Objective To establish the method for simultaneous determination of six index components in the water extract of Weile Prescription;To optimize the water extraction process.Methods UPLC-MS/MS was used with Waters CORTECS C18 column(2.1 mm×100 mm,1.6 μm)as the chromatographic conditions;the mobile phase was 0.1%formic acid water-acetonitrile with gradient elution;the flow rate was 0.25 mL/min;the column temperature was 40℃;the sample volume was 2 μL.Electrospray negative ion source,positive and negative ion switching multi-reaction monitoring(MRM)mode were detected.Taking the content of six index components(gallic acid,vitexin,paeoniflorin,naringin,hesperidin and glycyrrhizic acid)and extraction rate as evaluation indexes,the weight coefficient of each index was determined by G1-entropy weight method,and the optimum parameters of extraction process were determined by orthogonal experiment design with the amount of water,extraction time and extraction times as investigation factors.Results There was a good linear relationship of the six components in the water extract of Weile Prescription in the concentration range(r>0.999),and the average recovery rate was 96.83%-102.56%,RSD<4.0%.The best technological parameters were as follows:Chinese decoction pieces were soaked in 12 times of water for 2 h,and extracted twice,each time for 1.5 h.Conclusion The UPLC-MS/MS method established in the study for simultaneous determination of six components in Weile Prescription is rapid,simple and sensitive,and the optimized extraction process is stable and feasible,which provides experimental basis for the development and research of the preparation.

Background::Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a microbarrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.Methods::The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin β8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models. Results::Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.Conclusions::The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.

Objective:To develop and evaluate metal artifact removal systems (MARSs) based on deep learning to assess their effectiveness in removing artifacts caused by different thicknesses of metals in cone-beam CT (CBCT) images.Methods:A full-mouth standard model (60 mm×75 mm×110 mm) was three-dimensional (3D) printed using photosensitive resin. The model included a removable and replaceable target tooth position where cobalt-chromium alloy crowns with varying thicknesses were inserted to generate matched CBCT images. The artifacts resulting from cobalt-chromium alloys with different thicknesses were evaluated using the structural similarity index measure (SSIM) and peak signal-to-noise ratio (PSNR). CNN-MARS and U-net-MARS were developed using a convolutional neural network and U-net architecture, respectively. The effectiveness of both MARSs were assessed through visualization and by measuring SSIM and PSNR values. The SSIM and PSNR values were statistically analyzed using one-way analysis of variance (α=0.05).Results:Significant differences were observed in the range of artifacts produced by different thicknesses of cobalt-chromium alloys (all P<0.05), with 1 mm resulting in the least artifacts. The SSIM values for specimens with thicknesses of 1.0 mm, 1.5 mm, and 2.0 mm were 0.916±0.019, 0.873±0.010, and 0.833±0.010, respectively ( F=447.89, P<0.001). The corresponding PSNR values were 20.834±1.176, 17.002±0.427, and 14.673±0.429, respectively ( F=796.51, P<0.001). After applying CNN-MARS and U-net-MARS to artifact removal, the SSIM and PSNR values significantly increased for images with the same thickness of metal (both P<0.05). When using the CNN-MARS for artifact removal, the SSIM values for 1.0, 1.5 and 2.0 mm were 0.938±0.023, 0.930±0.029, and 0.928±0.020 ( F=2.22, P=0.112), while the PSNR values were 30.938±1.495, 30.578±2.154 and 30.553±2.355 ( F=0.54, P=0.585). When using the U-net-MARS for artifact removal, the SSIM values for 1.0, 1.5 and 2.0 mm were 0.930±0.024, 0.932±0.017 and 0.930±0.012 ( F=0.24, P=0.788), and the PSNR values were 30.291±0.934, 30.351±1.002 and 30.271±1.143 ( F=0.07, P=0.929). No significant differences were found in SSIM and PSNR values after artifact removal using CNN-MARS and U-net-MARS for different thicknesses of cobalt-chromium alloys (all P>0.05). Visualization demonstrated a high degree of similarity between the images before and after artifact removal using both MARSs. However, CNN-MARS displayed clearer metal edges and preserved more tissue details when compared with U-net-MARS. Conclusions:Both the CNN-MARS and U-net-MARS models developed in this study effectively remove the metal artifacts and enhance the image quality. CNN-MARS exhibited an advantage in restoring tissue structure information around the artifacts compared to U-net-MARS.

Objective:To develop and evaluate metal artifact removal systems (MARS) based on deep learning to assess their effectiveness in removing artifacts caused by different thicknesses of metals in cone-beam CT (CBCT) images.Methods:A full-mouth standard model (60 mm×75 mm×110 mm) was three-dimensional (3D) printed using photosensitive resin. The model included a removable and replaceable target tooth position where cobalt-chromium alloy crowns with varying thicknesses were inserted to generate matched CBCT images. The artifacts resulting from cobalt-chromium alloys with different thicknesses were evaluated using the structural similarity index measure (SSIM) and peak signal-to-noise ratio (PSNR). CNN-MARS and U-net-MARS were developed using a convolutional neural network and U-net architecture, respectively. The effectiveness of both MARSs were assessed through visualization and by measuring SSIM and PSNR values. The SSIM and PSNR values were statistically analyzed using one-way analysis of variance (α=0.05).Results:Significant differences were observed in the range of artifacts produced by different thicknesses of cobalt-chromium alloys (all P<0.05), with 1 mm resulting in the least artifacts. The SSIM values for specimens with thicknesses of 1.0, 1.5, and 2.0 mm were 0.916±0.019, 0.873±0.010, and 0.833±0.010, respectively ( F=447.89, P<0.001). The corresponding PSNR values were 20.834±1.176, 17.002±0.427, and 14.673±0.429, respectively ( F=796.51, P<0.001). After applying CNN-MARS and U-net-MARS to artifact removal, the SSIM and PSNR values significantly increased for images with the same thickness of metal (both P<0.05). When using the CNN-MARS for artifact removal, the SSIM values for 1.0, 1.5 and 2.0 mm were 0.938±0.023, 0.930±0.029, and 0.928±0.020 ( F=2.22, P=0.112), while the PSNR values were 30.938±1.495, 30.578±2.154 and 30.553±2.355 ( F=0.54, P=0.585). When using the U-net-MARS for artifact removal, the SSIM values for 1.0, 1.5 and 2.0 mm were 0.930±0.024, 0.932±0.017 and 0.930±0.012 ( F=0.24, P=0.788), and the PSNR values were 30.291±0.934, 30.351±1.002 and 30.271±1.143 ( F=0.07, P=0.929). No significant differences were found in SSIM and PSNR values after artifact removal using CNN-MARS and U-net-MARS for different thicknesses of cobalt-chromium alloys (all P>0.05). Visualization demonstrated a high degree of similarity between the images before and after artifact removal using both MARS. However, CNN-MARS displayed clearer metal edges and preserved more tissue details when compared with U-net-MARS. Conclusions:Both the CNN-MARS and U-net-MARS models developed in this study effectively remove the metal artifacts and enhance the image quality. CNN-MARS exhibited an advantage in restoring tissue structure information around the artifacts compared to U-net-MARS.

Objective:To clone the Helicobacter pylori(Hp)hp0169 gene and conduct the crystallographic study,and to clarify its secondary and tertiary structures.Methods:The hp0169 gene and its encoded protein sequence of the Hp NCTC26695 strain were retrieved from the UniProt database.Bioinformatics method was used to analyze the physicochemical properties of the Hp recombinant protease(HpPrtC)protein;SOPMA and DNAStrar softwares were used to predict the secondary structure characteristics of HpPrtC protein;SWISS-MODEL software was used to construct the tertiary structure of the HpPrtC protein;IEDB and ABCpred softwares were used to predict the antigenic epitopes of the B lymphocytes HpPrtC protein;SYFPEITMI website was used to predict the antigenic epitopes of the T lymphocytes of HpPrtC protein;the expert pool(EP)and random forest(RF)algorithms were used to predict the crystallizability of the HpPrtC protein;the HpPrtC recombinant protein was expressed in the prokaryotic system;the HpPrtC recombinant protein was purified by Ni2+affinity chromatography and size-exclusion chromatography;the crystallization conditions for HpPrtC were screened by crystallization kit.Results:The hp0169 gene contained 1 269 base pairs and encoded the protein of 422 amino acids,the theoretical isoelectric point was 7.64 and the relative molecular weight was 47 300.HpPrtC was a hydrophilic and soluble protein.The number of amino acids of alpha helices of HpPrtC accounted for 35.78%,beta sheets 18.72%,beta turns 6.87%,and random coils 38.63%.The antigen epitope analysis results showed that HpPrtC contained five dominant linear epitopes of B lymphocytes,three conformational epitopes,and multiple potential dominant epitopes of T lymphocytes.The homology modeling results showed that HpPrtC formed a dimer,and each monomer displayed a barrel structure surrounded by β sheets,alpha helices,and random coils.HpPrtC was predicted to have moderate crystallizability without signal peptides and transmembrane helices.Small clustered needle-like crystals of HpPrtC were obtained under the conditions of 0.2 mol·L-1 magnesium chloride,0.1 mol·L-1 tris(hydroxymethyl)amino methane(Tris),3.4 mol·L-1 hexanediol,and pH=8.5.Conclusion:HpPrtC is a hydrophilic protein that forms a dimeric structure and crystallizes into small clustered needle-like crystals under suitable conditions.HpPrtC contains dominant antigenic epitopes of the T lymphocytes and B lymphocytes and can serve as an antigen for the design of Hp vaccines to establish the multivalent fusion vaccines or multi-epitope vaccines;the results provide an experimental basis for the prevention and control of Hp.

Bladder cancer remains the 10th most common cancer worldwide. In recent years, metformin has been found to have potential anti-bladder cancer activ-ity while high concentration of IC50 at millimolar level is needed, which could not be reached by regular oral administration route. Thus, higher efficient agent is urgently demanded for clinically treating bladder cancer. Here, by conjugating artesunate to metformin, a novel artesunate-metformin dimer triazine derivative AM2 was designed and synthesized. The inhibitory effect of AM2 on bladder cancer cell line T24 and the mechanism underlying was determined. Anti-tumor activity of AM2 was assessed by MTT, cloning formation and wound healing assays. Decreasing effect of AM2 on lipogenesis was determined by oil red O staining. The protein expressions of Clusterin, SREBP1 and FASN in T24 cells were evaluated by Western blotting. The results show that AM2 significantly inhibited cell proliferation and migration at micromolar level, much higher than parental metformin. AM2 reduced lipogenesis and down-regulated the expressions of Clusterin, SREBP1 and FASN. These results suggest that AM2 inhibits the growth of bladder cancer cells T24 by inhibiting cellular lipogenesis associated with the Clusterin/SREBP1/FASN signaling pathway.

Zedoary tumeric (Curcumae Rhizoma, Ezhu in Chinese) has a long history of application and has great potential in the treatment of liver cancer. The antiliver cancer effect of zedoary tumeric depends on the combined action of multiple pharmacodynamic substances. In order to clarify the specific mechanism of zedoary tumeric against liver cancer, this paper first analyzes the mechanism of its single pharmacodynamic substance against liver cancer, and then verifies the joint anti liver cancer mechanism of its “pharmacodynamic group”. By searching the research on the antihepatoma effect of active components of zedoary tumeric in recent years, we found that pharmacodynamic substances, including curcumol, zedoarondiol, curcumenol, curzerenone, curdione, curcumin, germacrone, β-elemene, can act on multi-target and multi-channel to play an antihepatoma role. For example, curcumin can regulate miR, GLO1, CD133, VEGF, YAP, LIN28B, GPR81, HCAR-1, P53 and PI3K/Akt/mTOR, HSP70/TLR4 and NF-κB. Wnt/TGF/EMT, Nrf2/Keap1, JAK/STAT and other pathways play an antihepatoma role. Network pharmacological analysis showed that the core targets of the “pharmacodynamic group” for anti-life cancer are AKT1, EGFR, MAPK8, etc, and the core pathways are neuroactive live receiver interaction, nitrogen metabolism, HIF-1 signaling pathway, etc. At the same time, by comparing and analyzing the relationship between the specific mechanisms of pharmacodynamic substance and “pharmacodynamic group”, it is found that they have great reference significance in target, pathway, biological function, determination of core pharmacodynamic components, formation of core target protein interaction, in-depth research of single pharmacodynamic substance, increasing curative effect and so on. By analyzing the internal mechanism of zedoary tumeric pharmacodynamic substance and “pharmacodynamic group” in the treatment of liver cancer, this paper intends to provide some ideas and references for the deeper pharmacological research of zedoary tumeric and the relationship between pharmacodynamic substance and “pharmacodynamic group”.

Objective:To investigate the role and regulatory mechanism of triggering receptor expressed on myeloid cell 2 (TREM2) in mice lung ischemia/reperfusion injury (LIRI).Methods:Thirty-six healthy male C57BL/6 mice were divided into six groups according to the random number method ( n = 6): normal control group, and LIRI 2, 6, 12, 24, 48 hours group. Mice LIRI models were established by clamping the left hilum. The wet/dry weight ratio (W/D) of left lung tissue was measured. Lung injury was observed and evaluated by hematoxylin-eosin (HE) staining and electron microscopy. The levels of interleukins (IL-1β, IL-18) in lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expressions of TREM2 and caspase-1 were determined by polymerase chain reaction (PCR). The protein expressions of TREM2, caspase-1, Gasdermin-D (GSDMD) were determined by Western blotting. Results:At 2 hours after LIRI, lung injury began to appear, the lung ultrastructure changed, and the lung injury score increased; at 6 hours, the degree of lung injury was the most serious; after 12 hours, the lung injury gradually reduced and the lung injury score gradually decreased. Compared with the normal control group, lung W/D ratio and lung injury score of LIRI 2, 6, 12, 24, 48 hours groups were significantly higher, the differences were statistically significant (lung W/D ratio: 7.06±0.52, 8.34±0.17, 6.42±0.35, 5.34±0.25, 5.59±0.45 vs. 4.69±0.23; lung injury score: 5.50±0.54, 9.75±0.89, 5.88±0.84, 3.63±0.74, 4.13±0.64 vs. 1.13±0.35, all P < 0.05). Compared with the normal control group, the levels of IL-1β and IL-18 in lung tissue were significantly increased at 2 hours after LIRI, reached a peak at 6 hours [IL-1β (ng/L): 502.76±12.25 vs. 56.50±8.07, IL-18 (ng/L): 414.02±10.75 vs. 81.63±5.29, both P < 0.05], then decreased gradually, and were still significantly higher than the normal control group at 48 hours. The PCR and Western blotting showed that the expression of TREM2 was significantly lower than that in the normal control group at 2 hours after LIRI, and reached a valley at 6 hours [TREM2 mRNA (2 -ΔΔCt): 0.47±0.05 vs. 1.02±0.05, TREM2/GAPDH: 0.23±0.13 vs. 0.48±0.17, both P < 0.05], then gradually increased, and reached the peak at 24 hours [TREM2 mRNA (2 -ΔΔCt): 3.98±0.15 vs. 1.02±0.05, TREM2/GAPDH: 0.71±0.17 vs. 0.48±0.17, both P < 0.05]. The trend of expression of caspase-1 and GSDMD were opposite to that of TREM2, which increased at first and then decreased, and reached a peak at 6 hours after reperfusion [caspase-1 mRNA (2 -ΔΔCt): 2.20±0.13 vs. 1.01±0.02, caspase-1/GAPDH: 0.64±0.02 vs. 0.20±0.06, GSDMD/GAPDH: 1.23±0.01 vs. 0.87±0.02, all P < 0.05]. Conclusions:TREM2 might be involved in LIRI in mice. The mechanism may be related to the effect of TREM2 on caspase-1-mediated pyroptosis.

Objective:To test the reliability and validity of Urban empty-nesters Home Safety Risk Factor Assessment Scale (Uen-HAS), in order to provide an instrument for assessing the safety risk factors of urban empty-nesters.Methods:Totally 530 urban empty-nesters from Fuzhou City, Fujian Province were employed by convenient sampling method for household survey, from August to October 2018. Item analysis and validity and reliability test were conducted.Results:The final version of urban empty nest elderly home safety risk factor assessment scale (Uen-HAS) contains 5 scales, 12 sub-scales and 55 items. The Cronbach α coefficient of the scale was 0.896. The content validity index (S-CVI) of the total scale was 0.881. The correlations of the scores between each item and the total scale ranged from 0.509 to 0.812 with statistical significance ( P<0.05). Conclusion:The scale has good reliability and validity. It could be an instrument to access safety risk factors of urban empty nesters.

Objective:To investigate the protective effect and mechanism of dexamethasone in lung ischemia/reperfusion injury (LIRI) rats.Methods:① Part one experiment: 24 Sprague-Dawley (SD) rats were divided into four groups according to the random number method ( n = 6): standard ventilation group (N group), normal saline group (NS group), LIRI group, and dexamethasone+LIRI group (DEX group). The rat model of LIRI was established by clamping the left pulmonary hilum for 1 hour and reperfusing it for 2 hours. The DEX group was given dexamethasone 3 mg/kg 5 minutes before reperfusion, and NS group was injected with normal saline. Group N did not receive any treatment. The left lung tissue of the rats in each group were taken alive 2 hours after reperfusion. The lung tissue was harvested for lung wet/dry mass ratio (W/D) measurement. Hematoxylin-eosin (HE) staining and electron microscopy was used to observe the pathological changes of lung tissue and to assess the degree of injury. Ultrastructural changes of lung tissue were observed under electron microscope. The levels of tumor necrosis factor-α (TNF-α), interleukin (IL-1β, IL-6) in lung tissue were detected by enzyme linked immunosorbent assay (ELISA). The expressions of phosphorylated protein kinase B (p-AKT) was detected by Western Blot. ② Part two experiment: intervention with phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway inhibitor LY294002 to further explore the mechanism of dexamethasone in reducing lung injury induced by LIRI. Twenty-four SD rats were divided into four groups according to the random number method ( n = 6): N group, LIRI group, DEX group, and dexamethasone+LY294002+LIRI group (LY group). All the groups except the LY group were treated with membrane and intervention according to part one experiment. The LY group was injected with LY294002 0.3 mg/kg after injection of dexamethasone. The expressions of M1 macrophage polarization markers CD11c, CD16, and M2 macrophage polarization markers CD206, Arg1 were detected by immunohistochemistry. Results:① Part one experiment: compared with N group, the morphological and ultrastructural changes of lung tissue in the LIRI group were significantly changed, lung injury score, lung W/D ratio and TNF-α, IL-1β, IL-6 levels were significantly increased, and p-AKT expression was significantly decreased. Compared with the LIRI group, the morphological and ultrastructural changes of the lung tissue in the DEX group were significantly improved, and the lung injury score was reduced (5.00±0.89 vs. 8.83±0.75), lung W/D ratio and TNF-α, IL-1β, IL-6 levels were significantly decreased [lung W/D ratio: 6.25±0.56 vs. 8.27±0.72, TNF-α(ng/L): 93.28±16.42 vs. 205.90±25.30, IL-1β(ng/L): 130.10±10.81 vs. 209.10±19.20, IL-6 (ng/L): 195.80±21.17 vs. 310.50±20.77], p-AKT expression was significantly increased [p-AKT/AKT: (57.58±8.80)% vs. (36.62±9.25)%], and the differences were statistically significant (all P < 0.05). There was no significant difference in each index between NS group and N group. ② Part two experiment: compared with the N group, the expression of macrophage polarization markers CD11c, CD16, CD206 and Arg1 in the LIRI group were significantly increased. Compared with the LIRI group, the expressions of CD11c and CD16 in the lung tissue of the DEX group were significantly decreased, and the expressions of CD206 and Arg1 were significantly increased. The intervention of PI3K/AKT signaling pathway inhibitor LY294002 significantly blocked the effect of dexamethasone on LIRI-mediated macrophage polarization (CD11c immunohistochemical score: 7.20±0.36 vs. 5.00±0.34, CD16 immunohistochemical score: 8.20±0.48 vs. 7.40±0.64, CD206 immunohistochemical score: 5.80±0.59 vs. 7.40±0.28, Arg1 immunohistochemical score: 7.20±0.72 vs. 8.80±0.48, all P < 0.05). Conclusions:Dexamethasone pretreatment can alleviate the intrapulmonary inflammatory response and lung injury caused by LIRI in rats. The mechanism of action is related to the polarization direction of pulmonary macrophagesvia activation of the PI3K/AKT pathway by dexamethasone.