Activation of the MEK/ERK Pathway Mediates the Inhibitory Effects of Silvestrol on Nasopharyngeal Carcinoma Cells via RAP1A, HK2, and GADD45A.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor associated with Epstein-Barr virus (EBV) infection. Chemoradiotherapy is the mainstream treatment for locally advanced NPC, and chemotherapeutic drugs are an indispensable part of NPC treatment. However, the toxic side-effects of chemotherapy drugs limit their therapeutic value, and new chemotherapy drugs are urgently needed for NPC. Silvestrol, an emerging natural plant anticancer molecule, has shown promising antitumor activity in breast cancer, melanoma, liver cancer, and other tumor types by promoting apoptosis in cancer cells to a greater extent than in normal cells. However, the effects of silvestrol on NPC and its possible molecular mechanisms have yet to be fully explored. METHODS: Cell counting kit-8 (CCK-8), cell scratch, flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU), and Western blot (WB) assays were used to evaluate the effects of silvestrol on the cell viability, cell cycle, apoptosis, and migration of NPC cells. RNA sequencing (RNA-Seq) was used to study the effect of extracellular signal-regulated kinase (ERK) inhibitors on the cell transcriptome, and immunohistochemistry (IHC) to assess protein expression levels in patient specimens. RESULTS: Silvestrol inhibited cell migration and DNA replication of NPC cells, while promoting the expression of cleaved caspase-3, apoptosis, and cell cycle arrest. Furthermore, silvestrol altered the level of ERK phosphorylation. The ERK-targeted inhibitor LY3214996 attenuated silvestrol-mediated inhibition of NPC cell proliferation but not migration. Analysis of RNA-Seq data and WB were used to identify and validate the downstream regulatory targets of silvestrol. Expression of GADD45A, RAP1A, and hexokinase-II (HK2) proteins was inhibited by silvestrol and LY3214996. Finally, IHC revealed that GADD45A, RAP1A, and HK2 protein expression was more abundant in cancer tissues than in non-tumor tissues. CONCLUSIONS: Silvestrol inhibits the proliferation of NPC cells by targeting ERK phosphorylation. However, the inhibition of NPC cell migration by silvestrol was independent of the Raf-MEK-ERK pathway. RAP1A, HK2, and GADD45A may be potential targets for the action of silvestrol.

Successful surgical treatment of polybacterial gas gangrene confirmed by metagenomic next-generation sequencing detection: A case report.

BACKGROUND: We report on a case of Vibrio vulnificus (V. vulnificus) detected by metagenomics next-generation sequencing (mNGS) in a 53-year-old male patient with polymicrobial gas gangrene and successful treatment by surgery. This report raises awareness among dermatologists that when a patient is clinically suspected of a special type of pathogenic infection, the mNGS method should be preferred to identify the patient's pathogen infection as soon as possible and then take effective treatment in time to save patients' lives. CASE SUMMARY: A 53-year-old male who worked in the aquatic market complained of redness and swelling of the lower limbs, blisters and ulcers with fever for 3 d. We used mNGS to test the pathogens in ulcer secretions. The results were returned in 24 h and indicated: V. vulnificus, Fusobacterium necrophorum, Staphylococcus haemolyticus, Staphylococcus aureus, Streptococcus dysgalactiae and Klebsiella aerogenes. This patient was diagnosed with V. vulnificus infection. The emergency operation was performed immediately under combined lumbar and epidural anesthesia: Left leg expansion and exploration (August 10, 2021). After surgery, we continued to use piperacillin sodium tazobactam sodium 4.5 g every 8 h and levofloxacin 0.5 g for anti-infection treatment. The patient underwent further surgery under lumbar anesthesia on August 17, 2021 and August 31, 2021: Left leg deactivation and skin grafting, negative pressure closed drainage and right thigh skin removal. After treatment, the transplanted flap survived. CONCLUSION: We could confirm the diagnosis of Vibrio vulnificus infection within 24 h through mNGS detection and then immediately performed emergency surgery.

[Efficacy of dermal scaffold for promoting repair of acute full-thickness skin defects in pigs].

OBJECTIVE: To investigate the efficacy of Lando® dermal scaffold for promoting repair of acute full-thickness skin defects in pigs and explore the possible mechanism. METHODS: Three 5 cm×5 cm full-thickness skin defects were created on the left dorsal skin (control group) and another 3 on the right dorsal skin (treatment group) of each of 6 Tibetan pigs. The wounds in the treatment group were covered with a bilayer artificial skin (Lando) and the control wounds with vaseline gauze. In both groups, autogenous split-thickness skin were grafted to the wounds 2 weeks later (with the silicone rubber membrane removed before grafting in the treatment group). At 3 days and 2 and 10 weeks after the injury, the wounds were assessed for general condition and contraction, and tissue samples were collected from the wounds to examine the expressions of α-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1) using immunohistochemistry and the expressions of MMP-1 and TIMP-1 mRNA using RT-PCR. RESULTS: At 3 days after the injury, the wounds in the 2 groups showed no significant differences in the results of any examinations. At 2 weeks after the injury, the wounds in the treatment group showed rich and more smooth granulation tissues with more regular wound edges compared with the control wounds. At 2 and 10 weeks after the injury, the wound contraction rates in the treatment group were (30.5∓3.4)% and (39.2∓2.8)%, respectively, significantly lower than the rates of (51.8∓2.6)% (t=-29.840, P=0.000) and (60.7∓2.2)% (t=-50.213, P=0.000) in the control group. At 2 weeks, the wound tissues in the treatment group expressed significantly higher levels of α-SMA (t=15.921, P=0.000) and TGF-ß1 (t=29.995, P=0.000) than the control wounds, but at 10 weeks, the expressions of α-SMA (t=-41.823, P=0.000) and TGF-ß1 (t=-99.777, P=0.000) in the treatment group were significantly lower than those in the control group. Compared with those in the control group, the expression of MMP-1 mRNA in the treatment group was significantly lower at 2 weeks (t=-45.412, P=0.000) but significantly higher at 10 weeks (t=78.769, P=0.000), and the expression of TIMP-1 mRNA in the treatment group was significantly lower both at 2 weeks (t=-27.064, P=0.000) and at 10 weeks (t=-40.535, P=0.000). CONCLUSIONS: Lando® dermal scaffold can promote granulation tissue growth possibly in relation with increased TGF-ß1 and decreased MMP-1 expression in the wounds. This scaffold material also reduces wound contraction and lessens scar hyperplasia and contracture after wound healing, probably as a result of decreased α-SMA, TGF-ß1, and TIMP-1 and increased MMP-1 expressions.

[Clinical observation on repairing of wounds of skin graft donor site with acellular tissue engineering dermal matrix].

OBJECTIVE: To evaluate the clinical efficacy of acellular tissue engineering dermal matrix (ATDM) in repairing wounds of skin graft donor site. METHODS: Sixty patients with burn or chronic wounds hospitalized from January 2011 to April 2012 received autologous skin grafting. One wound [with size larger than 55 cm(2), and thickness of (0.33 ± 0.03) mm] out of multiple skin graft donor sites of every patient was selected, and it was divided into two parts in accordance with self-control principle. A part of wound close to the wound edge with diameter of 5 cm was taken as trial area (treated with ATDM), and the remaining wound was taken as control area (treated with vaseline gauze) according to the random number table. Blood and urine routine, liver and kidney function, and levels of IgG and IgM in blood of patients were measured one day before operation and on the 1st day after wound healing. Vital signs of patients were recorded on the operation day and the wound healing day. Gross condition of the wounds was observed during dressing change. Wound healing time was recorded. The healed wound was observed histologically. Data were processed with Log rank test or t test. RESULTS: Leucocyte count was lowered on the 1st day after wound healing [(7.1 ± 1.2)×10(9)/L] as compared with that one day before operation [(10.1 ± 1.5)×10(9)/L, t = -12.10, P < 0.01]. The differences were not statistically significant in red blood cell count, haemoglobin level, platelet count, urine routine, levels of indexes of liver and kidney function, levels of IgG and IgM in blood between one day before operation and the 1st day after wound healing, or in vital signs (including body temperature, pulse, respiration, systolic pressure, and diastolic pressure) between the operation day and the wound healing day (with t values from -1.43 to 1.88, P values all above 0.05). No adverse effects such as abnormal exudation, itching, redness and swelling, and exanthema were observed in the wound. The median wound healing time in trial area was 12 d (95% confidence interval: 11 - 13 d), which was significantly shorter than that in control area [17 d (95% confidence interval: 16 - 18 d), χ(2) = 24.9, P < 0.01]. The healed wound of trial area was closer to the normal skin than that of control area in the shape and distribution of Fb and vascular endothelial cell, and the shape of the basilar membrane and the epidermal layer. CONCLUSIONS: ATDM can accelerate healing of wound of skin graft donor site, and no adverse reactions were observed.

[Population pharmacokinetics research of clozapine in Chinese schizophrenic patients].

The goal of this study is to investigate the population pharmacokinetics of oral given clozapine in Chinese schizophrenic patients and to identify possible relationships between population parameters and covariates including demography factors and CYP1A2 genetic polymorphism, so as to create the population pharmacokinetics model to guide individual clinical delivery. Details of drug dosage history, sampling time and concentration of 626 data points from 183 patients were collected retrospectively. The 183 patients were randomly allocated either to the index group (n = 168) or to the validation group (n = 15). Population pharmacokinetic data analysis was performed using the nonlinear mixed-effects model (NONMEM) program on the index group. The values of apparent clearance (CL/F), apparent volume of distribution (V/F) and the constant of absorption rate were estimated. A number of covariates including demographic index, coadministration of other drugs and CYP1A2 genotypes were evaluated statistically for their influence on these parameters. The final population model related clearance with day-dose/BSA (DBSA) and smoke habit (SMOK). Predictive performance of the final model evaluated with the validation group showed insignificant bias between observed and model predicted concentrations. Typical value of CL/F (non-smoking group), V/F and the constant of absorption rate were 28.5 L x h(-1) (5.05%), 1 290 L (16.7%) and 2.26 h(-1) (fixed), inter-patient variability (CV) in CL/F and V/F was) 42.2% and 10.0%, respectively. It was observed that the values of CL/F in the two smoking groups were higher than that in the non-smoking group. The residual variability (SD) between observed and model-predicted concentrations was 45.8 microg x L(-1).

[Sequential management of residual wounds in burn patients].

OBJECTIVE: To seek a sequential method for the management of residual wounds in burn patients. METHODS: Three chronic residual wounds on each of 25 burn patients were either covered with vaseline gauze (A group), human tissue-engineered active skin (Active Skin, B group) or Active Skin after rinsing with fluid containing oxygen and vacuum assisted drainage ( C group) on wounds. The contents of (TNF)a in granulation tissue were assayed by enzyme linked immunosorbent assay (ELISA). Expression of metalloproteinase-13 (MMP-13) mRNA in granulation tissue was determined with reverse transcription polymerase chain reaction (RT-PCR). Moreover, quantity of wound bacteria in the wounds and wound healing rate were determined with usual method. RESULTS: The quantities of wound bacteria in C group on 3,6,9, 12 post-treatment day( PTD) were (5.30 +/- 1.60), (1.30 +/-0.80) , (1.70 +/- 0. 60)and (0.60 +/-0. 10)clone formation unit/ml( CFU/ml) , respectively, which were obviously lower than those in A and B groups. The contents of TNFa and expression of metalloproteinase-13 (MMP-13) mRNA in granulation tissue in C group on 6 PTD were [ (0. 650 +/- 0. 040) ng/mg and 0. 210 +/- 0. 010,] ,respectively, and they were evidently lower than those in A group [(1.550 +/-0. 370)ng/mg,1. 040 +/- 0. 050, P <0.01] and B group (0. 810 +/- 0.080) ng/mg, 0.640 +/- 0.030, P <0.01]. Meanwhile, the contents of (TNF)a and expression of MMP-13 mRNA in B group were also obviously lower than those in A group. The wound healing ratio in C group on 15 and 30 PTD were markedly higher than those in A or B group ( P <0.01). CONCLUSION: Covering the residual burn wounds with Active Skin after rinsing with fluid containing oxygen followed by vacuum assisted drainage can improve repairing of residual burn wounds.

[Effects of selective cyclooxygenase-2 inhibitor on proliferation and apoptosis of human bladder cancer cell line T24].

BACKGROUND & OBJECTIVE: Cyclooxygenase-2 (COX-2) is related closely to the tumorigenesis of bladder cancer, and COX-2 inhibitor has potential antitumor effect. This study was to investigate the effects of selective COX-2 inhibitors on the proliferation and apoptosis of human bladder cancer cell line T24. METHODS: The effects of selective COX-2 inhibitors SC-58125 and celecoxib, and nonselective COX inhibitor indomethacin on the proliferation of T24 cells were evaluated by MTT assay. Cell apoptosis was determined by flow cytometry (FCM), DNA ladder electrophoresis, and fluorescent microscopy with Hoechst33258 staining. The expression of apoptosis-related genes Bcl-2 and Bax were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Within concentrations of 12.5-200 micromol/L, SC-58125, celecoxib, and indomethacin could inhibit the proliferation of T24 cells to different extents. SC-58125 tended to be more effective than the other two. The 50% inhibition concentration (IC50) of SC-58125 was determined to be 25-50 micromol/L. The apoptosis of T24 cells was enhanced after exposure to SC-58125. When treated with 100 micromol/L SC-58125 for 6 and 12 h, the apoptosis rates of T24 cells were (7.95+/-1.88)% and (12.5+/-2.42)%, respectively, which were significantly higher than that of control cells (P<0.05). But the expression of Bcl-2 and Bax genes did not change. CONCLUSIONS: Selective COX-2 inhibitor could inhibit the proliferation and induce the apoptosis of T24 cells.

[Anti-inflammatory activity and healing-promoting effects of topical application of emu oil on wound in scalded rats].

OBJECTIVE: To investigate the effects of topical application of emu oil on wound healing in scalded rats. METHODS: In 144 male Wistar rats with 10%; total body surface superficial II degree scald treated on a random basis with physiological saline, povidone iodine and emu oil, respectively, the changes of the wound were observed and the wound tissue and blood samples harvested at different times after injury for evaluation of histopathological changes, total tissue water content (measured by wet:dry weight ratios), and tumor necrosis factor (TNF)-alpha levels in the wound tissue and plasma by enzyme-linked immunosorbent assay (ELISA). The general condition of the wound healing was also observed. RESULTS: After application of emu oil, the swelling and effusion of the burn wound were alleviated and evidences of wound infection or adverse effects were not observed. Pathological examination showed that emu oil could alleviate topical inflammation, which was particularly obvious on days 1 and 3 after injury as compared with the other two groups. On day 3 after injury, water content and TNF-alpha level in the tissues was markedly decreased with the application of emu oil (P<0.05), with a significant correlation between their changes (P<0.001) and shortened wound healing time (P<0.05). Pathological examination showed that emu oil could promote epithelialization and differentiation of various epidermal layers. CONCLUSION: Emu oil has topical anti-inflammatory activity in rats with superficial II degree scald, possibly in association with decreased levels of the proinflammatory cytokines in the tissues and can promote wound healing by inhibiting local secondary inflammation.

[Management of extensive burn accompanying severe inhalation injury].

OBJECTIVE: To review our clinical experiences in the treatment of extensive burn accompanied by severe inhalation injury. METHODS: A retrospective analysis was conducted of 18 such cases admitted from 2000 to 2003 in light of the curative rate, mortality rate and measures for effective management. RESULTS AND CONCLUSIONS: Fifteen patients (83.3%) were cured and 3 (16.7%) died. The curative rate of extensive burn accompanied by severe inhalation injury can be enhanced by combining various treatments, including early preventive tracheostomy, appropriate tracheobronchial lavage, humidification and application of ambroxol; protective lung ventilation, and administration of growth hormones should be done as early as possible.