf2 in the development of colitis-associated colorectal cancer (CACC) have not been well characterized. Nrf2 target genes as prognostic biomarkers in CACC remain to be explored. Thus, this work aimed to identify the molecular changes that occur during mouse CACC progression to facilitate the development of diagnostic and prognostic biomarkers. MAIN METHODS: The CACC model was established using azoxymethane (AOM) with dextran sulfate sodium salt (DSS) in BALB/c mice for 3 weeks to induce colitis-associated adenoma (CAA, early stage) and for 9 weeks to induce colitis-associated carcinoma (CAC, late stage). Using RNA-sequencing and bioinformatics analyses we examined the mRNA expression profiles of 6 groups: wild-type control (WT-C), WT-CAA, WT-CAC, Nrf2 knockout control (Nrf2KO-C), Nrf2KO-CAA, and Nrf2KO-CAC. KEY FINDINGS: In the AOM/DSS model of colitis-associated tumorigenesis, Nrf2-/- mice showed a phenotype similar to WT mice, but with significantly more tumors and a much higher percentage of adenocarcinomas. We identified 47 novel Nrf2 genes via gene expression profiling of tumor samples. Survival analysis showed that 23 of these genes were biomarkers of a poor prognosis in colon cancer patients. SIGNIFICANCE: Nrf2 target genes deserve exploration as prognostic and therapeutic targets for CRC.

Microwaves increase the effectiveness of systemic antibiotic treatment in acute bone infection: experimental study in a rat model.

BACKGROUND: Osteomyelitis is a challenge for orthopedic surgeons due to its protracted treatment process. Microwaves (MWs) can increase blood perfusion due to their thermal effect. Furthermore, MWs demonstrated significant bactericidal effects in vitro. In the present study, we assumed that the application of a 2450-MHz-frequency MW together with systemic antibiotic treatment would provide synergy for the treatment of acute osteomyelitis. METHODS: The medullary cavity of the right tibia was inoculated with 107 CFU of methicillin-sensitive Staphylococcus aureus (MSSA-ATCC 29213) in 40 rats, and the rats were randomly divided into four groups according to treatment: group I, saline (control); group II, saline + MW therapy; group III, systemic cefuroxime; and group IV, systemic cefuroxime + MW therapy. MWs were applied for 20 min per day to the infected limbs, and all rats were sacrificed on the 7th day. The severity of tibial osteomyelitis was assessed by quantitative culture analysis. RESULTS: Bacterial counts in groups III and IV were significantly reduced compared with those in the control (p = 0.001 and < 0.001, respectively). Furthermore, significant differences were detected between groups III and IV (p = 0.033). However, the difference between groups I and II was nonsignificant (p = 0.287). CONCLUSION: Our experimental model suggests that MW therapy provides a significant synergy for systemic antibiotic treatment. However, further clinical trials are required to safely use this treatment modality in patients.

Molecular investigation of "Candidatus Mycoplasma haemobos" in goats and sheep in central China.

Hemoplasma "Candidatus Mycoplasma haemobos" infections in cattle have been reported in East Asia, Europe, and South America, whereas same cases were documented in buffalo and cattle in Southern China. From April 2018 to May 2018, a mycoplasma epidemic was reported in the mountainous area of central China; Boophilus microplus has also been documented, causing severe haematuria in goats and sheep. The infected animals slowly recovered after diminazene aceturate and praziquantel treatment. To determine whether the hemoplasma caused this infection, 67 blood samples (42 from goats, 25 from sheep) and 132 B. microplus samples were collected for PCR and sequence analysis. The results showed that 19 out of the 42 goat blood samples, 10 out of the 25 sheep blood samples, and 70 out of the 132 B. microplus samples (53%) tested positive for "C. M. haemobos". This study provides molecular evidence of "C. M. haemobos" infections in goat and sheep, and that B. microplus harbours "C. M. haemobos".

Characterization of a Pestivirus H isolate originating from goats.

Natural Pestivirus H infections in cattle have been reported worldwide; however, only a few cases of Pestivirus H have been described in non-bovine ruminants such as goats. A new Pestivirus H HN1507 strain was isolated from an infected goat in 2015 and the genome sequence was determined. The full-length genome sequence was 12,556 nucleotides. Phylogenetic analysis, based on the complete genome and Npro fragments, revealed that the isolate belonged to Pestivirus H and was closely related to strains from Italy. Two unique amino acid substitutions were found in the C-terminal of the E2 protein. To the best of our knowledge, this is first report determining the complete genome of a Pestivirus H strain from goat.

The variation in urinary calcium levels in adult patients with fracture and surgical intervention.

BACKGROUND: Generally, a higher calcium diet is fed to fracture patients after surgery. However, recent studies have indicated that higher dietary calcium intakes increase the risk of urinary stones for fracture patients. Therefore, this study aimed to observe the variation in urinary calcium levels among fracture patients who underwent surgery, based on fracture type, fracture location, age and gender. METHODS: A total of 768 subjects were enrolled in this study from 2012 to 2015 and were divided into 2 groups: group A (fracture patients who underwent surgery) and group B (normal patients without fracture). Urine samples were collected for a 24-h period (24-h urine), at multiple specific time points before and after surgery for group A, or after hospitalisation for group B. Subsequently, urine calcium was detected and the changes were evaluated according to fracture location, fracture type, age and gender, as well as the distribution of hypercalciuria. RESULTS: Compared with group B, the level of urine calcium in group A significantly increased at different time points during the study period (P < 0.05). There were significant differences in the changes in urine calcium levels according to fracture location, fracture type and age, but not gender. Further, there were more patients with hypercalciuria in group A at the different time points, compared with group B. CONCLUSION: Variation in urinary calcium among fracture patients that underwent surgery was of a regular pattern and hypercalciuria was also found in these patients. Therefore, a high-calcium diet and calcium supplements should be used with caution in this patient population.

Outcomes of cement beads and cement spacers in the treatment of bone defects associated with post-traumatic osteomyelitis.

BACKGROUND: Cement spacers (Masquelet technique) have traditionally been used for the treatment of segmental bone defects. However, no reports have used cement spacers for the treatment of small/partial segmental bone defects associated with osteomyelitis and compared the outcomes with cement beads. METHODS: We retrospectively analysed 40 patients with post-traumatic osteomyelitis of the tibia who underwent treatment, which was performed in two stages. In the first stage, thorough debridement was performed, and bone defects were filled with either antibiotic-impregnated cement beads (bead group, 18 patients) or spacers (spacer group, 22 patients). In the second stage, the cement beads or spacers were removed (for the spacer group, the induced membrane formed by the spacer was preserved) and the bone defects were filled with cancellous autografts. RESULTS: All patients in the bead group had small/partial segmental bone defects after debridement, while 3 patients in the spacer group had large/segmental bone defects. The mean volume of bone defects of the spacer group (40.4 cm3) was significantly larger than that of the bead group (32.4 cm3). The infection control rate (88.9%,16/18 vs 90.9%, 20/22), bone healing time (8.5 months vs 7.5 months) and complication rates (22.2%, 4/18 vs 27.2%, 6/22) were comparable between bead group and spacer group. CONCLUSION: The results of this study suggest that cement spacers may have an infection control rate comparable to cement beads in the treatment of bone defects associated with post-traumatic osteomyelitis. Furthermore, cement spacers could be used for the reconstruction of small/partial segmental bone defects as well as for large/segmental bone defects, whereas cement beads were not suitable for the reconstruction of large/segmental bone defects.

BDNF regulates the expression and secretion of VEGF from osteoblasts via the TrkB/ERK1/2 signaling pathway during fracture healing.

Brain­derived neurotrophic factor (BDNF), a member of the neurotropic family, is expressed in osteoblast­like cells of a fracture callus, however, its role in fracture healing remains to be fully elucidated. Osteoblasts isolated from Sprague Dawley rats were stimulated by BDNF in a dose­ and time­dependent manner. Immunoblotting and immunofluorescence was used to detect the expression and distribution of targeted proteins. The concentration of vascular endothelial growth factor (VEGF) released in medium was determined using an ELISA. PD98059 and K252a were used to investigate the signaling pathways that may be involved. The present study demonstrated that BDNF was involved in fracture repair by controlling the expression and secretion of VEGF from osteoblasts, which predominantly drives angiogenesis during fracture healing. Tropomyosin­related kinase B (TrkB), the specific receptor of BDNF, was shown to be expressed at high levels in the osteoblasts. Following BDNF stimulation, TrkB and extracellular signal­regulated kinase 1/2 (ERK1/2) were rapidly activated. The inhibition of TrkB by K252a decreased the expression and secretion of VEGF, and suppressed the phosphorylation level of ERK1/2. PD98059, an antagonist of ERK1/2, elicited the same effects on VEGF from the BDNF­stimulated osteoblasts, however, it did not affect the phosphorylation of TrkB. In conclusion, during fracture healing, BDNF was found to stimulate the expression and secretion of VEGF from osteoblasts via the TrkB/ERK1/2 signaling pathway.

Acupuncture combined with methylcobalamin for the treatment of chemotherapy-induced peripheral neuropathy in patients with multiple myeloma.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) seriously affects the quality of life of patients with multiple myeloma (MM) as well as the response rate to chemotherapy. Acupuncture has a potential role in the treatment of CIPN, but at present there have been no randomized clinical research studies to analyze the effectiveness of acupuncture for the treatment of CIPN, particularly in MM patients. METHODS: The MM patients (104 individuals) who met the inclusion criteria were randomly assigned into a solely methylcobalamin therapy group (500 µg intramuscular methylcobalamin injections every other day for 20 days; ten injections) followed by 2 months of 500 µg oral methylcobalamin administration, three times per day) and an acupuncture combined with methylcobalamin (Met + Acu) group (methylcobalamin used the same way as above accompanied by three cycles of acupuncture). Of the patients, 98 out of 104 completed the treatment and follow-ups. There were 49 patients in each group. The evaluating parameters included the visual analogue scale (VAS) pain score, Functional Assessment of Cancer Therapy/Gynecologic Oncology Group-Neurotoxicity (Fact/GOG-Ntx) questionnaire scores, and electromyographic (EMG) nerve conduction velocity (NCV) determinations. We evaluated the changes of the parameters in each group before and after the therapies and made a comparison between the two groups. RESULTS: After 84 days (three cycles) of therapy, the pain was significantly alleviated in both groups, with a significantly higher decrease in the acupuncture treated group (P < 0.01). The patients' daily activity evaluated by Fact/GOG-Ntx questionnaires significantly improved in the Met + Acu group (P < 0.001). The NCV in the Met + Acu group improved significantly while amelioration in the control group was not observed. CONCLUSIONS: The present study suggests that acupuncture combined with methylcobalamin in the treatment of CIPN showed a better outcome than methylcobalamin administration alone. TRIAL REGISTRATION: China Clinical Trials Register (registration no. ChiCTR-INR-16009079 , registration date August 24, 2016).

Thromboxane Governs the Differentiation of Adipose-Derived Stromal Cells Toward Endothelial Cells In Vitro and In Vivo.

RATIONALE: Autologous adipose-derived stromal cells (ASCs) offer great promise as angiogenic cell therapy for ischemic diseases. Because of their limited self-renewal capacity and pluripotentiality, the therapeutic efficacy of ASCs is still relatively low. Thromboxane has been shown to play an important role in the maintenance of vascular homeostasis. However, little is known about the effects of thromboxane on ASC-mediated angiogenesis. OBJECTIVE: To explore the role of the thromboxane-prostanoid receptor (TP) in mediating the angiogenic capacity of ASCs in vivo. METHODS AND RESULTS: ASCs were prepared from mouse epididymal fat pads and induced to differentiate into endothelial cells (ECs) by vascular endothelial growth factor. Cyclooxygenase-2 expression, thromboxane production, and TP expression were upregulated in ASCs on vascular endothelial growth factor treatment. Genetic deletion or pharmacological inhibition of TP in mouse or human ASCs accelerated EC differentiation and increased tube formation in vitro, enhanced angiogenesis in in vivo Matrigel plugs and ischemic mouse hindlimbs. TP deficiency resulted in a significant cellular accumulation of ß-catenin by suppression of calpain-mediated degradation in ASCs. Knockdown of ß-catenin completely abrogated the enhanced EC differentiation of TP-deficient ASCs, whereas inhibition of calpain reversed the suppressed angiogenic capacity of TP re-expressed ASCs. Moreover, TP was coupled with Gαq to induce calpain-mediated suppression of ß-catenin signaling through calcium influx in ASCs. CONCLUSION: Thromboxane restrained EC differentiation of ASCs through TP-mediated repression of the calpain-dependent ß-catenin signaling pathway. These results indicate that TP inhibition could be a promising strategy for therapy utilizing ASCs in the treatment of ischemic diseases.

Mechanical property and biological performance of electrospun silk fibroin-polycaprolactone scaffolds with aligned fibers.

The mechanical strength, biocompatibility, and sterilizability of silk fibroin allow it to be a possible candidate as a natural bone regenerate material. To improve mechanical character and reinforce the cell movement induction, silk fibroin (SF)-polycaprolactone (PCL) alloy was fabricated by electrospinning techniques with a rotating collector to form aligned fibrous scaffolds and random-oriented scaffolds. The scanning electron microscope image of the scaffold and the mechanical properties of the scaffold were investigated by tensile mechanical tests, which were compared to random-oriented scaffolds. Furthermore, mesenchymal stem cells were planted on these scaffolds to investigate the biocompatibility, elongation, and cell movement in situ. Scanning electron microscopy shows that 91% fibers on the aligned fibroin scaffold were distributed between the dominant direction ±10°. With an ideal support for stem cell proliferation in vitro, the aligned fibrous scaffold induces cell elongation at a length of 236.46 ± 82 µm and distribution along the dominant fiber direction with a cell alignment angle at 6.57° ± 4.45°. Compared with random-oriented scaffolds made by artificial materials, aligned SF-PCL scaffolds could provide a moderate mesenchymal stem cell engraftment interface and speed up early stage cell movement toward the bone defect.