Objective:To develop a modified University of Wisconsin preservation solution (UW solution) containing α 2-adrenergic receptor agonists (dexmedetomidine) and noble gases (argon) and investigate its protective effect on the isolated amputated skeletal muscle of rats. Methods:Sixty male SD rats were selected to establish a hindlimb cold preservation/perfusion model and were divided into blank control group, hypothermic storage group, UW solution perfusion group, and modified UW solution perfusion group using a random number table, with 15 rats in each group. Simultaneously, a cold preservation model of rat skeletal muscle myoblasts (L6 cells) was established and the rats were also divided into four groups in the same way. Animal models were prepared in different ways: In the blank control group, the hindlimbs received no special treatment; In the hypothermic storage group, the amputated hindlimbs were stored in a dry centrifuge tube at 4℃ for 18 hours; In the UW solution perfusion group, the amputated hindlimbs were perfused with UW solution and then stored in a centrifuge tube containing UW solution at 4℃ for 18 hours; In the modified UW solution perfusion group, the amputated hindlimbs were perfused with modified UW solution (containing 0.1 nmol/L dexmedetomidine and 50% volume fraction of argon) and then stored in a centrifuge tube containing the modified UW solution at 4℃ for 18 hours. Cell models were treated as follows: In the blank control group, L6 cells were cultured under standard conditions; In the hypothermic storage group and UW solution group, L6 cells were treated with conventional culture medium or UW solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours; In the modified UW solution group, L6 cells were treated with the modified solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours. After sample collection, skeletal muscle morphology, tissue edema and ultrastructure features were assessed by HE staining, wet-to-dry weight ratio, and transmission electron microscopy, respectively. Additionally, L6 cell morphology was examined by light microscopy. L6 cell viability was determined by cell counting kit-8 (CCK-8) assay (expressed as absorbance A value). Expression levels of glutathione peroxidase 4 (GPX4) protein in both skeletal muscle tissue and L6 cells were evaluated by immunofluorescence staining and Western blot, respectively.Results:After 18 hours of in vitro preservation of rat isolated amputated limbs, the following results were obtained: (1) HE staining results showed that the muscle fiber morphology of the modified UW solution perfusion group was close to that of the blank control group. Moreover, the area ratio of skeletal muscle cells in the modified UW solution perfusion group was significantly higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). (2) The wet-dry weight ratio results showed that there was no statistically significant difference among the modified UW solution perfusion group, the blank control group and UW solution group ( P>0.05), with significantly lower ratios in all three groups than that in the hypothermic storage group ( P<0.05). (3) Transmission electron microscopy results revealed that the modified UW solution perfusion group showed no statistically significant differences in ultrastructural metrics, including myofiber diameter, sarcomere length, mitochondrial short-axis/long-axis ratio, and mitochondrial cristae count, compared with those in the blank control group ( P>0.05), and performed significantly better than both the hypothermic storage group and UW solution perfusion group ( P<0.05). (4) Morphological observation of L6 cells showed that the cellular morphology was regular in the modified UW solution perfusion group, close to that in the blank control group, while it was severely damaged in the hypothermic storage group. Moreover, the cells were reduced in number and partially damaged in the UW solution group. The sequence of cell viability expressed as absorbance A value was blank control group >modified UW solution perfusion group > UW solution perfusion group > hypothermic storage group, with statistically significant differences among the four groups ( P<0.05). (5) Immunofluorescence staining showed that there was no statistically significant difference in fluorescence intensity of GPX4 protein expression between the modified UW solution perfusion group and blank control group ( P>0.05), while the fluorescence intensity was higher in the modified UW solution perfusion group than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Western blot analysis showed that the relative expression level of GPX4 in the modified UW solution group was significantly lower than that in the blank control group ( P<0.05), but higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Conclusion:The modified UW solution can stabilize the expression level of GPX4 protein, thereby inhibiting ferroptosis and alleviating cold preservation injury in both rat amputated isolated limb skeletal muscle tissue and L6 cells.

Objective To explore the sex differences in drug metabolism of sufentanil in Chinese patients based on the mutation classification of cytochrome P450 3A(CYP3A)enzymes.Methods According to the possible effects of combined cytochrome P450 3A4 gene*1G locus(CYP3A4*1G)and cytochrome P450 3A5 gene*3 locus(CYP3A5*3)mutation groups on Chinese population,we added different weights to CYP3A4*1G and CYP3A5*3 polymorphisms and classified patients into 3 groups:GroupⅠ,patients carried either the CYP3A4*1G/*1G allele or both CYP3A4*1/*1G allele and CYP3A5*3/*3 allele;Group Ⅱ,patients with both CYP3A4*1/*1G allele and CYP3A5*1/*3 allele;Group Ⅲ,patients with either the CYP3A4*1/*1 allele or both CYP3A4*1/*1G allele and CYP3A5*1/*1 allele.A single-dose,double-blind,stratified random sampling was performed,and 255 patients undergoing endoscopic surgery in the First Affiliated Hospital of Army Medical University were finally subjected.According to the results of genetic testing,an independent statistician,before operation,randomly selected 30 patients from each stratified group to form a study cohort(male-female ratio of 1∶1)and named each group A,B or C.Clinical investigators and subjects kept double-blind to the results of grouping and genetic testing.After entering the operating room,the subjected 90 patients received a single dose of sufentanil followed by collection of blood samples at 10 time points including 2 min before and from 2 to 120 min after administration.After the surgery,we determined the plasma drug concentration,calculated the pharmacokinetic parameters,and compared the metabolic differences between different genders in each group and unblinded the study.Results The cohort best fitted the two-compartment pharmacokinetic model,and groups A,B and C corresponded to group Ⅰ,Ⅱand Ⅲ,respectively.In different patient groups based on mutatron types of CYP3A enzymes the females had lower plasma drug concentration-time curves at each time point,higher systemic clearance(P≤0.01)and smaller area under the plasma concentration-time curve from zero to infinity(P＜0.05)when compared with the males.In addition,in group Ⅰ,the elimination rate of central compartment and movement rate of drug from central compartment to peripheral compartment were obviously greater in the females than the males(P＜0.05),while the distribution half-life(P＜0.05)and elimination half-life(P＜0.01)were notably longer in the males than the females.In both group Ⅱ and group Ⅲ,the males obtained larger total area under the plasma concentration-time curve than the females(P＜0.05).Conclusion There are sex differences in the drug metabolism of sufentanil in Chinese patients.Women show faster distribution and higher clearance of sufentanil while men present greater drug exposure.Preoperative CYP3A genotyping and intraoperative personalized medication are of great significance to ensure the safety in clinical practice.

Objective:To develop a modified University of Wisconsin preservation solution (UW solution) containing α 2-adrenergic receptor agonists (dexmedetomidine) and noble gases (argon) and investigate its protective effect on the isolated amputated skeletal muscle of rats. Methods:Sixty male SD rats were selected to establish a hindlimb cold preservation/perfusion model and were divided into blank control group, hypothermic storage group, UW solution perfusion group, and modified UW solution perfusion group using a random number table, with 15 rats in each group. Simultaneously, a cold preservation model of rat skeletal muscle myoblasts (L6 cells) was established and the rats were also divided into four groups in the same way. Animal models were prepared in different ways: In the blank control group, the hindlimbs received no special treatment; In the hypothermic storage group, the amputated hindlimbs were stored in a dry centrifuge tube at 4℃ for 18 hours; In the UW solution perfusion group, the amputated hindlimbs were perfused with UW solution and then stored in a centrifuge tube containing UW solution at 4℃ for 18 hours; In the modified UW solution perfusion group, the amputated hindlimbs were perfused with modified UW solution (containing 0.1 nmol/L dexmedetomidine and 50% volume fraction of argon) and then stored in a centrifuge tube containing the modified UW solution at 4℃ for 18 hours. Cell models were treated as follows: In the blank control group, L6 cells were cultured under standard conditions; In the hypothermic storage group and UW solution group, L6 cells were treated with conventional culture medium or UW solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours; In the modified UW solution group, L6 cells were treated with the modified solution, stored in argon-filled sealed bags at 4℃ for 8 hours, and then rewarmed and cultured for 6 hours. After sample collection, skeletal muscle morphology, tissue edema and ultrastructure features were assessed by HE staining, wet-to-dry weight ratio, and transmission electron microscopy, respectively. Additionally, L6 cell morphology was examined by light microscopy. L6 cell viability was determined by cell counting kit-8 (CCK-8) assay (expressed as absorbance A value). Expression levels of glutathione peroxidase 4 (GPX4) protein in both skeletal muscle tissue and L6 cells were evaluated by immunofluorescence staining and Western blot, respectively.Results:After 18 hours of in vitro preservation of rat isolated amputated limbs, the following results were obtained: (1) HE staining results showed that the muscle fiber morphology of the modified UW solution perfusion group was close to that of the blank control group. Moreover, the area ratio of skeletal muscle cells in the modified UW solution perfusion group was significantly higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). (2) The wet-dry weight ratio results showed that there was no statistically significant difference among the modified UW solution perfusion group, the blank control group and UW solution group ( P>0.05), with significantly lower ratios in all three groups than that in the hypothermic storage group ( P<0.05). (3) Transmission electron microscopy results revealed that the modified UW solution perfusion group showed no statistically significant differences in ultrastructural metrics, including myofiber diameter, sarcomere length, mitochondrial short-axis/long-axis ratio, and mitochondrial cristae count, compared with those in the blank control group ( P>0.05), and performed significantly better than both the hypothermic storage group and UW solution perfusion group ( P<0.05). (4) Morphological observation of L6 cells showed that the cellular morphology was regular in the modified UW solution perfusion group, close to that in the blank control group, while it was severely damaged in the hypothermic storage group. Moreover, the cells were reduced in number and partially damaged in the UW solution group. The sequence of cell viability expressed as absorbance A value was blank control group >modified UW solution perfusion group > UW solution perfusion group > hypothermic storage group, with statistically significant differences among the four groups ( P<0.05). (5) Immunofluorescence staining showed that there was no statistically significant difference in fluorescence intensity of GPX4 protein expression between the modified UW solution perfusion group and blank control group ( P>0.05), while the fluorescence intensity was higher in the modified UW solution perfusion group than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Western blot analysis showed that the relative expression level of GPX4 in the modified UW solution group was significantly lower than that in the blank control group ( P<0.05), but higher than that in the hypothermic storage group and UW solution perfusion group ( P<0.05). Conclusion:The modified UW solution can stabilize the expression level of GPX4 protein, thereby inhibiting ferroptosis and alleviating cold preservation injury in both rat amputated isolated limb skeletal muscle tissue and L6 cells.

Background::China is one of the countries with the largest burden of gastrointestinal and liver diseases (GILD) in the world. The GILD constitutes various causes of mortality and disability. The study aimed to investigate the trend of GILD in China using the Global Burden of Diseases Study 2019 (GBD 2019) data resources from 1990 to 2019.Methods::The data on the age-standardized mortality rates (ASMR) and disability-adjusted life years (DALYs) for GILD in China from 1990 to 2019 were collected from the GBD 2019 data resources. Furthermore, the ranking of the main causes of deaths and DALYs, as well as the trends of ASMR, DALYs, years of life lost (YLLs), and years of life lost due to disability (YLDs) per 1,000,000 in GILD were reported.Results::The ASMR and DALYs for stomach cancer, liver cancer, and esophageal cancer, which ranked top three among the GILDs from 1990 to 2019, were gradually decreasing. Significant decreases in the ASMR and DALYs were found in diarrheal diseases and acute hepatitis (A, E, and C). However, noteworthy increases were found in those of colon and rectum cancer (CRC) and pancreatic cancer. Trend of DALYs, mortality, and YLLs rates for most of GILD were decreasing from 1990 to 2019, except the burden of CRC and pancreatic cancer with an increasing trend. The DALYs, mortality and YLLs of most GILD diseases showed decreasing trends from 1990 to 2019, except the burden of CRC and pancreatic cancer with an increasing trends.Conclusions::The result of the GBD 2019 showed that the rates of most GILDs decreased in China; however, gastrointestinal and liver cancer, such as stomach cancer still held the top ranking. Furthermore, the shift from infectious diseases to non-communicable causes among GILD burden is occurring.

Purpose To investigate the clinicopathological characteristics,diagnosis,and differential diagnosis of invasive mucinous adenocarcinoma(IMA)and mixed invasive mucinous and non-mucinous adenocarcinoma(mIMA).Methods The clinical data were collected in 36 patients with primary IMA and 17 patients with mIMA,and the expression of TTF-1,CK7,CK20,SATB2,CDX2,EGFR,HNF4a,etc.was detected by immunohistochemical EnVision two-step method.The Sanger se-quencing and the FISH were used for KRAS mutation and NRG1 gene rearrangement detection.The clinicopathological character-istics were analyzed with review of relevant literature.Results There were 9 cases(25.0％)and 3(8.3％)cases of papillary and micropapillary structures in IMA,while 13 cases(76.5％)(P＜0.001)and 9 cases(52.9％)(P=0.001)were present in mIMA.There were 5 cases(13.9％)of high nuclear grade of IMA and 10 cases(58.8％)of high nuclear grade of mIMA(P=0.002).TTF-1 had a positive rate of 37.5％in IMA,but 60.0％and 80.0％in the mucinous adenocarcinoma and non-mucinous adenocarcinoma components of mIMA(P=0.021),respectively.The positive rates of CK7,CK20,and CDX2 in IMA were 90.6％,21.9％,and 9.4％,and the positive rates in mucinous adenocarcinoma and non-mucinous adenocarcinoma components of mIMA were 100％,20％,20％and 100％,6.7％,6.7％,respectively and no SATB2 expression was found in all cases.There was no significant difference in the expres-sion of total EGFR and two EGFR mutation-specific antibodies(L858R,DEL19)between IMA and mIMA.There were 3 cases of mucinous adenocarcinoma with L858R positive in mIMA,and 2 of them were negative for non-mueinous adenocarcinoma.In another case,the non-mueinous adenocarcinoma component of mIMA expressed DEL19,but the mucinous adenocarcinoma component was not expressed.The positive rate of HNF4a in IMA was 72.0％(18/25),and those of HNF4a in mucinous adenocarcinoma and non-mucinous adenocarcinoma in mIMA were 41.7％(5/12)and 33.3％(4/12),respectively(P=0.048).KRAS gene sequencing was carried out in 19 cases of IMA,among which 9 cases(47.4％)had mutations,G12D and G12V were most commonly detected,and 4 cases of mIMA were sequenced,but none of them showed KRAS mutations.FISH detection showed that 2 cases(7.1％)IMAs had NRG1 translocation rearrangement.Conclusion Pulmonary mIMA is more aggressive than IMA.For example,mIMA has significantly more papillary structure,micropapillary structure,and high nu-clear grade cases than IMA.The differences in immunohisto-chemical expression and KRAS mutation between the two are sta-tistically significant.

Objective To evaluate the safety and clinical efficacy of transcatheter arterial chemoembolization (TACE) in treating primary hepatic carcinoma (PHC) complicated by tumor thrombus in inferior vena cava and right atrium (IVC-RA).Methods A total of 17 patients with PHC complicated by tumor thrombus in IVC-RA were included in this study.After the tumor-feeding arteries were confirmed with selective arteriography,TACE was carried out.The used embolization materials included chemotherapy drug-lipiodol emulsion and particle type embolic materials,and the target arteries included branches of hepatic artery,right inferior phrenic artery,branches of left gastric artery,etc.All patients were periodically followed up,and further treatment would be conducted if it was necessary.Results A total of 45 interventional procedures were performed in the 17 patients and all procedures were successful without any significant complication.Explicit blood supply arteries of IVC-RA tumor thrombus were observed in all the 17 patients,including hepatic artery branches (n=12) and extra-hepatic arteries (n=9),which included left gastric artery (n=1) and right inferior phrenic artery (n=8).CT reexamination showed that lipiodol deposition in IVC-RA tumor thrombus was found in 15 patients.In the 17 patients,the median survival time was 12 months,and the one-year and 2-year overall survival rates were 52.9％ and 29.4％ respectively.Conclusion IVC-RA tumor thrombus has rich blood supply,and its main blood supply arteries include hepatic artery and right inferior phrenic artery.For the treatment of PHC associated with IVC-RA tumor thrombus,TACE is safe and effective.

Objective To explore the safety of Habib VesOpen bipolar radiofrequency ablation (RFA) catheter used in the treatment of portal vein tumor thrombus (PVTT). Methods A total of 10 miniature pigs were randomly divided into 3 groups. Group A(n=6):RFA of normal portal vein was directly performed;group B (n=2): balloon obstruction of the portal vein was performed first, which was followed by RFA for the fresh thrombus in the portal vein; group C (n=2): PVTT model was established first, and RFA of the portal vein was carried out when the portal thrombus became organized. MRI examination was employed at one, 3 and 4 weeks after RFA; the animals were sacrificed 4 weeks after RFA and pathological examination of portal vein was performed. Results Pigs of group A received portal vein RFA under the condition of 5 W power for 0.6-3.6 min. No obvious abnormality was detected by MRI and pathological examination , which were performed one month after the treatment. In the pigs of group B , MRI performed after RFA showed that the damage of portal vein area was more serious than that in the pigs of group A;abdominal MRI examination performed at one, 3 and 4 weeks after RFA showed that the portal venous edema was gradually decreased;pathological examination at one month after RFA demonstrated serious injury of adjacent liver tissue. Pigs of group C received portal vein RFA under the condition of 7 W power for 1.5 min; no obvious edema of the ablated area was observed on MRI performed after RFA , and pathological examination revealed organized thrombus necrosis and va scular endothelial cell damage. Conclusion When Habib VesOpen bipolar RFA catheter is used for the treatment of PVTT, the RFA power and time should be properly selected according to the severity of PVTT. In order to ensure a safer procedure, high power and short ablation time should be used when the severity of PVTT is mild, while low power and longer ablation time are recommended when the PVTT is more severe.

Objective To investigate the effets of three mood stabilizers on ouabain?induced ERK1/2 phosphorylation in astrocytes. Methods As?trocytes were treated with different agents and divided into different groups accordingly,namely,the control group with saline,the group with oua?bain,the group with mood stabilizers(lithium carbonate,carbamazepine,sodium valproate)and the group with ouabain+mood stabilizers. The phosphorylation of ERK1/2 in each group was analyzed by Western blot. Results Compared with saline and mood stabilizer groups,the phosphoryla?tion of ERK1/2 was increased in the ouabain group,with statistical significance(P<0.05). There was no significant difference in ERK1/2 phosphoryla?tion between the group with ouabain+mood stabilizers and the control or mood stabilizer group. Conclusion The three kinds of mood stabilizers can inhibit ouabain?induced ERK1/2 phosphorylation in astrocytes.

Antibiotics, Antineoplastic ; administration & dosage ; Carcinoma, Hepatocellular ; therapy ; Chemoembolization, Therapeutic ; methods ; Doxorubicin ; administration & dosage ; Drug Carriers ; Follow-Up Studies ; Humans ; Liver Neoplasms ; therapy ; Male ; Microspheres ; Treatment Outcome

BACKGROUND:The main component of cartilage, type Ⅱ col agen gene expression in chondrocyte is positively correlated with SOX9 concentration in a dose-dependent manner.
OBJECTIVE:To observe the variation of SOX9 and type Ⅱ col agen mRNA content at different periods in the differentiation process (osteogenic, chondrogenic, adipogenic induction) of mesenchymal stem cel s, and to explore the correlation of SOX9 expression and type Ⅱ col agen.
METHODS:Bone marrow mesenchymal stem cel s were isolated from 4-week-old Kunming mice, and cultured in vitro to passage 3. The cel phenotype was identified with flow cytometry. Cel s were divided into three groups and subjected to three kinds of induction conditions favorable for adipogenic, chondrogenic and osteogenic differentiation, and each group was observed at three time points. In addition, the non-induced cel s were used as a control group. The total RNA of cel s was extracted at 3, 7, 14 days after induction, and SOX9 and type Ⅱ col agen mRNA was quantified with reverse transcription-polymerase chain reaction. The induced cel s were stained by immunofluorescence to observe the differentiation and perform statistical analysis.
RESULTS AND CONCLUSION:Passage 3 bone marrow mesenchymal stem cel s grew wel , and cel phenotype was confirmed as stem cel s by flow cytometry. The staining results showed that, the cel s differentiated into chondrocytes, adipocytes and osteoblasts. The SOX9 mRNA levels in the induced cel s were the highest in chondrogenic differentiation group, then in osteogenic differentiation group, and the lowest in adipogenic differentiation group. Type Ⅱ col agen mRNA levels in the induced cel s were the highest in chondrogenic differentiation group, then in adipogenic differentiation group, and the lowest in osteogenic differentiation group. SOX9 expression in chondrogenic differentiation group increased at 3 and 7 days, and then decreased at 14 days. While type Ⅱ col agen expression increased at 3, 7, 14 days. SOX9 mRNA levels increased as the osteogenic differentiation, while type Ⅱ col agen expression gradual y decreased. There was no significant difference in the SOX9 mRNA expression between adipogenic differentiation group and control group (P>0.05), while type Ⅱ col agen expression was not regularly changed. Experimental findings suggest that, critical effect of SOX9 in chondrogenic differentiation is better than that in osteogenic and adipogenic differentiation. SOX9 is associated with type Ⅱcol agen, which may alter along with the SOX9 in the early chondrogenic differentiation;SOX9 may play a fine-tuning role in the process of chondrogenic and osteogenic differentiation.