OBJECTIVE To explore the application of HL60-pNL3.2 reporter gene assay in pyrogen detection of live attenuated varicella vaccine, inactivated influenza vaccine and lyophilized live attenuated hepatitis A vaccine. METHODS According to the publication draft named as "in vitro pyrogen test(reporter gene assay)" in Chinese Pharmacopoeia, maximum valid dilution(MVD) of three kinds of vaccines was calculated, and the interference test was conducted to explore whether these vaccines interfere with the pyrogen determination. According to the general rule 9101 from Chinese Pharmacopoeia 2020, the assay in the pyrogen detection of the three vaccines was validated, using the methods to determine the pyrogen content of three vaccines. RESULTS The MVD of live attenuated varicella vaccine, inactivated influenza vaccine and lyophilized live attenuated hepatitis A vaccine respectively were 1 000, 200 and 1 000 times. The recovery rates of endotoxin(LPS) in the three kinds of vaccine were between 52.5% and 110.1%. The methodological validation results showed that the method could be used for the determination of pyrogens in three vaccines, and the correlation coefficient(R2) between LPS concentration and its chemical light intensity(RLU value) was > 0.98, the recovery rates were between 76.4% and 192.8% when LPS concentration was between 1 and 250 EU·mL–1. The coefficient of variation of measured results was less than 25% when LPS concentration was more than 5 EU·mL–1. The limit of detection and the limit of quantitation were between 0.05 and 0.13 EU·mL–1. Then, the pyrogen of these vaccines was determined by HL60-pNL3.2 reporter gene assay, and the pyrogen concentration all was less than contaminant limit concentration. CONCLUSION HL60-pNL3.2 reporter gene assay has the advantages of no animal, simple and rapid operation, with wide pyrogen spectrum and can quantitative pyrogen, which can be used to detect pyrogen of live attenuated varicella vaccine, inactivated influenza vaccine and lyophilized live attenuated hepatitis A vaccine.

AIM:To investigate the effect of hypericin(Hyp)on the heart of rats with myocardial ischemia-re-perfusion injury(MIRI),and to explore its mechanism.METHODS:Thirty SPF male SD rats were divided into 5 groups:sham group,MIRI group,low-dose Hyp(L-Hyp)group(MIRI+L-Hyp group),high-dose Hyp(H-Hyp)group(MIRI+H-Hyp group),and positive control trimetazidine(TMZ)group(MIRI+TMZ group),with 6 rats in each group.Apart from the sham group,the MIRI model was established by ligating the anterior descending branch of the left coronary artery and then recanalizing it in the remaining four groups of rats.The success of the modeling was determined by monitor-ing the electrocardiogram.We assessed the cardiac function in rats using echocardiography.TTC staining was employed to measure the area of myocardial infarction in rats,and HE staining was utilized to observe the morphological traits of rat myocardium.We assayed the activities of lactate dehydrogenase(LDH)and superoxide dismutase(SOD)and the levels of malondialdehyde(MDA)in rat serum using biochemical kits.ELISA kits were applied to assess the contents of cardiac troponin I(cTnI)and reactive oxygen species(ROS)in rat serum.Western blot analysis was perfomed to measure the pro-tein expression levels of AMPK,p-AMPK,Nrf2,and HO-1 in rat myocardial tissues.RESULTS:The rats in MIRI group exhibited increased myocardial tissue injury,larger myocardial infarction areas,decreased left ventricular ejection fraction(LVEF),and reduced left ventricular fractional shortening(LVFS)compared with sham group,as shown by echo-cardiography.Additionally,there were increases in LDH activity,cTnI,MDA and ROS levels,along with significant de-creases in SOD activity,and p-AMPK,Nrf2 and HO-1 protein levels(P<0.05).Compared with MIRI group,the rats in MIRI+L-Hyp,MIRI+H-Hyp and MIRI+TMZ groups showed decreased myocardial histopathological damage and reduced myocardial tissue infarction area,increased LVEF and LVFS,and lowered serum levels of LDH activity,cTnI,MDA and ROS,while SOD activity,p-AMPK,Nrf2 and HO-1 protein levels were elevated(P<0.05).CONCLUSION:Hypericin attenuates myocardial ischemia-reperfusion injury in rats,possibly by modulating the AMPK/Nrf2/HO-1 signaling path-way.

The material basis and mechanism of Chaenomelis Fructus in the treatment of rheumatoid arthritis(RA) were explored by network pharmacology, and the potential anti-RA targets of Chaenomelis Fructus were verified by molecular docking and animal experiments. The active components and targets of Chaenomelis Fructus were searched against the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform. GeneCards, DisGeNET, and OMIM were used to obtain RA-related targets. The common targets shared by Chaenomelis Fructus and RA were considered as the potential targets of Chaenomelis Fructus in the treatment of RA. Cytoscape 3.9.0 was employed to establish a "traditional Chinese medicine-active component-common target-disease" network. The protein-protein interaction(PPI) network was established by STRING, and the core genes were visualized by RStudio 4.1.0. DAVID was used for Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment to predict and visualize the involved signaling pathways. Molecular docking was carried out with the active components screened out as ligands and RA core genes as the targets. Finally, the prediction results were verified by animal experiments. Four main active components of Chaenomelis Fructus were obtained, which corresponded to 137 targets. Chaenomelis Fructus and RA shared 37 common targets. GO annotation yielded 239 terms(P<0.05), and KEGG pathway enrichment analysis screened out 94 signaling pathways(P<0.05), mainly involving interleukin-17(IL-17), tumor necrosis factor, Toll-like receptor, and nuclear factor-kappa B(NF-κB) signaling pathways. Molecular docking results showed that the main active components of Chaenomelis Fructus bound well with the core targets of RA. The results of animal experiments proved that Chaenomelis Fructus can alleviate joint swelling in the mice with RA. The results of ELISA showed that Chaenomelis Fructus lowered the levels of interleukin-6(IL-6) and interleukin-1β(IL-1β). Western blot showed that Chaenomelis Fructus down-regulated the protein level of vascular endothelial growth factor A(VEGFA). Chaenomelis Fructus exerts anti-inflammatory effect and reduces pannus formation by regulating the core targets such as VEGFA, IL-1β, and IL6 in the treatment of RA. The findings of this study provide new ideas for the future treatment of RA with Chaenomelis Fructus.
Animals ; Mice ; Network Pharmacology ; Vascular Endothelial Growth Factor A ; Molecular Docking Simulation ; Arthritis, Rheumatoid/genetics* ; Tumor Necrosis Factor-alpha ; NF-kappa B ; Drugs, Chinese Herbal/therapeutic use* ; Medicine, Chinese Traditional

BACKGROUND: The basis of individualized treatment should be individualized mortality risk predictive information. The present study aimed to develop an online individual mortality risk predictive tool for acute-on-chronic liver failure (ACLF) patients based on a random survival forest (RSF) algorithm. METHODS: The current study retrospectively enrolled ACLF patients from the Department of Infectious Diseases of The First People's Hospital of Foshan, Shunde Hospital of Southern Medical University, and Jiangmen Central Hospital. Two hundred seventy-six consecutive ACLF patients were included in the present study as a model cohort (n = 276). Then the current study constructed a validation cohort by drawing patients from the model dataset based on the resampling method (n = 276). The RSF algorithm was used to develop an individual prognostic model for ACLF patients. The Brier score was used to evaluate the diagnostic accuracy of prognostic models. The weighted mean rank estimation method was used to compare the differences between the areas under the time-dependent ROC curves (AUROCs) of prognostic models. RESULTS: Multivariate Cox regression identified hepatic encephalopathy (HE), age, serum sodium level, acute kidney injury (AKI), red cell distribution width (RDW), and international normalization index (INR) as independent risk factors for ACLF patients. A simplified RSF model was developed based on these previous risk factors. The AUROCs for predicting 3-, 6-, and 12-month mortality were 0.916, 0.916, and 0.905 for the RSF model and 0.872, 0.866, and 0.848 for the Cox model in the model cohort, respectively. The Brier scores were 0.119, 0.119, and 0.128 for the RSF model and 0.138, 0.146, and 0.156 for the Cox model, respectively. The nonparametric comparison suggested that the RSF model was superior to the Cox model for predicting the prognosis of ACLF patients. CONCLUSIONS The current study developed a novel online individual mortality risk predictive tool that could predict individual mortality risk predictive curves for individual patients. Additionally, the current online individual mortality risk predictive tool could further provide predicted mortality percentages and 95% confidence intervals at user-defined time points.
Acute-On-Chronic Liver Failure ; Humans ; Prognosis ; Proportional Hazards Models ; ROC Curve ; Retrospective Studies

This study explored the in vivo effects and mechanisms of the modern classical prescription Supplemented Gegen Qinlian Decoction Formula(SGDF) against diabetic kidney disease(DKD). Sixty rats were randomly divided into the normal group, model group, SGDF group, and rosiglitazone(ROS) group. The modified DKD rat model was established by employing the following three methods: exposure to high-fat diet, unilateral nephrectomy, and intraperitoneal injection of streptozotocin(STZ). After modeling, rats in the four groups were treated with double distilled water, SGDF suspension, and ROS suspension, respectively, by gavage every day. At the end of the 6 th week of drug administration, all the rats were sacrificed for collecting urine, blood, and kidney tissue, followed by the examination of rat general conditions, urine and blood biochemical indicators, glomerulosclerosis-related indicators, podocyte pyroptosis markers, insulin resistance(IR)-related indicators, and key molecules in the insulin receptor substrate(IRS) 1/phosphatidylinositol-3-kinase(PI3 K)/serine threonine kinase(Akt) signaling pathway. The results showed that SGDF and ROS improved the general conditions, some renal function indicators and glomerulosclerosis of DKD model rats without affecting the blood glucose(BG). Besides, they ameliorated the expression characteristics and levels of podocyte pyroptosis markers, alleviated IR, and up-regulated the protein expression levels of the key molecules in IRS1/PI3 K/Akt pathway to varying degrees. In conclusion, similar to ROS, SGDF relieves DKD by targeting multiple targets in vivo. Specifically, it exerts the therapeutic effects by alleviating podocyte pyroptosis and IR. This study has preliminarily provided the pharmacological evidence for the research and development of new drugs for the treatment of DKD based on SGDF.
Animals ; Diabetes Mellitus ; Diabetic Nephropathies/drug therapy* ; Drugs, Chinese Herbal ; Insulin Resistance ; Podocytes ; Pyroptosis ; Rats

Objective To develope a deep learning algorithm for pathological classification of chronic gastritis and assess its performance using whole-slide images (WSIs). Methods We retrospectively collected 1,250 gastric biopsy specimens (1,128 gastritis, 122 normal mucosa) from PLA General Hospital. The deep learning algorithm based on DeepLab v3 (ResNet-50) architecture was trained and validated using 1,008 WSIs and 100 WSIs, respectively. The diagnostic performance of the algorithm was tested on an independent test set of 142 WSIs, with the pathologists' consensus diagnosis as the gold standard. Results The receiver operating characteristic (ROC) curves were generated for chronic superficial gastritis (CSuG), chronic active gastritis (CAcG), and chronic atrophic gastritis (CAtG) in the test set, respectively.The areas under the ROC curves (AUCs) of the algorithm for CSuG, CAcG, and CAtG were 0.882, 0.905 and 0.910, respectively. The sensitivity and specificity of the deep learning algorithm for the classification of CSuG, CAcG, and CAtG were 0.790 and 1.000 (accuracy 0.880), 0.985 and 0.829 (accuracy 0.901), 0.952 and 0.992 (accuracy 0.986), respectively. The overall predicted accuracy for three different types of gastritis was 0.867. By flagging the suspicious regions identified by the algorithm in WSI, a more transparent and interpretable diagnosis can be generated. Conclusion The deep learning algorithm achieved high accuracy for chronic gastritis classification using WSIs. By pre-highlighting the different gastritis regions, it might be used as an auxiliary diagnostic tool to improve the work efficiency of pathologists.
Algorithms ; Deep Learning ; Gastritis/diagnosis* ; Humans ; ROC Curve ; Retrospective Studies

Objective To explore the dosimetric differences of radiotherapy plan for cervical cancer with 4 different fluence smoothing (FS) parameters using Monaco treatment planning system (Monaco TPS). Methods Fifteen patients with ⅠB2 stage cervical cancer in our hospital were enrolled in this study. And a 2 Volumetric Modulated Arc Therapy (VMAT) plan for each patient were completed by Monaco 5.11 TPS according to the X-Ray Voxel Monte Carlo (XVMC) method. For each plan was optimized by FS function, with the level of Off, Low, Medium and High. To compare the difference of plan optimization time, conformity index (CI), Homogeneity index (HI), D_mean, D_min, D_2% of PTV，dose to the organ at risk (OAR)，the number of Segments# and MU#，estimated total delivery time (ETDT), quantum Efficiency (QE) of the plans, the formation of Segments# with the same angle and verification of inserting 729 two-dimensional matrix into PTW octavius 4D module of different FS function levels, with the precondition of the Prescription isodose curve covering 95% of the target area. The data was analysed by multivariate factor analysis with the application of SPSS, and P < 0.05 was considered as statistically significant. And the Planned revenue score of different FS levels was also calculated. Results Except for the D_min of PTV (the lowest value is (32.09 ± 0.26) Gy for the Off group, and the highest value is (35.98 ± 0.42) Gy for the High group), V₄₀ of the rectum (the lowest value in the Medium group is 55.88% ± 2.02%, and the highest value in the High group was 61.90% ± 2.98%) and bladder (the lowest value was 45.01% ± 2.08% in the Medium group, and the highest value is 50.45% ± 1.98% in the High group), the V₂₀ (the lowest value High group was 49.05% ± 1.98%, the highest value Off group was 56.52% ± 1.75%) of femoral head (P < 0.05), there was no significant difference of the dose assessment results for PTV and OARs in 4 different FS function levels. In the High level, the ETDT, QE and MU# were showed better than other groups evidently, however, the number of Segments# showed no significant difference. The plan validation results was increased with the improvement of FS function level, and the level of High was considered to be the optimal. To compare the score of overall benefits of the plan, the level of Medium (−17.18 ± 0.05) got the highest score, and the Low group (−17.58 ± 0.05) and the High group (−17.42 ± 0.06) have similar scores, and Off group (−18.81 ± 0.08) has the lowest score. Conclusion Different FS levels of the Monaco 5.11 TPS can optimize the radiotherapy plan for cervical cancer, but the level of Medium is considered to be the most applicable.

Biopsy ; Deep Learning ; Humans ; Male ; Neoplasm Grading ; Prostatectomy ; Prostatic Neoplasms

To observe the multi-targeted therapeutic effects of Huangkui Capsules(HKC)on insulin resistance(IR)and urine microalbumin in the early diabetic kidney disease(DKD)patients. The case data from the 83 DKD patients at G2 and A2 stage were collected respectively and analyzed retrospectively. According to the different treatment,all patients were divided into the control(A)group(40 cases)and the treated(B)group(43 cases). Among them,the A group patients were received "routine basic treatment";the B group patients were received "routine basic treatment+HKC". For the 2 group patients,firstly,the baseline parameters before receiving the treatment were compared respectively,and then,the changes of the total scores of traditional Chinese medicine(TCM) syndromes and the indicators of IR,urine protein,renal function,blood lipids and safety after receiving the treatment for 8 weeks were compared,respectively. Furthermore,for the all patients,the correlation analysis between IR and urine protein or IR and the total scores of TCM syndromes was carried out,respectively. The results showed that,for the B group patients received "routine basic treatment",their total scores of TCM syndromes,urine protein indicators including urine microalbumin(micro-UAlb) and urine microalbumin/urinary creatinine(UACR),IR indicators including fasting serum insulin(FIN)and homeostasis model assessment of insulin resistance(HOMA-IR)were significantly improved,respectively. For the all DKD patients,before and after the treatment,the main IR indicators(FIN and HOMA-IR)were positively correlated with urine protein indicators(micro-UAlb and UACR). The main IR indicators(FIN and HOMA-IR) were also positively correlated with the total scores of TCM syndromes. In addition,2 treatments had no significant effects on renal function,blood lipids and safety indicators in the all DKD patients. Overall, "routine basic treatment+HKC" can ameliorate IR and reduce urine microalbumin in the early DKD patients. Its therapeutic targets may be not only proteinuria,but also IR,which is the upstream risk factor of proteinuria.
Albuminuria ; Capsules ; Diabetes Mellitus ; Diabetic Nephropathies ; Drugs, Chinese Herbal ; Humans ; Insulin ; Insulin Resistance ; Kidney ; Retrospective Studies

Fucoidan(FPS) is an effective component of the Chinese patent medicine named Haikun Shenxi, which treats schronic renal failure in clinics, and has the potential anti-aging effects. However, it is still unclear whether FPS can improve renal aging, especially the molecular mechanism of its anti-aging. The human proximal renal tubular epithelial cells(HK-2) in vitro were divided into normal group(N), D-gal model group(D), low dose of FPS group(L-FPS), high dose of FPS group(H-FPS) and vitamin E group(VE), and treated by the different measures, respectively. More specifically, the HK-2 cells in each group were separately treated by 1 mL of 1% fetal bovine serum(FBS) or D-galactose(D-gal, 75 mmol·L~(-1)) or D-gal(75 mmol·L~(-1))+FPS(25 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+FPS(50 μg·mL~(-1)) or D-gal(75 mmol·L~(-1))+VE(50 μg·mL~(-1)). After the treatment for 24 h, firstly, the effects of D-gal on senescence-associated β-galactosidase(SA-β-gal) staining characteristics and klotho, P53 and P21 protein expression le-vels, as well as adenosine monophosphate activated protein kinase(AMPK)-uncoordinated 51-like kinase 1(ULK1) signaling pathway activation in the HK-2 cells were detected, respectively. Secondly, the effects of FPS and VE on SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal were investigated, respectively. Finally, the effects of FPS and VE on microtubule-associated protein 1 light chain 3(LC3) protein expression level and AMPK-ULK1 signaling pathway activation in the HK-2 cells exposed to D-gal were examined severally. The results indicated that, for the HK-2 cells, the dose of 75 mmol·L~(-1) D-gal could induce the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels. That is causing cells aging. FPS and VE could both ameliorate the changes of SA-β-gal staining characteristics and klotho, P53 and P21 protein expression levels in the HK-2 cells exposed to D-gal. That is anti-cells aging, here, the functions of FPS and VE are similar. D-gal could not only induce cell aging but also increase LC3Ⅱ, phosphorylated-AMPK(p-AMPK) and phosphorylated-ULK1(p-ULK1) protein expressions, and activate autophagy-related AMPK-ULK1 signaling pathway. FPS and VE could both improve the changes of LC3Ⅱ, p-AMPK and p-ULK1 protein expression levels in the HK-2 cells exposed to D-gal. That is inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. On the whole, for the human proximal renal tubular epithelial cells aging models induced by D-gal, FPS similar to VE, can ameliorate renal cells aging by possibly inhibiting autophagy-related AMPK-ULK1 signaling pathway activation. This finding provides the preliminary pharmacologic evidences for FPS protecting against renal aging.
Aging ; Autophagy ; Epithelial Cells ; Humans ; Polysaccharides ; Signal Transduction