Objective To observe the effects of different ligation sites and fasting method on a C57BL/6J mouse model of partial bile duct ligation(pBDL)-induced cholestasis,to establish a pBDL modeling method with a high modeling rate,typical symptoms,and good stability.Methods C57BL/6J mice were subjected to selective ligation of the left hepatic bile duct(L-pBDL)and left-to-median bile duct junction ligation(ML-pBDL)for modeling,and the effects of different pBDL ligation method on serum alanine aminotransferase,aspartate aminotransferase,alkaline phosphatase(ALP),total bilirubin,total bile acid,and liver histopathology were observed.The effects of different fasting method on symptoms and liver injury in the ML-pBDL model were also observed after fasting for 12 and 16 h before surgery,and for 4 h after surgery.Results(1)The incidence of jaundice in the ML-pBDL group was 52.94%and the survival rate within 3 weeks after surgery was 64.71%,while the incidence of jaundice in the L-pBDL group was 11.76%and the survival rate within 3 weeks after surgery was 82.35%.Compared with those in the sham surgery group,serum liver function indicators were significantly increased in the L-pBDL and ML-pBDL groups(P＜0.01),and ALP activity was significantly higher in the ML-pBDL group than in the L-pBDL group(P＜0.05).Compared with mice in the L-pBDL group,mice in the ML-pBDL group had more severe liver fibrosis at 3 weeks post-surgery(P＜0.01).(2)In addition,the incidence of jaundice in the 16 h fasting group was 93.33%and the survival rate within 3 weeks after surgery was 73.77%,while the incidence of jaundice in the 12 h fasting group was 42.86%and the survival rate within 3 weeks after surgery was 71.42%.Compared with those in the normal group,ALP activity,alanine aminotransferase/aspartate aminotransferase ratio,total bile acid level,and proportion of collagen fiber area were all significantly increased in the 16 h and 12 h fasting groups(P＜0.05).Although the observed indicators were higher in the 16 h fasting group compared with those in the 12 h fasting group,the difference was not significant(P＞0.05).Mice in the 12 h and 16 h fasting groups both showed significant bile duct hyperplasia and liver fibrosis(P＜0.01),with more severe liver fibrosis in the 16 h fasting group(P＜0.01).Conclusions Both L-pBDL and ML-pBDL ligation method can be used to establish a mouse model of cholestasis;however,symptoms in the L-pBDL model only exhibit transient damage characteristics,while the liver lesions in the ML-pBDL model are typical and stable.Prolonging the preoperative fasting time can improve the modeling rate and stability of the ML-pBDL model and produce more-typical pathological symptoms.

Objective To establish a rat model of hyperuricemic nephropathy(HN)using a multifactorial induction method of potassium oxazinate combined with adenine and yeast feed to observe the intervention effect of Qiling granules(QLG).Methods Fifty-eight SPF-grade male SD rats were selected,and 10 rats were randomly allocated to the normal control(NC)group.The remaining rats were induced by multiple factors to establish HN rat models.After 2 weeks of modeling,submandibular blood samples were taken to detect serum UA,CREA,BUN,TG,and TC.Forty HN rats with bleeding clearance UA and body weight close to the mean were selected.They were randomly divided into a model(M)group,QLG low dose(QLG-L)groups,QLG high dose(QLG-H)group,and a positive control(PC)group,with 10 rats in each group,using a stratified randomization method.Each group was given corresponding drugs by gavage daily,and after continuous administration for 4 weeks,submandibular blood samples were taken to detect serum UA,CREA,BUN,TG,and TC.After euthanasia of the rats,liver tissue was taken to detect XOD and ADA activity.Renal tissue was taken for HE and Gomori hexamine silver staining,and the protein expression of GLUT9,OAT1,VCAM-1,and TGF-β in the kidneys was observed using immunohistochemistry and Western blot method.Results Compared with the NC group,the M group's serum levels of UA,CREA,BUN,TC,and TG,as well as liver XOD and ADA activities,were significantly increased(P＜0.01).The renal tissue of the model rats showed significant pathological changes.The area of renal tubules positive for urate and the expression of GLUT9,VCAM-1,and TGF-β proteins in the kidneys were significantly increased(P＜0.01,P＜0.05),while the expression of OAT1 was significantly reduced(P＜0.01).Compared with the M group,each treatment group showed significantly reduced serum UA levels,liver XOD,ADA activity,and renal VCAM-1 protein expression(P＜0.01,P＜0.05).The serum CREA and BUN levels and renal TGF-β protein expression of rats in the QLG-L group were significantly reduced(P＜0.05,P＜0.01).The serum CREA and BUN levels and renal GLUT9 protein expression of rats in the QLG-H group were also significantly reduced(P＜0.01,P＜0.05).The urate deposition and renal injury caused by each treatment were reduced to varying degrees,but there were no significant differences among groups(P＞0.05).Conclusions A stable HN rat model can be induced by gavage of potassium oxyzinate and adenine in combination with yeast feed.QLG can effectively treat HN by improving UA metabolic disorders,reducing the renal inflammation and urate deposition that cause renal damage in HN model rats.Its mechanism of action is related to a reduction in serum UA,CREA,BUN,and TG levels;liver XOD and ADA activities;and the expression of GLUT9,OAT1,VCAM-1,and TGF-β proteins in the kidneys.

With the gradual improvement of medical informatization and the vigorous development of medical and health big data，the exploration and practice of real -world research are becoming more and more mature ，and real -world data have become an important source of evidence for post marketing re -evaluation of drugs . As an important high -quality real -world medical data ， electronic medical record data is an indispensable data source for post marketing re -evaluation of drugs . Most of the existing guidelines and norms of real -world research are designed from the perspective of prospective research ，and do not propose specific measures and methods in the implementation of retrospective research ，especially for the operation suggestions on the technical level of using conventionally collected electronic medical record data . In combination with the operational process framework formulated by the existing guidelines and norms ，this paper creatively adds the operating procedures for dataE- validation，data integration ，data verification ，and throughout quality control ，data management and storage of retrospectiveelectronic medical record data ，and describes the data analysis methods and key points involved in carrying out multi -center retrospective real -world research using electronic medical record data ，taking the post marketing safety research of drugs as an example. Finally，the full process operation procedure applicable to the use of multi -center retrospective electronic medical record data is established .

Objective: Yupingfeng Powder (YPF), a kind of preventative patent medicine, is chosen for treatment of coronavirus disease 2019 (COVID-19) due to its high frequency application in respiratory tract diseases, such as chronic obstructive pulmonary disease, asthma, respiratory tract infections, and pneumonia, with the advantage of reducing the relapse rate and the severity. However, the active components of YPF and the mechanisms of components affecting COVID-19 are unclear. This study aimed to determine active constituents and elucidate its potential mechanisms. Methods: Ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q/TOF-MS) and liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS) were used to determine the components and absorbable constituents of YPF. Secondly, TCMSP, Drugbank, Swiss and PharmMapper were used to search the targets of absorbable bioactive constituents of YPF, and the targets of COVID-19 were identified based on GeneCards and OMIM databases. STRING database was used to filter the possible inter-protein interactions. Thirdly, Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis were performed to identify molecular function and systemic involvement of target genes. Results: A total of 61 components of YPF and 36 absorbable constituents were identified through UPLC-Q/TOF-MS. Wogonin, prim-O-glucosylcimifugin, 5-O-methylvisamminol, astragaloside IV and 5-O-methylvisamminol (hydroxylation) were vital constituents for the treatment of COVID-19, and RELA, TNF, IL-6, MAPK14 and MAPK8ere recognized as key targets of YPF. The major metabolic reactions of the absorbed constituents of YPF were demethylation, hydroxylation, sulfation and glucuronidation. GO and KEGG pathway analysis further showed that the most important functions of YPF were T cell activation, response to molecule of bacterial origin, cytokine receptor binding, receptor ligand activity, cytokine activity, IL-17 signaling pathway, Chagas disease, lipid and atherosclerosis, etc. Conclusion: The approach of combining UPLC-Q/TOF-MS with network pharmacology is an effective tool to identify potentially bioactive constituents of YPF and its key targets on treatment of COVID-19.

Objective To investigate the relatio nship between the DNA methylation status of gluco-corticoid receptor (GR) gene promoter and mRNA expression level of GRα gene of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Methods Fifteen new onset SLE patients and fifteen healthy controls were enrolled in this study. The DNA methylation status of GR gene promoter 1 of PBMCs was detected through bisulfite sequencing polymerase chain reaction (PCR). The mRNA expression of GRα, DNA methyltransferases, growth arrest and DNA damage-induced 45α (GADD45α) of PBMCs was detected using the quantitative real-time polymerase chain reaction method. T-test and χ2-test were used to detect the differences between the two groups, Pearson's correlation coefficient was used to analyze the linear correlation between two variables. Results Compared with healthy controls, the mRNA expression of GRα was signi-ficantly declined in SLE patients (10±5, 17±7, t=2.69, P<0.05), and the mRNA expression of DNMT1 and GADD45α was significantly elevated in SLE patients (t=3.11, P<0.05 and t=2.98, P<0.05). The overall mean methylation status of the 142 CpG islands of the four promoters was significantly elevated in SLE patients [(16±8)%vs (11±6)%, t=2.75, P<0.05]. The global methylation status of PBMCs in SLE patients was obviously lower than healthy controls (t=4.39, P<0.05). Conclusion Hypermethylation of GRα promoter may result in GRαgene low expression in PBMCs of patients with SLE.

Objective To study the application of implantable telemetry and whole-body plethysmography to observe the changes of circadian rhythm in conscious rats and evaluate the pharmacological safety of doxofylline, and to provide a basis for the future application of this technological system for drug safety evaluation.Methods Eight healthy SPF Sprague-Dawley rats were used in this study, 4 males and 4 females.The rats were implanted with telemetry transmitters by surgery to establish a telemetry system combined with plethysmography to observe the changes of 24 h physiological parameters and circadian rhythm in conscious rats at 14 d after operation, including heart rate (HR), blood pressure, the time interval from the Q wave to point A in the ECG of the aortic pressure wave (QA interval), respiration, activity, body temperature and pulmonary function parameters.The rats were divided into 3 groups: normal control group, doxofylline 40 mg/kg and 80 mg/kg groups, and the performance was validated by aerosolizing saline, doxofylline 40 mg/kg and 80 mg/kg inhalation, respectively, to observe the changes in physiological parameters after the drug administration.Results The physiological parameters of rats showed obvious changes in circadian rhythms at 14 d after operation.Compared with the normal control group, the doxofylline 40 mg/kg-treated group showed significantly increased changes of HR, tidal volume (TV), minute ventilation (MV), 50% expiratory flow (EF50), peak inspiratory flow (PIF) and peak expiratory flow (PEF) (P<0.01), significantly decreased respiratory frequency, QA interval and enhance pause (Penh) (P<0.05, P<0.01), but no significant differences in the blood pressure, activity and body temperature (P>0.05).Compared with the normal control group, the group treated with doxofylline 80 mg/kg had significantly increased HR, blood pressure, TV, MV, EF50, PIF and PEF (P<0.01), significantly decreased respiratory frequency, QA interval and Penh not (P<0.01), but not significantly changed activity and body temperature (P>0.05).Conclusions The application of implantable telemetry and whole-body plethysmography in this study does not obviously affect the circadian rhythm, and can sensitively monitor the relevant cardiovascular and respiratory parameters in conscious rats.It can be used in drug safety pharmacological research of cardiovascular and respiratory systems in conscious rats.

Objective To observe the pathological changes of heart failure caused by transverse aortic constriction in rats.Methods Partial thoracotomy was performed to the second rib and the transverse aortic constriction was performed between the innominate and left carotid arteries to establish a model of heart failure in 24 rats.The same operation was performed on another 8 rats, except for the ligation of the transverse aorta.Echocardiographic assessment, hemodynamic measurement, myocardial histopathological examination and NT-proBNP measurement were performed to the sham group at 12 weeks and model group at 4 weeks, 8 weeks, 12 weeks after the operation.Result At 4 weeks after the operation, NT-proBNP, EF, FS and -dp/dtmax of the model group was significantly increased and LVESV, +dp/dtmax of the model group were significantly decreased (P<0.05).At 8 weeks after the operation, EF and-dp/dtmax of the model group were increased and +dp/dtmax of the model group was significantly decreased (P<0.05).At 12 weeks after the operation, NT-proBNP, EF and +dp/dtmax of the model group were decreased, and LVESV, LVEDV and -dp/dtmax of the model group were increased (P<0.05).The cardiomyocytes became hypertrophic and lined up in disorder at 4 weeks after the operation.Pathologic examination of the myocardial tissue showed connective tissue proliferation and inflammatory cell infiltration at 8 weeks after the operation, and cardiomyocyte apoptosis and collagen fiber deposition at 12 weeks after the operation.Conclusions Transverse aortic constriction induces heart failure in rats.The pathological processes are compensatory hypertrophy at 4 weeks after the operation, initial reaction of decompensation at 8 weeks after the operation, and heart failure at 12 weeks after the operation.

Objective To observe the changes of mRNA expression related to kidney yang deficiency and the ul-trastructure of hypothalamus in Lewis rats, and further study the constitution of kidney yang deficiency in Lewis rats.Meth-ods Ten 7-8-week old SPF male Lewis rats and twenty 7-8-week old SPF male Wistar rats were used in this study.The rats received subcutaneous injection of hydrocortisone to establish kidney yang deficiency model.The expressions of TNF-α, IFN-γ, IL-10, CRH, MR and GR mRNA in the hypothalamus of the two groups were detected and the ultrastructural changes of hypothalamus and adrenal gland were observed to compare the differences between the two groups, and to ex-plore the mechanism of kidney yang deficiency constitution in the Lewis rats.Results Compared with the normal Wistar rats, the expressions of GR and IL-2 mRNA in the hypothalamus were significantly increased ( P<0.01 ) , while the ex-pressions of CRH, TNF-α, IFN-γand IL-10 mRNA were significantly decreased in the Lewis rats (P<0.01).The zona fasciculata of the adrenal cortex was slightly thinner, the mitochondria were slightly swollen and the amount of mitochondria was decreased.The nuclei of hypothalamic neurons were larger, and the mitochondria, neuron synapses and secretory vesi-cles in the presynaptic neurons were decreased.Conclusions There are abnormal expression of immune-related cytokines and CRH mRNA, and ultrastructural changes in the hypothalamus and adrenal gland of Lewis rats with kidney yang defi-ciency.Such changes may be related to the functional imbalance of the hypothalamus-pituitury-adrenal axis.

ObjectiveToinvestigatethekidneyyangdeficiencyconstitutionandhypothalamus-pituitary-adrenal ( HPA) axis dysfunction in Lewis rats .Method Two kidney yang deficiency models were established by subcutaneous injection of hydrocortisone and by adrenalectomy to induce hypothalamic -pituitary-adrenal ( HPA) axis dysfunction in Lewis rats.Wistar rats were used as control and compared with the two types of Lewis rat models of kidney yang deficiency .The degree of kidney yang deficiency of the Lewis rats was evaluated by examination and detection of the general signs , behavior , and neuroendocrine function , and so on .Result Compared with the normal Wistar rats , the body weight of Lewis rats was significantly higher than that of Wistar rats at the same age .The body temperature , urine volume and grip strength were significantly lower than those of Wistar rats ( P<0.01 for all ) .The memory ability of the Lewis rats was slightly decreased.The liver, kidney and adrenal indexes were significantly decreased in the Lewis rats (P<0.01).The levels of serum ACTH, CRH, cGMP, Cort and urine 17OHCS were significantly decreased (P<0.01).Conclusion A very slight deficiency of adrenal cortex function of Lewis rats is caused by genetic inheritance , and Lewis rats have the constitution of congenital kidney yang deficiency ( criticality or prophase of kidney yang deficiency ) .

Objective To compare the effects of forming atherosclerosis by conducting ballon injury operation after 1th, 2th and 3th week of Vitamin D3(VD3) i.p., exploring the best method for atherosclerosis modeling .Methods 36 male rats were selected for balloon-injured carotid artery .SD rats were divided into 4 groups randomly:control group ( n=6), Model group1 (n=10), Model group2(n=10), Model group3 (n=10).Control group were fed up with common diet.Model groups were fed up with high-fat diet and injected 4.0 ×105 IU/kg VD3 through enterocoelia in the beginning , followed by the balloon-injured left carotid artery operation after 1th, 2th and 3th week respectively and 1.0 ×105 IU/kg VD3injection at 0th, 2th week after operation.The rats were killed at 4th week after operation.The serum levels of TG, TC, HDL-C and LDL-C were checked .ELISA was used to detect the content of hsCRP , IL-6 and TNFα.HE staining was used to observe the pathological changes in the thoracic aorta , and the thoracic aorta thickness , plaque area ( PA) , cross-sectional area of vessel ( CVA) and the ratio of PA to CVA ( PA/CVA) were analyzed .Results After 4 weeks of operation , levels of TC and LDL-C were significantly increased in Model group 2 and 3 compared with that of the control group ( P<0.05).Furthermore, contents of hsCRP, IL-6 and TNFαof model groups were also seriously higher than that of the control group (P<0.05), and that of Model group 3 were the highest.Typical AS plagues were observed in different degrees in model groups, and thoracic aorta thickness and PA/CVA were obviously increased than that of control group (P<0.05). Model group 3 turned out masses of lipid foam cells accumulated , and PA, CVA and PA/CVA were significantly increased than that of Model group2 or 3.Conclusion The AS model can be established successfully in rats with ballon injury after 3 weeks of high-fat diet plus VD3 i.p., which is the ideal method to induced atherosclerosis model .