Objective To explore the detailed underlying molecular and signaling mechanisms in the effects of icariin on bone formation by an in vitro cell model. Methods The proliferation of MC3T3-E1 osteoblast-like cells was evaluated by MTT, and gene expression of cell cycle related proteins in MC3T3-E1 cells after icariin treatment was detected by real-time PCR. The phosphorylation of MAPK signals, including ERK, P38, and JNK was determined by Western blot, and then the inhibitors of MAPK signals were used to treat cells with icariin alone or together to determine the role of MAPKs in the process of icariin treatment on MC3T3-E1 cell proliferation. Alkaline phosphatase and Alizarin red staining were used to detect the formation of mineralization nodules, and gene expressions of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in osteoblasts after being treated by icariin were evaluated by real-time PCR. ICI182780, and nilutamide was used to decide the participation of estrogen and androgen receptor signals in the process of icariin treatment on the differentiation and mineralization of MC3T3-E1 cells. Results Treatment with icariin promoted MC3T3-E1 cell growth in a time- and dose-dependent manner. This treatment also revealed that icariin increased the expression of mRNAs encoding both cyclin E and PCNA, positive regulators of cell growth, but decreased levels of mRNAs encoding Cdkn2b, a negative regulator of cell cycle progression. When MC3T3-E1 cells were cultured in a differentiated condition, icariin enhanced mineralized nodule formation and increased the expression of mRNAs encoding alkaline phosphatase, type Ⅰ collagen, and osteocalcin. Treatment with icariin significantly induced phosphorylation of both ERK and JNK and this phosphorylated effect occurred very rapidly within 5 minutes and reached peak at 15 minutes. Furthermore, the stimulated effects of icariin on proliferation and gene expression of cyclin E, PCNA, and Cdkn2b in MC3T3-E1 cells were dramatically attenuated by treatment with both U0126 and SP600125, inhibitors of MAPKs. Interestingly, such stimulating effects of icariin were at least partly reduced by treatment with ICI182780, an inhibitor of estrogen receptor. Icariin induced mineralized nodule formation and gene expression of alkaline phosphatase, type Ⅰ collagen, and osteocalcin in MC3T3-E1 cells were also partly reduced when the cells were treated with ICI182780. Conclusions Our findings indicate that the anabolic effect of icariin on bone formation is, at least partly, mediated through the MAPK signaling pathway in order to modulate osteoblast proliferation and differentiation.

Objective To assess the coordinated regulation and the molecular mechanisms of 17β-estradiol and 1,25-dihydroxyvitamin D3 [ 1,25-( OH ) 2 D3 ] on the proliferation and differentiation of osteoblastic MC3T3-E1 cells.Methods MC3T3-E1 cells were cultured in phenol-red free α-MEM medium supplemented with 10％ FBS,MTT assay was performed to determine the effects of 17β-estradiol and 1,25-( OH )2 D3 on MC3T3-E1 cells proliferation.After cells were treated with different agents,cell cycle related genes [ cyclin E,proliferation cell nuclear antigen ( PCNA ),and cyclin-dependent kinase inhibitor 2b ( Cdkn2b ) ] and markers of osteoblastic differentiation [ type Ⅰ collagen ( COL Ⅰ ),alkaline phosphatase ( ALP),osteopontin ( OPN ) ] were detected with SYBR green-based quantitative PCR.ALP activity was detected with BCIP/NBT method.Results 17β-estradiol could promote proliferation of MC3T3-E1 cells,which was accordant to its ability to increase cyclin E and PCNA and to inhibit Cdkn2b mRNA expression in MC3T3-E1 cells.However,1,25-( OH)2D3 had no effect on the proliferation of MC3T3-E1 cells and also did not enhance the proliferation of MC3T3-E1 cells stimulated by 17β-estradiol.On the other hand,17β-estradiol promoted the gene expression of differentiation markers Col Ⅰ,ALP,and OPN,and 1,25-(OH) 2 D3 synergistically increased the expression of these genes with 17 β-estradiol.Conclusion As two of the most important hormones which regulate bone metabolism,estrogen and vitamin D may coordinately promote osteoblast differentiation,but may not regulate osteoblasts proliferation synergistically.

Objective:To retrospectively investigate the efficiency of Jinlong capsule combined with transarterial chemoemboli-zation for the treatment of advanced pancreatic carcinoma. Methods: Sixty advanced pancreatic carcinoma patients between January 2009 and May 2013 were randomly assigned into the experimental group (n=30, Jinlong capsule combined with transarterial chemoem-bolization) and the control group (n=30, transarterial chemoembolization). Both groups were subjected to superselective pancreas artery chemotherapy with GEMZAR at a dose of 1.0 g/m2. Results:The overall response rate (CR+PR) was 53.3%and the control rate was 70%(CR+PR+SD) (CR 0, PR 16, SD 5) in the experimental group, whereas those in the control group were 36.7%and 56.7%(CR 0, PR 11, SD 6), respectively. The short-term therapeutic efficacy was higher in the experimental group than in the control group (P<0.05). No significant difference in adverse reactions, including digestive reactions and myelosuppression, almost I-II degree, was observed be-tween the two groups (P>0.05). No significant difference in the Karnofsky performance status was observed between the two groups af-ter the treatment (P<0.05). The 24-month survival rate of the experimental group was 50.0%, whereas that of the control group was 33.3%(P<0.05). Conclusion:The application of Jinlong capsule combined with transarterial chemoembolization for the treatment of advanced pancreatic carcinoma showed good clinical effects. The patients also showed good tolerance to this treatment.

Objectives To evaluate the safety and efficacy of endovascular angioplasty for extracranial vertebral artery ( VA ) stenosis caused by atherosclerosis. Methods We analyzed retrospectively data of the 24 patients with extracranial vertebral artery stenosis who had been placed endovascular angioplasty from April 2006 to March 2010. According to Mori classification, there were 21 type A and 3 type B among all cases.The artery stenosis rate was 60％ -95％ , the average was 79％ ± 10％. Results Twenty-four balloon mounted stents were placed, the successful rate was 100％. Postoperatively the stenosis rate decreased to 4％ ± 6％. Patients were followed up from 3 to 36 months, the average was 22 months. Symptomes disappeared in 15 out of 17 patients. Postoperative restenosis on the treatment site with transient brain ischemia occurred in one patient. The symptoms in another patient of multiple cerebral infarction with ataxia and episodic vertigo were not relieved, although the patient didn't suffer from apoplectic seizure after the intervention. Postoperative color Doppler ultrasound revealed an over 50％ residual stenosis in 5 patients. The postoperative restenosis rate was 20. 8％. According to Malek scoring, 22 patients were scored 1 point, 1 patient scored 2 and one scored 4. Conclusions Endovascular angioplasty with stent placement is a safe and effective treatment. The restenosis rate could be futher reduced by technology improvement.

Objective To investigate the value of Q-analysis real-time elasticity in differentiating between benign and malignant thyroid nodules. Methods Eighty-six thyroid nodules in 62 patients with pathologic diagnosis were included in this study and were examined using Q-analysis real-time elasticity. The real-time elasticity features were observed and the quantitative index including the whole elasticity rate and the local elasticity rate were compared between benign and malignant nodules. Results There were 51 benign and 35 malignant nodules according to histopathological examination. The Q-analysis curve of real-time elasticity of benign nodules was smoother and with lower peak, compared with that of malignant nodules. The whole elasticity rate of malignant nodules were significantly higher than that of benign nodules (3.59±0.84 vs 2.32±0.56, P=0.000). And the local elasticity rate of malignant nodules were significantly higher than that of benign nodules (3.96±1.32 vs 2.39±0.58, P=0.000). The cutoff point of whole elasticity rate for the differential diagnosis was 3.25 with sensitivity, specificity and diagnostic accuracy as 71.4%, 96.1% and 86.0% respectively. The cutoff point of local elasticity rate for the differential diagnosis was 3.45 with sensitivity, specificity and diagnostic accuracy as 68.6%, 96.1% and 84.9% respectively. The diagnostic efficiency of whole elasticity rate and local elasticity rate had no significant difference (P=0.591).Conclusions Q-analysis real-time elasticity could provide the real-time elasticity features of thyroid nodules. The whole and local elasticity rate as the quantitative index contributed to the differential diagnosis of benign and malignant thyroid nodules.

Objective To evaluate the effect of mild hypothermia on the renal ischemia-reperfusion injury (IRI), and the expression profile of RNA-binding motif protein 3(RBM3) and its downstream effector molecules during this process. Methods Eighteen healthy SD male rats were randomly divided into the normal control (NC) group, IRI group and mild hypothermia pretreat (MHP) group, with 6 rats in each group. Serum creatinine level was measured to evaluate the renal function. Hematoxylin-eosin (HE) staining was performed to assess the renal tissue injury. Western blot was used to determine the relative expression levels of RBM3, Yes-associated protein 1(YAP1), nuclear factor E2-related factor 2(Nrf2), B cell-lymphoma-2(Bcl-2) and Bcl-2-associated X protein (Bax) in the kidney tissues. Immunohistochemical staining was employed to further detect the expression levels of RBM3 and YAP1 proteins. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was adopted to detect the cell apoptosis of kidney tissues. Malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were evaluated to determine the oxidative stress level of kidney tissues. Results Compared with the NC group, the serum creatinine level, the pathological injury score of kidney tissues and the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably lower, the apoptosis rate was remarkably elevated, the MDA content was significantly increased and the SOD activity was dramatically reduced in the IRI and MHP groups (all P < 0.05). Compared with the IRI group, the serum creatinine level and the pathological injury score of kidney tissues were significantly decreased, the expression levels of RBM3, YAP1 and Nrf2 proteins were significantly up-regulated, the Bcl-2/Bax ratio was considerably higher, the apoptosis rate was significantly decreased, the MDA content was significantly decreased and the SOD activity was considerably elevated in the MHP group (all P < 0.05). Conclusions Mild hypothermia may exert protective effect upon renal IRI and it could alleviate cell apoptosis and oxidative stress injury induced by IRI, probably by up-regulating the expression level of RBM3 and its downstream effector molecules of YAP1 and Nrf2.

Objective To evaluate the safety and efficacy of endovascular intervention to revise peripheral bypass problems through prosthetic approach.Methods Among 17 cases undergoing graft bypass anastomotic stenosis and graft thrombosis was identifled in 16 cases(inflow or outflow obstructive lesions in 10),inflow obstructive lessions in 1(without anastomotic and graft thrombosis).All revision procedures were taken under local anesthesia,16 patients were treated by means of surgical thrombectomy followed by endovascular intervention through prosthesis itself in addition to one who had no thrombectomy.The graft patency and clinical outcome were observed.Resuits Thirteen stents were implanted in 13patients with distal anastomotic stenosis and 1 with proximal anastomotic stenosis including 10 stentings/PTAs in iliac popliteal,posterior tibial or anterior tibial arteries.One stent was implanted in 1 patient with common femoral stenosis.Stenting were not used(abandoned)in 2 patients,of which one underwent a foot amputation and calf gangrene occurred a week later,and the other had a redo of grafting.Follow-up time is 1-35 months.with an average of 12±4 months.One had a below-knee amputation two months after intervention,the other had symptoms recurred and treated with a redo 3 months afterwards.the third died of myocardial infarction six months later.Grafts remained patent in the rest 13 patients at follow-up.Conclusions Endovascular intervention through prosthesis is a safe and effective method,which offers an alternative means to treat anastomotic stenosis.inflow or outflow obstructive lesions.

Objective: To report an investigation of a family cluster of coronavirus disease 2019 ( COVID-19 ) in Ningbo, so as to provide reference for the prevention and control measures. Methods: According to the COVID-19 Prevention and Control Program ( fourth version ) , an epidemiological investigation was conducted to collect the demographic information, clinical features and exposure history, to find the close contacts, and to figure out the source and route of infection. Results: Twelve confirmed cases and one asymptomatic case were reported. The attack rate was 16.05%. Among them, five were males and eight were females; the age ranged from 11 to 85 years old, with a median of 39 years old; most had mild symptoms. The incubation period was 2-13 days, with a median of 6.5 days. The first case ( Case 1 ) developed the symptoms on January 22, and had close contact with Zhang, an asymptomatic case, on January 20. Zhang was related to a cluster in the Buddhist assembly on January 19. Case 1, who caused the spread of the epidemic among family members, participated in several family visits and dinners from January 22 to 27 with other 24 families, resulting in six secondary cases and six third-generation cases. There were 54 close contacts except the family members, no infection was found. Conclusion This family cluster may result from the close contact with an asymptomatic case, and then spread within families through having dinners and living together.

OBJECTIVETo study the changes in the levels of authophagy-related proteins ATG4A and p-ATG4A in nervous tissue after treated with tri-ortho-cresyl phosphate and explore the possible pathogenesis of OPIDN.

METHODSIn the first experiment, thirty hens were randomly divided into control group and 1 d, 5 d, 10 d and 21d treated groups, hens in treated groups were treated with TOCP by gavage at a single dosage of 600 mg/kg. In the second experiment, other thirty hens were also randomly divided into control group and 1 d, 5 d, 10 d and 21 d treated groups, hens in treated group were pretreated with PMSF by subcutaneous at a single dosage of 90 mg/kg. 24 h later, hens in intervention group was treated with TOCP by gavage at a single dosage of 600 mg/kg. The hens were killed at the corresponding time points, and collected their tibial nerves. The levels of ATG4A and p-ATG4A were measured by immunoblotting.

RESULTScompared with the control group, the levels of ATG4A decreased by36%, 43.7% and 41% at 1d, 5d and 10d in the intoxication groups (P < 0.05), the levels of p-ATG4A decreased by 22.5%, 25%and 21%at 1d, 5d and 10d in the intoxication group (P < 0.05). However, compared with the control group, there is no significant change in the levels of ATG4A and p-ATG4A in PMSF-pretreated groups.

CONCLUSIONThe intoxication of TOCP influence the levels of autophagy-related proteins ATG4A and p-ATG4A, which might be associated with the inhibition of autophagy activity in neurons of OPIDN.

Animals ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; drug effects ; Chickens ; Female ; Nerve Tissue ; physiology ; Phosphorylation ; drug effects ; Tibial Nerve ; Tritolyl Phosphates ; toxicity

Objective:To evaluate the oncologic outcomes of different laparoscopic radical hysterectomy.Methods:From January 2011 to December 2014, the laparoscopic operation cases of cervical cancer at stage Ⅰb1, Ⅰb2, Ⅱa1 and Ⅱa2, including the histologic subtypes of squamous-cell carcinoma, adenocarcinoma and adenosquamous carcinoma, were collected in five clinical centers. The data were divided into two groups according to the surgical procedures, that is, modified laparoscopic-vaginal radical hysterectomy (mLVRH) and total laparoscopic radical hysterectomy (TLRH). The overall survival rate (OS), disease-free survival rate (DFS) at 5 years were retrospectively analyzed in this study.Results:There were 674 cases in total, including 377 cases of mLVRH, 297 cases of TLRH. (1) The OS at 5 years: the mLVRH was 96.1% and the TLRH was 92.0%, and the mLVRH was higher than that of TLRH （ P=0.010）. Stratify analysis， including stage of disease （Ⅰb1 and Ⅱa1）， histologic subtypes （squamous-cell carcinoma, adenocarcinoma）， lymph node metastasis， revealed that, ① Stage of disease: in stage Ⅰb1, the OS at five years of mLVRH was higher than that in TLRH group （98.6% vs 93.6%, P=0.012）. In stage Ⅱa1, there was significant difference between the two groups, the OS at five years of mLVRH and TLRH were 93.6% and 77.6% （ P=0.007）. ② Histologic subtypes: for the OS at five years of squamous-cell carcinoma, mLVRH and TLRH were 96.1% and 92.3%, and there was significant difference （ P=0.046）； for adenocarcinoma, the OS at five years were 91.0% and 88.6%， and there was no difference between two groups （ P=0.230）. ③ Lymph node metastasis： the mLVRH and TLRH with lymph node metastasis, the OS at five years were 98.6% and 96.4%; the mLVRH and TLRH without lymph node metastasis, the OS at five years were 89.3% and 80.8%. There were no significant differences between the two groups,respectively ( P=0.156， P=0.093）. (2) The DFS at 5 years: there was no significant difference between mLVRH and TLRH (94.1% vs 90.9%, P=0.220). Stratify analysis for stage of disease, the mLVRH group was higher than that in the TLRH group in stage Ⅰb1 (97.0% vs 92.8%, P=0.039). However, for stage Ⅱa1, there was no significant difference between mLVRH and TLRH group （88.2% vs 75.8%, P=0.074）. Conclusions:The results of this retrospective study indicated that different laparoscopy surgical procedures had diverse oncologic outcomes. The OS at 5 years of the mLVRH is superior to the TLRH. The DFS at 5 years in Ⅰb1 stage, the mLVRH is higher than the TLRH. Therefore, the modified laparoscopy is still an alternative surgery for early cervical cancer patients when following the principle of no-tumor-exposure.