Karacoline is a compound found in the plant Aconitum kusnezoffii Reichb. Although Aconitum kusnezoffii Reichb is widely used for the treatment of pain, very few studies have been carried out on the use of karacoline due to its potential toxicity. In this study, we selected key matrix metalloproteinases (MMPs), collagen II, and aggrecan as targets due to their association with intervertebral disc degeneration (IDD). Using these targets, we then used network pharmacology to predict a series of molecules that might exert therapeutic effects on IDD. Of these molecules, karacoline was predicted to have the best effect. Tumor necrosis factor (TNF)-αis known to promote the degeneration of the extracellular matrix in IDD. We therefore applied different concentrations of karacoline (0, 1.25, or 12.88μM) along with 100 ng/mL TNF-αto rat nucleus pulposus cells and found that karacoline reduced the expression of MMP-14 in IDD by inhibiting the nuclear factor (NF)-κB pathway, while collagen II and aggrecan expression was increased. This suggested that extracellular matrix degradation was inhibited by karacoline (P<0.05). Our data therefore reveal a new clinical application of karacoline and provide support for the use of network pharmacology in predicting novel drugs.

Aim To investigate the effect of puerarin on proliferation, differentiation, and mineralization of MC3T3-E1 cells and the expression of TRPM3 mRNA.Methods Proliferation, differentiation, and mineralization of puerarin in MC3T3-E1 cells were determined using CCK-8 assay, alkaline phosphatase(ALP) activity assay, and Alizarin Red Staining, respectively.Effect of puerarin on cell cycle and intracellular calcium concentration of MC3T3-E1 cells was detected by flow cytometry.RT-PCR was used to detect the effect of puerarin on TRPM3 mRNA expression.Resuls 0.1, 1, 10 μmol·L-1 puerarin significantly promoted the proliferation of MC3T3-E1 cells, reduced the proportion of cells in G1 phase, increased the proportion of G2 and S phase, of which 0.1 μmol·L-1 concentration effect was the most significant.Compared with control group, the ALP activity and mineralized nodule area of 0.1 μmol·L-1 puerarin group were significantly increased.The expression of TRPM3 mRNA and the intracellular calcium concentration were significantly decreased in 0.1 μmol·L-1 puerarin group.Conclusion Puerarin can promote the proliferation, differentiation, and mineralization of MC3T3-E1 cells, and reduce the expression level of TRPM3 mRNA and intracellular calcium concentration.

Objective To investigate the expressions of apoptosis associated protein 3 (APR3) and nuclear factor 3 of activated T-cell (NFAT3) in the tissue of epithelial ovarian tumors and its correlation with the clinicopathological features.Methods 92 patients with epithelial ovarian tumor were collected,23 cases with malignant tumor,24 cases with borderline tumor,45 cases with benign tumor.The expressions of APR3 and NFAT3 were detected by immunohistochemical methods,and the differences of different types of epithelial ovarian tumor were compared.The correlation of the expressions of APR3 and NFAT3 with the clinicopathological features of epithelial ovarian tumor was analyzed.The correlation of the expressions of APR3 with the expressions of NFAT3 in epithelial ovarian tumor was analyzed.Results The positive expression rate of APR3 in patients with malignant epithelial ovarian tumors (78.26％) was significantly higher than borderline tumors (41.67 ％) and benign tumors (22.22 ％),the differences were statistically significant (x2 =5.864,7.632,all P ＜ 0.05).The expression of APR3 in patients with malignant epithelial ovarian tumors was significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis and ascites (x2 =7.425,7.262,8.421,5.031,all P ＜ 0.05).The positive expression rate of NFAT3 in patients with malignant epithelial ovarian tumors (56.52％) was significantly higher than borderline tumors (29.17％) and benign tumors(17.78％),the differences were statistically significant (x2 =6.829,7.547,all P ＜0.05).The expression of NFAT3 in patients with malignant epithelial ovarian tumors was significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis (x2 =5.253,6.367,8.021,all P ＜ 0.05).The expressions of APR3 and NFAT3 in patients with malignant epithelial ovarian tumors were positively correlated (r =0.032,P ＜ 0.05).Conclusion The expressions of APR3 and NFAT3 in the tissue of malignant epithelial ovarian tumor obviously increase,are significantly correlated with differentiation,clinical stage,lymph node and abdominal organs metastasis and are positively correlated,and it may be correlated with the development and progression of malignant epithelial ovarian tumor.

Objective To investigate the timing of early naso-jejunum nutritional support on severe acute pancreatitis(SAP) patients and its feasibility and safety. Methods 72 patients with SAP were enrolled and randomized into experiment group and control group according to random number. EN (enteral nutrition) was administrated in the experiment group when the patients' circulation were relatively stable, abdominal distention decreased and the bowel sound could be heard; while EN was administrated in the control group when the patients' vital signs were stable, and peristalsis recovered with exhaust and sense of hunger, without obvious abdominal distention. The cultivation of secretions, hospital stay and overall cost of hospitalization were evaluated in the two groups of patients. Results The throat swabs, sputum culture, mid-portion urine culture of the two group were not significantly different. But the fecal culture in the experiment group was significantly lower than those of the control group (5.6% vs. 33.3%, P ＜ 0. 01); the hospital stay and overall cost of hospitalization in the experiment group was (26.0 ± 15.2)d and (78910 ± 77734)Yuan, which were significantly lower than those in the control group [(32.9 ± 22.3) d and (149528 ± 145936) Yuan, P ＜0.05]. Conclusions Appropriately bringing forward EN is safe and feasible, which can shorten clinical course and reduce the cost of hospitalization.

Aim To study proliferation capacity of cell and the target relationship between microRNA and Runx2 after effect of puerarin on osteoblasts MC3 T3-E1 . Methods The proliferation capacity of cell was detected by MTT after effect of puerarin on osteoblasts MC3 T3-E1 . The vitality of osteoblasts was detected by activity of alkaline phosphatase. The expression level of mRNA and protein of Runx2 were detected by real-time quantitative PCR and Western blot. The result of miRNA expression spectrum was compared with the predicted result to determine the Runx2-targeting miR-NAs. The expression levels of miRNAs possiby targeted to Runx2 were detected by real-time quantitative PCR. The RhoE 3′UTR vector and RhoE mut 3′UTR vector were constructed. miRNA-204 mimics and miRNA-204 NC were synthetised. The target genes were verified by dual luciferase report gene assay. Results After osteo-blasts treated with puerarin, proliferation capacity and activity of cells were enhanced , expression levels of mRNA and protein of Runx2 were both increased , the expression levels of miRNA-204 and miRNA-344 f-5 p were declined, the expression levels of miRNA-2861 was increased,the expression levels of miRNA-23a-5p, miRNA-770-5 p and miRNA-871-5 p showed no obvious change. According to the results of dual luciferase re-porter gene method after cell transfection of 48 h, only set of 3′UTR Runx2+mimics the miRNA-204 of fluo-rescein protein expression level decreased significantly, showing only the miRNA-204 inhibits Runx2 3′UTR report gene expression. Conclusion Puerarin pro-motes the proliferation of osteoblasts and regulates the miRNAs which possibly target to Runx2 .

Objective To establish and evaluate the CaV1?1?R528H gene knock?in mouse model of thyrotoxic hy?pokalemic periodic paralysis. Methods Thirty?six 8?week?old male CaV1?1?R528H gene knock?in mice and thirty?six 8?week?old wild?type male C57BL/6J mice were used in this study. Using three?factor two?level 2 × 2 × 2 factorial design ( the three factors including mutation, thyroxine and insulin, and two levels were with or without) , the mice were divided into 8 groups. The thyroxine groups were intraperitoneally injected with levothyroxine in a dose of 350 μg/kg once per day for 12 consecutive days to produce thyrotoxicosis. The insulin groups were intraperitoneally injected with short?acting insulin in a dose of 0?8 U/kg after the last administration of levothyroxine, and the potassium levels of different groups were meas?ured and recorded before (0 min) and after insulin injection (30 min, 60 min). Results (1) Compared with the control group, the following phenomena including irritability, dull coat, increased diet and water intake, and slow body weight gain, were observed in the thyrotoxic mice. Thyroid function tests showed that the levels of T3 and T4 in the thyrotoxic mice were significantly higher than those in the corresponding control mice (P<0?05), and the TSH level was significantly low?er than that of the corresponding control mice (P<0?05 ). (2) After administration of insulin or thyroxine alone, the po?tassium levels in the mutant and wild?type mice were not significantly different. However, after combined administration of thyroxine and insulin, the potassium levels in the mutant group were significantly lower than those in the wild?type mice at 30 min and 60 min ( P<0?05 for both). (3) The main effects and interactions:Mutation factor or thyroxine factor alone did not influence on the potassium level, only insulin showed hypokalemic effect (P<0?05). There were interactions be?tween thyroxine and mutation, and between insulin and mutation (P<0?05), but no interaction between thyroxine and in?sulin. Conclusions (1) A thyrotoxicosis state in mice is successfully developed in this study. (2) An CaV1?1?R528H gene knock?in mouse model of thyrotoxic hypokalemic periodic paralysis is successfully established.

Objective To study the relationship between serum cystatin C(Cys C)level and the development of insulin resistance(IR)in elderly patients with type 2 diabetes mellitus (T2DM).Methods A cross-sectional survey research involving 757 elderly patients with T2DM was performed and patients were divided into groups according to Cys C and IR levels.Serum 1evels of fasting insulin (FBI),fasting blood glucose(FPG),fasting C-peptide (FCP),glycosylated hemoglobin A1 c (H bA1 c),homeostasis model assessment of insulin resistance (IR) (HOMA2 IR-C),homeostasis model assessment of insulin secretion,homeostasis model assessment of insulin sensitivity(HOMA2-％ S-C),micro-albuminuria(mALB)and serum lipid were measured and compared among the groups.Possible influence factors were adjusted,and the correlation between Cys C levels and IR was analyzed.The influencing factors on IR were also analyzed.Results There were statistically significant differences in ages,course of T2 DM,FBI,FCP,HbA1c,urinary mALB,IR,insulin secretion,insulin sensitivity,morbidity rate of coronary heart disease,hypertension and diabetic nephropathy among the three groups(allP＜0.05).Insulin sensitivity was decreased with the increase of Cys C level,while others were increased.Among 757 patients,the level of serum Cys C was positively correlated with FCP,HOMA2-IR C levels,and was negatively correlated with HOMA2-％ S-C levels(P＜0.05).Multiple logistic regression analysis showed that the higher level of Cys C was independent risk factors for IR in elderly T2DM patients(OR=3.41,95％CI:2.22-5.22,P＜0.05).Conclusions Serum Cys C level is closely related with IR in elderly T2DM,and the elevated level of serum Cys C is one of independent risk factors for elderly T2DM.

Objective:To evaluate the value of MAGiC STIR sequence inassessing bone marrow edema (BME) of the sacroiliac joint (SIJ) in ankylosing spondylitis (AS) patients.Methods:Twenty two AS patients from the Affiliated Fifth Hospital of Sun Yat-sen University from September 2019 to January 2020 underwent MR scanning, consisting of T 2WI fat-suppressed (FS) sequence and MAGiC sequence. Each SIJ was divided into four quadrants for analysis on oblique coronal MR image. Two radiologists [middle-higher seniority levels (radiologist 1 and radiologist 2) reviewed image sets of two sequences [T 2WI FS and MAGiC short T 1 inversion recovery (STIR) sequence] independently at separate times, using Hermann-Braun scoring system for degree of BME and summing numbers of quadrants based on BME findings. Disagreements were resolved to reach an agreement for final results.Assessments were repeated two weeks later by radiologist 2. Intra-class correlation coefficients (ICC) were calculated to assess the inter-and intra-observer agreement for the degree of BME and numbers of positive BME quadrants in the MAGiC STIR sequence, respectively. The differences between the two sequences for the degree of BME and numbers of positive BME quadrants were analyzed with Wilcoxon signed-rank test and Chi-square test, respectively. Taking the results of T 2WI FS sequence as a reference standard, the ROC curve was drawn, sensitivity, specificity, positive predictive value and negative predictive value in the detection of BME were calculated for the MAGiC STIR sequence. Results:Nineteen AS patients including 38 sacroiliac joints were enrolled in our study, being divided into 152 quadrants. Inter-and intra-observer agreement for the degree of BME and inter-observer agreement for numbers of positive BME quadrants in the MAGiC STIR sequence were all good (ICC=0.936, 95%CI 0.912-0.953, P<0.001; ICC=0.910, 95%CI 0.878-0.934, P<0.001; ICC=0.876, 95%CI 0.833-0.909, P<0.001). The difference between the two sequences for the degree of BME in quadrants was statistically significant ( Z=-3.132, P=0.002). But there was no statistically significant difference between sequences for numbers of quadrants in detecting BME (χ2=0.244, P=0.622). Area under the ROC curve of MAGiC STIR sequence in diagnosis of BME was 0.920 ( P<0.001). Sensitivity, specificity, positive predictive value and negative predictive value for the MAGiC STIR sequence in the detection of BME were 82.0%, 95.1%, 89.1% and 91.5%, respectively. Conclusion:MAGiC STIR sequence can be used to evaluate bone marrow edema of SIJ in AS patients with high sensitivity and specificity.

Objective To establish nucleic acid testing techniques for detecting Nipah virus (NiV) and Hendra virus (HeV), and to test the NiV and HeV in peripheral blood collected from domestic pigs, cows and goats in Chongqing. Methods Peripheral blood samples of 580 domestic pigs, 250 cows, 180 goats were collected from Chongqing since June 2007 to June 2008. The lymphocytes were separated by density gradient centrifugation and total RNA was extracted using Trizol method for detection of NiV and HeV with one-step real-time RT-PCR. Sequence identification and analysis were performed for positive PCR prod-ucts. Virus isolation and culture were adopted for positive samples, and epidemiologic reports were submit-ted. Results Nucleic acid detections searching for NiV and HeV were successfully performed in animal blood samples collected from Chongqing. "Takeoff points" were not found in fluorescence amplification curves of all samples. Curves kept the same slope, and assays were judged as negative. Conclusion Until now, Neither NiV or HeV infection has been found in domestic animals blood samples collected from Chongqing, which suggest a lower possibility of outbreaks of Nipah disease and Hendra disease in Chongqing in the near future.

Objective To observe the epidemiology characterization of Borna disease virus (BDV) in animal brain in Ili, Xinjiang, and to find out the potential infection of the Borna disease virus to prevent its outbreak. Methods The BDV p24 gene of animal brain tissues in Ili including 200 horses, 75 donkeys and 100 shepherd dogs was detected by fluorescence quantitative nest reverse transcriptase polymer-ase chain reaction(FQ-nRT-PCR). GFP-p24,pMD-19 plasmid contamination was excluded from positive products. Clone sequencing was used to analyze the homology of gene and amino acid sequence. Results BDV p24 gent was found in 3 Ili horses, 4 Ili donkeys and 9 shepherd dogs, and the positive ratio is 1.5%, 5.3% and 9.0%, respectively. The GFP-p24,pMD-19 were not found in BDV p40 gene and plasmid stand-ard. The sequence of BDV p24 amplification production was totally the same as He/80 virus strain. Conclu-sion Natural infection of BDV may exist in the animals(horses, donkeys and dogs)in Ili, and the epidem-ic strain of BDV in this area was homological as He/80 virus strain.