Chronic visceral pain is a persistent and debilitating condition arising from dysfunction or sensitization of the visceral organs and their associated nervous pathways. Increasing evidence suggests that imbalances in central nervous system function play an essential role in the progression of visceral pain, but the exact mechanisms underlying the neural circuitry and molecular targets remain largely unexplored. In the present study, the ventral tegmental area (VTA) was shown to mediate visceral pain in mice. Visceral pain stimulation increased c-Fos expression and Ca²⁺ activity of glutamatergic VTA neurons, and optogenetic modulation of glutamatergic VTA neurons altered visceral pain. In particular, the upregulation of NMDA receptor 2A (NR2A) subunits within the VTA resulted in visceral pain in mice. Administration of a selective NR2A inhibitor decreased the number of visceral pain-induced c-Fos positive neurons and attenuated visceral pain. Pharmacology combined with chemogenetics further demonstrated that glutamatergic VTA neurons regulated visceral pain behaviors based on NR2A. In summary, our findings demonstrated that the upregulation of NR2A in glutamatergic VTA neurons plays a critical role in visceral pain. These insights provide a foundation for further comprehension of the neural circuits and molecular targets involved in chronic visceral pain and may pave the way for targeted therapies in chronic visceral pain.
Animals ; Male ; Visceral Pain/metabolism* ; Up-Regulation/physiology* ; Ventral Tegmental Area/metabolism* ; Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors* ; Neurons/drug effects* ; Mice, Inbred C57BL ; Mice ; Proto-Oncogene Proteins c-fos/metabolism* ; Chronic Pain/metabolism* ; Glutamic Acid/metabolism*

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 investigate the risk factors of in-hospital mortality and establish a risk prediction model for patients receiving venoarterial extracorporeal membrane oxygenation(VA-ECMO).Methods We retrospectively collected the data of 302 patients receiving VA-ECMO in ICU of 3 hospitals in Guangdong Province between January,2015 and January,2022 using a convenience sampling method.The patients were divided into a derivation cohort(201 cases)and a validation cohort(101 cases).Univariate and multivariate logistic regression analyses were used to analyze the risk factors for in-hospital death of these patients,based on which a risk prediction model was established in the form of a nomogram.The receiver operator characteristic(ROC)curve,calibration curve and clinical decision curve were used to evaluate the discrimination ability,calibration and clinical validity of this model.Results The in-hospital mortality risk prediction model was established based the risk factors including hypertension(OR=3.694,95%CI:1.582-8.621),continuous renal replacement therapy(OR=9.661,95%CI:4.103-22.745),elevated Na2+ level(OR=1.048,95%CI:1.003-1.095)and increased hemoglobin level(OR=0.987,95%CI:0.977-0.998).In the derivation cohort,the area under the ROC curve(AUC)of this model was 0.829(95%CI:0.770-0.889),greater than those of the 4 single factors(all AUC<0.800),APACHE Ⅱ Score(AUC=0.777,95%CI:0.714-0.840)and the SOFA Score(AUC=0.721,95%CI:0.647-0.796).The results of internal validation showed that the AUC of the model was 0.774(95%CI:0.679-0.869),and the goodness of fit test showed a good fitting of this model(χ2=4.629,P>0.05).Conclusion The risk prediction model for in-hospital mortality of patients on VA-ECMO has good differentiation,calibration and clinical effectiveness and outperforms the commonly used disease severity scoring system,and thus can be used for assessing disease severity and prognostic risk level in critically ill patients.

Objective:To establish high performance liquid chromatography (HPLC) wavelength switching method to simultaneously determine the contents of chlorogenic acid, hydroxysafflor yellow A, ferulic acid, Nicotiflorin, Osthole and columbianadin in Tongluo Zhibi Prescription.Methods:The column was XBridge C18 column (250 mm × 4.6 mm, 5 μm); the mobile phase was acetonitrile (A)-0.1% phosphate water (B); gradient eluted, with flow rate: 1 ml/min, column temperature: 30 °C, detection wavelength 330 nm (0-14 min detection of chlorogenic acid, 15-80 min detection of ferulic acid, Nicotiflorin, Osthole, and columbianadin), 403 nm (14-15 min detection of hydroxysafflower yellow pigment A).Results:Chlorogenic acid, hydroxyrhodopsin A, ferulic acid, kaempferol 3-O-rutinoside, serpentin, and dihydroeurobicarpus angelicus acid ester showed good linearity ( R2 &ge; 0.999 8) within 0.029 7-1.485 0, 0.030 0-1.500 0, 0.009 9-0.495 0, 0.017 5-0.875 0, 0.028 4-1.420 0, 0.013 7-0.685 0 μg, respectively. The precision, stability (24 h), repeatability relative standard deviation ( RSD) were all <2%. The average spiked recoveries were all in the range of 95%-105%, and the RSDs were all in the range of 0.32%-1.67%. In 10 batches of test samples of Tongluo Zhibi Prescription, the content of the above six components, including chlorogenic acid, was determined to be 0.221 60, 0.314 30, 0.085 10, 0.032 95, 0.043 87, 0.026 21 mg/g in the following order. Conclusion:The established HPLC wavelength switching method is fast, simple and accurate, which can be used for simultaneous determination of the content of the above six components in Tongluo Zhibi Prescription, which provides reference for quality monitoring and new dosage form research of Tongluo Zhibi Prescription.

Objective To investigate the risk factors of in-hospital mortality and establish a risk prediction model for patients receiving venoarterial extracorporeal membrane oxygenation(VA-ECMO).Methods We retrospectively collected the data of 302 patients receiving VA-ECMO in ICU of 3 hospitals in Guangdong Province between January,2015 and January,2022 using a convenience sampling method.The patients were divided into a derivation cohort(201 cases)and a validation cohort(101 cases).Univariate and multivariate logistic regression analyses were used to analyze the risk factors for in-hospital death of these patients,based on which a risk prediction model was established in the form of a nomogram.The receiver operator characteristic(ROC)curve,calibration curve and clinical decision curve were used to evaluate the discrimination ability,calibration and clinical validity of this model.Results The in-hospital mortality risk prediction model was established based the risk factors including hypertension(OR=3.694,95%CI:1.582-8.621),continuous renal replacement therapy(OR=9.661,95%CI:4.103-22.745),elevated Na2+ level(OR=1.048,95%CI:1.003-1.095)and increased hemoglobin level(OR=0.987,95%CI:0.977-0.998).In the derivation cohort,the area under the ROC curve(AUC)of this model was 0.829(95%CI:0.770-0.889),greater than those of the 4 single factors(all AUC<0.800),APACHE Ⅱ Score(AUC=0.777,95%CI:0.714-0.840)and the SOFA Score(AUC=0.721,95%CI:0.647-0.796).The results of internal validation showed that the AUC of the model was 0.774(95%CI:0.679-0.869),and the goodness of fit test showed a good fitting of this model(χ2=4.629,P>0.05).Conclusion The risk prediction model for in-hospital mortality of patients on VA-ECMO has good differentiation,calibration and clinical effectiveness and outperforms the commonly used disease severity scoring system,and thus can be used for assessing disease severity and prognostic risk level in critically ill patients.

Objective:To investigate the effects of tantalum coating on adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. Methods:In this study, BMSCs were extracted from 6 6-week-old rats and cultured in vitro to the third generation. Tantalum coating was manufactured on Ti6Al4V by chemical vapor deposition. The cells were identified by flow cytometry before they were induced with different mediums for osteogenesis, chondrogenesis and adipogenesis. The adhesion, proliferation and osteogenic differentiation of BMSCs were detected with fluorescence staining, Cell Counting Kit-8 (CCK8) assay and Q-PCR, respectively. Recorded and compared were the adhesion rate, proliferation rate, and expression of osterix (OSX), Runt-related transcription factor 2 (RUNX2), osteonectin (OSN) and osteopontin (OPN) of BMSCs on the surface of titanium alloy round plates (the Ti6Al4V group) and of tantalum coating round plates (the Ta group). Results:The flow cytometry revealed CD44 (94.55%), CD90 (95.01%) and CD34 (0.06%). Alkaline phosphatase (ALP) staining was positive after osteogenic induction for 14 days; Alizarin red staining showed calcified nodules after osteogenic induction for 21 days; oil red O staining was positive after adipogenic induction for 21 days; alcian blue staining found chondrogenic ability after chondrogenic induction for 21 days. Laser confocal microscopy showed that the BMSCs grew in patches aggregated and closely linked on the surface of titanium alloy round plates (in the Ti6Al4V group) and of tantalum coating round plates (in the Ta group). More BMSCs adhered on the tantalum coating plates than on the titanium alloy plates and exhibited better ductility. The proliferation rates of BMSCs on tantalum coating were significantly faster than those on titanium alloy after 1, 3, 5 and 7 days of co-culture in vitro ( P<0.05).Q-PCR showed that tantalum coating promoted the expression of OSN and OPN after 7 days of culture significantly higher than titanium alloy did ( P<0.05).After 21 days of co-culture in vitro, tantalum coating enhanced the expression of OSX, RUNX2, OSN and OPN significantly higher than titanium alloy did ( P<0.05). Conclusion:Compared to titanium alloy which is used for conventional orthopedic implants, tantalum coating can observably promote adhesion, proliferation and osteogenic differentiation of BMSCs.

Objective To discuss the prevention and management method for chylous fistula after neck lymph node dissec‐tion .Methods Totally 1 793 cases of neck lymph node dissection in this department from January 2005 to September 2014 were retrospectively analyzed .The clinical data in the cases of chylous fistula occurred after operation were summarized .Results Twenty one cases of chylous fistula occurred ,accounting for 1 .17% ,in which 13 cases were cured by the local compressed bandaging and continuous negative pressure drainage;5 cases adopted the conventional method for 2-3 d ,but under the ineffective condition ,then they were treated by combining with somatostatin pumping (somatostatin 6 mg+0 .9% normal saline 48 mL ,2 mL/h ,lasting for 24 h ,for successive 2-3 d) and finally cured;3 cases were cured after reoperation .Conclusion Prevention is the best treatment for chylous fistulas ,local compression bandage plus continuous negative pressure drainage is the main method for treatment of chylous fistulas after neck dissection .The combined therapy with somatostatin can increase the close rate of chylous fistulas;for the patients with long persistent time ,large drainage volume and invalid conservative therapy should adopt the remedial measure of operation .

Objective:To study the change rule of decocting quantity of the effective components in Amomi fructus and Amomi fructus rotundus with decocting time to determine whether or not decocted later and optimal decocting time. Methods:The herbs were extracted by the traditional water decoction, and at different time points, sampling was carried out. Using camphor and eucalyptol as the index components, the change rule of decocting quantity of the effective components with the decoction time under the condition of single and combined decoction was investigated. Results:When the decoction time of Amomi fructus was within the range of 3-6 min, the total amount of camphor in the decoction reached relatively high value, and the total amount lost more than 45% when the decoction time exceeded 10 min. Amomi fructus rotundus boiled for a short time below 2 min, and when the decoction time was more than 5 min, more than 50% eucalyptol lost. Conclusion:Amomi fructus and Amomi fructus rotundus should be decocted later with decocting time within 3-6min and below 2 min, respectively. The analytical method is reliable and precise in the quality control of relative decoction.

Objective To develop an instrument that detects the endurance of an animal hanging on a bar during medical and pharmaceutical scientific experiments .Methods One pair of opto sensors were used for signal conversion .A solenoid valve device was used for water level .A 51-Series microcontroller was used to control the experimental setup and record the results .Results We have developed a microcontroller-based experimental instrument that can automatically detects the endurance of an animal hanging on a bar during medical and pharmaceutical scientific experiments .Our experimental results with 28 mice indicated that the applicability of the instrument is good .Conclusion The instrument provides an innovative tool for detecting the endurance of an animal .