We used CRISPR/Cas9 to delete plin1 of 3T3-L1 preadipocyte, to observe its effect on lipolysis in adipocytes and to explore regulatory pathways. We cultured 3T3-L1 preadipocytes, and the plin1 knockout vectors were transfected by electroporation. Puromycin culture was used to screen successfully transfected adipocytes, and survival rates were observed after transfection. The optimized "cocktail" method was used to differentiate 3T3-L1 preadipocytes. The glycerol and triglyceride contents were determined by enzymatic methods. The changes in lipid droplet form and size were observed by Oil red O staining. The protein expression of PLIN1, PPARγ, Fsp27, and lipases was measured by Western blotting. RT-PCR was used to measure the expression of PLIN1 and lipases mRNA. After the adipocytes in the control group were induced to differentiate, the quantity of tiny lipid droplets was decreased, and the quantity of unilocular lipid droplets was increased and arranged in a circle around the nucleus. Compared with the control group, the volume of unilocular lipid droplets decreased, and the quantity of tiny lipid droplets increased after induction of adipocytes in the knockout group. The expression of PLIN1 mRNA and protein in the adipocytes was significantly inhibited (P<0.05); glycerol levels increased significantly (0.098 4±0.007 6), TG levels decreased significantly (0.031 0±0.005 3); mRNA and protein expression of HSL and ATGL increased (P<0.05); PPARγ and Fsp27 expression unchanged in adipocytes. The above results indicate that the knockout of plin1 enhances the lipolysis of 3T3-L1 adipocytes by exposing lipids in lipid droplets and up-regulating lipases effects.
3T3-L1 Cells ; Adipocytes ; metabolism ; Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Lipase ; metabolism ; Lipolysis ; genetics ; Mice ; Perilipin-1 ; genetics ; metabolism

Triple negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that is characterized by the lack of estro-gen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2), thereby making it difficult to treat. Owing to the aggressive clinical behavior of TNBC and the lack of recognized molecular targets for therapy, patients with TNBC have shown poorer outcomes than those with other subtypes of breast cancer. Chemotherapy is the primary established systemic treatment for TNBC. However, various novel therapeutic targets have come into focus with the advances in molecular characterization of TNBC. In recent years, several targeted drugs have undergone clinical trials and have shown certain curative effects with relatively mild adverse reactions. The Food and Drug Administration has approved some of these drugs. In the current review, we have summa-rized the advances in the targeted therapy of TNBC.

OBJECTIVE： To investigate the effects and its mechanism of calcium phosphate bone cement （CPC） loading total flavonoids of Davallia mariesii on osteogenic differentiation of induced membrane in rats. METHODS： Drug-loading CPC and drug-loading polymethyl methacrylate （PMMA） cement were prepared with the contents of Qianggu capsules （total flavonoids of D. mariesii as active ingredient） using CPC and PMMA cement as carrier. Totally 64 male SD rats were randomly divided into drug-loading CPC group， drug-loading PMMA cement group， no-drug CPC group， no-drug PMMA cement group， with 16 rats in each group. The femur of rats was separated and osteotomized to prepare bone defect model， and then the corresponding bone cement was implanted. Four weeks after modeling， the induced membranes of rats were cut and protected. Bone cement was taken out and autogenous cancellous bone was implanted. At the 4th week after modeling， X-ray photographs were taken on the hind limb bones of rats. At the 4th week after modeling and 6th week after bone grafting， induced membranes and new bone were taken from the bone defect area of rats respectively. HE staining was used to observe the morphology of induced membrane， and the width of bone rabecular and the number of osteoblasts of new bone tissue were measured. Immunohistochemistry was used to detect the protein expression of BMP-2 and VEGF in induced membrane. Western blotting assay was used to detect the protein expression of Smad1， Smad4 and Smad7 in new bone. RESULTS： Compared with other 3 groups， the degradation of bone cement in drug-loading CPC group was more obvious in the bone defect areas， which showed that the formation of induced membrane was observed and the bone defect areas were smaller； capillary endothelial cells were abundant and orderly arranged in the induced membranes， and the width of bone trabeculae and the number of osteoblasts in the new bone tissue increased significantly （P＜0.05）； the protein expression of BMP-2 and VEGF in the induced membrane， the protein expression of Smad1， Smad4 and Smad7 in new bone were increased significantly （P＜0.05）. CONCLUSIONS： CPC loading total flavonoids of D. mariesii promotes the formation of induced membrane osteoblast in bone defect model rats， which may be associated with regulating osteoblast differentiation by activating BMP-2/Smad pathway； at the same time， it can promote bone healing by promoting the differentiation of vascular endothelial cells， accelerating the formation of capillary network and increasing the expression of vascular endothelial cells.

OBJECTIVE： To study the mechanism of Prunus persica-Carthamus tinctorius couplet medicine in the treatment of osteonecrosis of the femoral head （ONFH）. METHODS： The network pharmacology was adopted. The active components of P. persica -C. tinctorius couplet medicine and ONFH target were screened through TCM systematic pharmacological analysis platform target （TCMSP）， DRAR-CPI， hnuman gene database （GeneCards） and online medelian inheritance in man （OMIM） using oral availability of compounds （OB）＞30％ and drug like （DL）＞0.18 as standard. Network topology attribute analysis software Cytoscape 3.6.0 was utilized to construct the active components-ONFH targets network. Target protein interaction network was established on the basis of STRING database， and top 5 target proteins in the list of connectivity were screened， and molecular docking server was used to predict the combination activity of active components from P. persica -C. tinctorius couplet medicine. The biological processes of target gene ontology （GO） and metabolic pathways in Kyoto encyclopedia of genes and genomes （KEGG） were enriched and analyzed by DAVID. RESULTS： A total of 44 active components were screened from P. persica -C. tinctorius couplet medicine， including baicalin， quercetin， etc.， and 78 targets related to ONFH including VEGF， VEGI， CRP， etc. Through analysis of molecular docking server， binding activity of active components of P. persica -C. tinctorius couplet medicine to target protein was strong. GO and KEGG pathway enrichment analysis showed that biological process of P. persica -C. tinctorius couplet medicine for ONFH was related with negative regulation of apoptosis process and positive regulation of nuclear factor-κB transcription factor， mainly through regulating secretory glycoprotein signaling pathway， melanogenesis signaling pathway， VEGF signaling pathway， signaling pathway of basal cell carcinoma， adenosine-activated protein kinase signaling pathway. CONCLUSIONS： This study preliminarily validates the major targets and pathways of P. persica -C. tinctorius couplet medicine for ONFH， which lay a foundation for further study on their pharmacological action.

Objective To investigate the safety and feasibility of thoracoscopic lobectomy without mechanical suture. Methods The data of 28 consecutive patients (a non-mechanical suture group, 16 males and 12 females at age of 61.23±11.10 years) who underwent non-mechanical suture anatomic thoracoscopic lobectomy performed by the same surgeon from March 2015 to March 2018 were analyzed retrospectively, and 28 patients (18 males and 10 females at age of 59.45±13.39 years) who underwent completely anatomic thoracoscopic lobectomy with endoscopic stapler (a mechanical suture group) in the same period were matched. The clinical effectiveness of the two groups was compared. Results The operation time between the non-mechanical suture group (136.30±53.46 min) and the mechanical suture group (109.63±44.61 min) showed a statistical difference (P<0.05). While in term of intraoperative bleeding volume (65.00 mlvs. 50.00 ml), postoperative thoracic drainage time (3.73 days vs. 3.56 days), thoracic drainage volume (538.60 ml vs. 563.70 ml), postoperative hospital stay (5.58 days vs. 5.35 days) and postoperative complication rate (5/28 vs. 6/28), there was no statistical difference between the two groups. Hospitalization expense was significantly different between the two groups (35 438.30 yuan vs. 51 693.60 yuan). Conclusion Non-mechanical suture thoracoscopic anatomic lobectomy is safe and feasible, and can significantly reduce the medical cost but prolong the operation time.

Objective To investigate the effects of enteral immunonutrition supplemented with omega-3 polyunsaturated fatty acid (ω-3 PUFA) on the incidences and severity of ventilator associated pneumonia com- plications, inflammatory response, and the prognosis in patients with severe traumatic brain injury (sTBI) un-dergoing ventilator therapy. Methods From January 2015 to June 2017, 64 patients of sTBI were selected and randomly divided into experimental group (ω-3 PUFA, n=32) and control group (n=32). White blood cells (WBC) and the proportion of neutrophils (N%) were evaluated by cell analyzer. Meanwhile, the serum levels of C-reactive protein (CRP) and procalcitonin (PCT) were tested with enzyme linked immunosorbent assay. After 14-days treatment, the Glasgow coma scale (GCS) score, APACHE Ⅱ score, clinical pulmonary infec-tion score (CPIS), pulmonary function and prognoses were also compared between the two groups. Results As compared with the control group, the experimental group had lower incidences of ventilator associated pneumonia (66% vs. 56%, P=0. 048), reduced degree of lung infection and lower CPIS (8. 25±0. 60 vs. 7. 47±0. 53); higher lung function indexes [FVC: (2. 89±0. 19) L vs. (3. 46±0. 22) L, P=0. 010;FEV1: (2. 35±0. 16) L vs. (2. 84±0. 24) L, P=0. 040; FEV1/FVC %: (49. 11±3. 34)% vs. (56. 00± 2. 64)%, P=0. 038) ]; lower serum levels of inflammatory responses [WBC: (11. 83±0. 74) ×109/L vs. (9. 51±0. 90) ×109/L, P=0. 029; N%: (79. 11±1. 51)% vs. (72. 71±1. 16)%, P=0. 041; CRP:(85. 15±8. 42) mg/L vs. (63. 96±5. 72) mg/L, P=0. 001; PCT: (6. 43±0. 47) μg/L vs. (4. 83±0. 39) μg/L, P=0. 013] 14 days after enteral immunonutrition supplemented with ω-3 PUFA. As compared with the control group, the experimental group received better prognosis with GCS scores increasing ( 8. 69 ± 0. 41 vs. 9. 52±0. 59, P=0. 038), APACHE Ⅱ scores decreasing (14. 74±1. 01 vs. 12. 68±0. 89, P=0. 049), the time of mechanical ventilation [ (13. 23±1. 17) d vs. (10. 88±1. 24) d, P=0. 024] and the hospitalization days [ (23. 29±2. 45) d vs. (18. 42±1. 96) d, P=0. 012] reduced on the 14th day, mechanical ventilation withdraw rate within 14 days increasing [24/32 (75%) vs. 27/32 (84%), P=0. 030] on the 14th day. Conclusion Enteral immunonutrition supplemented with ω-3 PUFA can effectively reduce the incidence of ventilator associated pneumonia, alleviate the degree of infection and the inflammatory response in patients with sTBI undergoing ventilator therapy possibly improving condition and prognosis, which is worthy of being widely used.

Objective To evaluate the effect of recurrent laryngeal nerve detector on the operation of upper or middle-thoracic esophageal carcinoma. Methods A total of 60 patients with resectable esophageal carcinoma in Beijing Tsinghua Changgung Hospital from January 2015 to December 2017 were recruited. These patients were randomly divided into experimental group and control group by using random number table method.The experimental group used recurrent laryngeal nerve detector to assist in the nerve exploration and separation. The control group was treated by routine operation method, and the operation effect of the two groups was compared. Results In the experimental group, the time spent on the confirmation of recurrent laryngeal nerve was significantly shorter than that in the control group [right side: 1.50 min (1.00, 1.63 min) vs. 5.50 min(4.88, 6.50 min), Z= -6.715, P < 0.05; left side: 1.75 min (1.50, 2.00 min) vs. 7.85 min (6.50, 9.00 min), Z= -6.726, P< 0.05]. The rate of recurrent laryngeal nerve injury in the experimental group was significantly lower than that in the control group [3.3 % (1/30) vs. 20.0 % (6/30), χ 2= 4.043, P < 0.05]. Conclusion The use of recurrent laryngeal nerve detector in the neck anastomosis of upper or middle-thoracic esophageal carcinoma can significantly shorten the time spent on confirming of the recurrent laryngeal nerve and reduce the rate of recurrent laryngeal nerve injury.

Objective:To investigate the application of Coronary Artery Disease-Reporting and Data System (CAD-RADS) in the diagnosis of coronary atherosclerotic heart disease(CAD) and its risk factors,and to clarify the effective strength of different risk factors in the diagnosis of CAD by using CAD-RADS.Methods:All the data of 266 patients,who were initially suspected with CAD and underwent CT angiography,were collected and diagnosed by using CAD-RADS and were divided into CAD group(n=174) and non-CAD group(n=69).The informations of age,gender,hypertension,hyperlipemia,diabetes,smoking,serum uric acid (UA) levels,and plasma fibrinogen (FIB) levels of the patients in two groups were analyzed;single factor analysis and multivariate Logstic regression analysis were used to analyze the risk factors of CAD diagnosed by CAD-RADS.Results:Compared with non-CAD group,the ratios of male,hypertension,diabetes,smoking of the patients in CAD group were increased (P<0.05),and the age and the level of UA of the patients in CAD group were also increased (P<0.05).The Logistic regression analysis results showed that age and diabetes were the independent risk factors for CAD diagnosed by CAD-RADS.Conclusion:There are many independent risk factors for CAD diagnosed by CAD-RADS,and age and diabetes are the most correlated risk factors for CAD.

A simple and selective HPLC method for simultaneous determination and quantification of anthraquinones, lignans and flavonoids in Xiao-Cheng-Qi Tang (XCQT), Hou-Po-San-Wu Tang (HPSWT) and Hou-Po-Da-Huang Tang (HPDHT) was developed and validated. An Agilent Zorbax SB-C 18 (4.6 mm x 250 mm, 5 µm) column with the mobile phase of acetonitrile and 0.5% acetic acid aqueous solution in gradient elution mode was used. The flow rate was 1.0 mL · min(-1) at 30 °C, and injection volume was 10 µL. The detection wavelength was set at 254 nm and 294 nm simultaneously for the quantitative analysis. The current HPLC assay was validated for linearity, intra-day and inter-day precisions, accuracy, recovery and stability. The method was applied to the content comparison of the gallic acid, cinnamic acid, sennoside A, sennoside B, rhein, emodin, aloe-emodin, chrysophanol, physcion, magnolol, honokiol, narirutin, naringin, hesperidin, neohesperidin, hesperetin, naringenin and nobiletin in XCQT, HPSWT and HPDHT. The good linear equations of eighteen constituents were obtained within the investigated ranges (r > 0.998). The recovery of the method was 94.28%-99.89% and the precision was less than 5%. The sample was stable within 16 h. There were some differences between the contents of anthraquinones, lignans and flavonoids in analogous formulae about XCQT. XCQT contained the greatest abundance of anthraquinones and flavonoid, HPSWT contained the greatest abundance lignans. In conclusion, the methods are simple, low-cost, precise, accurate and reliable for the determination of eighteen constituents in analogous formulae about XCQT, and these results provide methodological support for its quality control.

The pharmacodynamic (PD) and pharmacokinetic (PK) properties of Huangqin Tang (HQT) were investigated in yeast-induced febrile rats. Blood sample and rectal temperature data of the rats were collected at different times after single oral administration of HQT at 20 g x kg(-1). The plasma concentrations of paeoniflorin, baicalin, wogonoside, baicalein, wogonin, oroxylin A, glycyrrhizic acid and glycyrrhetinic acid were quantified by a sensitive liquid chromatography-tandem mass spectrometric (LC-MS) method. The blood concentrations of PGE2, 1L-1β and TNF-α were detected by radioimmunoassay (RIA). All pharmacokinetic parameters were processed by non-compartmental analysis using WinNonlin software. The potential relationship between the mean concentration of eight constituents and the antifebrile efficacy was investigated by calculating Pearson correlation coefficients. It was found that HQT had significant antifebrile efficacy in yeast-induced febrile rats, but had no effect to normal rats. The antifebrile effect of HQT can be attributed to the inhibition of PGE2, 1L-1β and TNF-α. The constituents (baicalin, wogonoside, baicalein, wogonin, oroxylin A, glycyrrhizic acid and glycyrrhetinic acid) in febrile rats had delayed absorption and elimination, a longer residence time in the body, and higher C(max) and AUC than those in normal rats. Febrile condition could affect the pharmacokinetic behaviour of HQT in vivo; the flavonoids with the same backbone showed the similar fate in the body; baicalein and wogonin had a strong positive correlation (R > 0.66, P ≤ 0.02) with the antifebrile efficacy determined. Together, these constituents demonstrated different pharmacokinetic properties in the febrile body.