OBJECTIVE: To establish the drug-resistant cell lines of hepatocellular carcinoma (HCC) induced by sorafenib, and to screen out the high expression genes in drug-resistant cell lines of HCC induced by sorafenib, then to explore the genes related to sorafenib resistance in hepatocellular carcinoma. METHODS: The human PLC and Huh7 cell lines were obtained, then the PLC and Huh7 drug-resistant cell lines were induced with sorafenib by using intermittent induction in vitro. CCK8 assay was used to detect the IC50 value of sorafenib for evaluation of drug sensitivity of hepatocellular carcinoma cell lines in PLC and Huh7. All the up regulated genes in PLC and Huh7 drug-resistant cell lines induced by sorafenib were screened out using high-throughput cDNA sequencing (RNA-Seq), Ualcan database was used to analyze the correlations between the up regulated genes in PLC and Huh7 drug-resistant cell lines induced and four clinical biological characteristics of hepatocellular carcinoma, including the gene expressions between normal samples and tumor samples, tumor stage, tumor grade, and patient overall survival, to find the genes that might be involved in the mechanism of sorafenib resistance of hepatocellular carcinoma. RESULTS: All the up regulated genes detected by the using high-throughput cDNA sequencing (RNA-Seq) in PLC and Huh7 drug-resistant cell lines were further screened out by following conditions:(1) genes co-expressed in PLC and Huh7 drug-resistant cells induced by sorafenib, (2) the fold change was more than 4 times and the difference was statistically significant (P <0.05), the top 12 up regulated genes in PLC and Huh7 drug-resistant cell lines were found, which were TPSG1, CBX4, CLC, CLEC18C, LGI4, F2RL1, S100A6, HABP2, C15ORF48, ZG16, FOLH1, and EPCAM. Compared with the correlations between the twelve genes and the clinical biological characteristics by Ualcan database, the potentially significant gene CBX4 was screened out. CONCLUSION The human PLC and Huh7 drug-resistant cell lines of hepatocellular carcinoma induced by sorafenib were successfully established. CBX4, the gene related to sorafenib resistance in hepatocellular carcinoma, was screened out by the high-throughput cDNA sequencing (RNA-Seq) and further analysis using Ualcan database, which is providing a powerful basis for further research on the mechanism of sorafenib resistance of hepatocellular carcinoma.
Antineoplastic Agents/therapeutic use* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Humans ; Ligases ; Liver Neoplasms/drug therapy* ; Polycomb-Group Proteins ; Serine Endopeptidases ; Sorafenib/therapeutic use*

OBJECTIVE: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot (RDS), was developed based on a classical prescription of traditional Chinese medicine (TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019 (COVID-19), have been shown. However, the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS. METHODS: To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein-protein interaction (PPI) network, and cluster analysis; further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS. RESULTS: For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent-target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target-constituent-disease network. Twelve disease pathways attributed to the Lung-Large Intestine meridians, with six and five attributed to the Kidney-Urinary Bladder and Stomach-Spleen meridians, respectively. One-hundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-2 3-chymotrypsin-like protease (3CL), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3CL inhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode, the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches. CONCLUSION RDS acts primarily in the Lung-Large Intestine, Kidney-Urinary Bladder and Stomach-Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS's dual effects of health-strengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.
Antiviral Agents ; chemistry ; therapeutic use ; Betacoronavirus ; chemistry ; enzymology ; Coronavirus Infections ; drug therapy ; prevention & control ; virology ; Cysteine Endopeptidases ; chemistry ; Drugs, Chinese Herbal ; chemistry ; therapeutic use ; Humans ; Mass Spectrometry ; Medicine, Chinese Traditional ; Molecular Docking Simulation ; Pandemics ; prevention & control ; Pneumonia, Viral ; drug therapy ; prevention & control ; virology ; Protein Interaction Maps ; Viral Nonstructural Proteins ; chemistry

OBJECTIVE: This study aims to use Arginine-gingipain A gene vaccine (pVAX1-rgpA) to immunize adult Beagle dogs and to evaluate its effect during peri-implantitis progression and development. METHODS: Plasmid pVAX1-rgpA was constructed. The second and third bilateral mandible premolars of 15 adult Beagle dogs were extracted, and the implants were placed immediately. After 3 months, the animals were randomly divided into groups A, B, and C. Afterward, the animals were immunized thrice with plasmid pVAX1-rgpA, with heat-killed Porphyromonas gingivalis, or pVAX1, respectively. IgG in the serum and secretory IgA (sIgA) in saliva were quantitatively analyzed by enzyme-linked immunosorbent assay before and after 2 weeks of immunization. Peri-implantitis was induced with cotton ligatures fixed around the neck of implants. Probing depth (PD) and bleeding on probing were recorded. All animals were sacrificed after ligaturation for 6 weeks. Decalcified sections with thickness of 50 μm were prepared and dyed with methylene blue to observe the bone phenotype around implants. RESULTS: Levels of serum IgG and sIgA in saliva were higher in groups A and B after immunization than before the process (P<0.05) and higher than those in group C (P<0.05). However, no difference was observed between groups A and B (P>0.05). At 4 and 6 weeks after ligaturation, PD of the ligatured side in group C was higher than that in groups A and B (P<0.05). On the other hand, no difference was identified between groups A and B (P>0.05). Bone loss in group A was significantly lower than that of the other groups (P<0.05). Abundant inflammatory cells and bacteria were present in the bone loss area around the implants in the three groups, as identified through hard tissue section observation. However, group C presented the most number of inflammatory cells and bacteria in the bone loss area around the implants. CONCLUSIONS IgG and sIgA can be generated by immunity with rgpA DNA vaccine, which can significantly slow down bone loss during experimental peri-implantitis in dogs.
Adhesins, Bacterial ; therapeutic use ; Alveolar Bone Loss ; Animals ; Arginine ; Cysteine Endopeptidases ; therapeutic use ; Dental Implants ; Dogs ; Peri-Implantitis ; prevention & control ; Porphyromonas gingivalis ; chemistry ; Vaccines ; therapeutic use

OBJECTIVETo observe the effects of Huannao Yicong Formula (, HYF) on learning and memory and it's regulating effect on γ-secretase related anterior pharynx defective 1 (APH-1), presenilin enhancer-2 (PEN-2) signaling pathway, so as to discuss and further clarify the mechanism of HYF on Alzheimer's disease.

METHODSSixty APP/PS1 transgenic mice, randomly allocated into 4 groups, the model group, the donepezil group (0.65 mg/kg), HYF low-dose group (HYF-L, 5.46 g/kg) and HYF high-dose group (HYF-H, 10.92 g/kg), 15 for each group. Another 15 C57BL/6J mice with the same age and same genetic background were allocated into the control group, proper dosage of drugs or distilled water were given by intragastric administration once daily for 12 weeks. After 12 weeks of administration, the learning and memory abilities of mice in each group was evaluated by the morris water maze test, amyloid precursor protein (APP), Aβand Aβlevels in hippocampus were detected by enzyme-linked immunosorbent assay, γ-secretase was detected by dual luciferase assaying, the levels of APH-1a, hypoxia-inducible factor 1α (HIF-1α), cAMP response element-binding protein (CREB) and PEN-2 and their mRNA expression was measured by Western blot and real-time polymerase chain reaction.

RESULTSHYF can ameliorate learning and memory deficits in APP/PS1 transgenic mice by decreasing the escape latency, improving the number of platform crossing and swimming speed (P<0.01, P<0.05). HYF can decrease the levels of APP, Aβ, Aβand the activity of γ-secretase in hippocampus of Alzheimer's disease model mice. HYF can down-regulate the levels of CREB and PEN-2 and the expression of their mRNA.

CONCLUSIONHYF can improve the learning and memory ability by inhibiting the activity of γ-secretase through the CREB/PEN-2 signaling pathway, and this may be one of the therapeutic mechanisms of HYF in Alzheimer's disease.

Amyloid Precursor Protein Secretases ; metabolism ; Amyloid beta-Protein Precursor ; metabolism ; Animals ; Cyclic AMP Response Element-Binding Protein ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Endopeptidases ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Gene Expression Regulation ; drug effects ; Hippocampus ; drug effects ; metabolism ; pathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Immunohistochemistry ; Learning ; drug effects ; Male ; Memory Disorders ; drug therapy ; genetics ; Mice, Inbred C57BL ; Mice, Transgenic ; Presenilin-1 ; metabolism ; Presenilin-2 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo investigate the effect and molecular mechanisms of different doses of 8-hydroxy dihydroberberine (Hdber) for the treatment of hyperlipidemia in rats.

METHODSA rat model of hyperlipidemia was established by feeding rats a high-fat diet for 4 weeks in 70 rats of 80 animals, and 10 rats were randomly selected as control group. The hyperlipidemic rats were then randomly divided into the following groups: a model group (MOD); a berberine group [BBR, 156 mg/(kg day)]; Hdber groups, which were treated with different doses of Hdber [78, 39 and 19.5 mg/(kg day)]; and a simvastatin group [SIM, 4 mg/(kg day)]. The corresponding therapy was administered to the rats of each treatment via gastric tubes. Normal animals were used as a control group. The blood levels of various lipids, including total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, free fatty acid (FFA), apolipoprotein AI(Apo-AI) and apolipoprotein B (Apo-B) were examined. The protein expressions of low-density lipoprotein receptor (LDL-R), sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and proprotein convertase subtilisin/kexin type 9 (PCSK-9) in liver tissues were determined by Western blot analysis.

RESULTSCompared with the control group of rats, the model group demonstrated a deteriorated blood lipid profile and exhibited increased expression levels of PCSK-9 protein in their liver tissues (P<0.01). In addition, the high-fat diet decreased the expression levels of LDL-R, SREBP-2 and HMGCR proteins in murine liver tissues. However, the addition of berberine or Hdber reversed the blood lipid profile changes (P<0.05 or P<0.01), decreased the expression levels of PCSK-9 proteins (P<0.01), and increased the expression levels of LDL-R proteins in the hyperlipidemic rats (P<0.01). These compounds did not significantly influence the expression levels of SREBP-2 and HMGCR proteins in the hyperlipidemic rats.

CONCLUSIONSHdber is effective in the treatment of hyperlipidemia in rats. The therapeutic mechanisms of Hdber may be associated with increasing the expression of LDL-R protein and decreasing the expression of PCSK-9 protein in liver tissues.

Animals ; Apolipoprotein A-I ; blood ; Apolipoproteins B ; blood ; Berberine ; analogs & derivatives ; pharmacology ; therapeutic use ; Hydroxymethylglutaryl CoA Reductases ; metabolism ; Hyperlipidemias ; blood ; drug therapy ; Lipids ; blood ; Liver ; drug effects ; metabolism ; Male ; Proprotein Convertase 9 ; Rats, Wistar ; Receptors, LDL ; metabolism ; Serine Endopeptidases ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism

BACKGROUNDNon-small cell lung cancer (NSCLC) is one of the most common malignant tumors. Despite the advances in therapy over the years, its mortality remains high. The aim of this study was to evaluate the expression of small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) in NSCLC tissues and its role in the regulation of vascular endothelial growth factor (VEGF) expression. We also investigated the association between the expression level of SENP1 and the clinicopathological features and survival of the patients.

METHODSA SENP1 small interfering RNA (siRNA) was constructed and transfected into the NSCLC cells. VEGF gene expression was analyzed by real-time polymerase chain reaction (RT-PCR). Immunohistochemistry staining was used to assess the expression of SENP1 in 100 NSCLC patients and its association with the clinicopathological features and survival was analyzed.

RESULTSVEGF expression was significantly higher in NSCLC tissues than in normal lung tissues. Inhibition of SENP1 by siRNA was associated with decreased VEGF expression. SENP1 was over-expressed in 55 of the 100 NSCLC samples (55%) and was associated with a moderate and low histological tumor grade (3.6%, 38.2%, and 58.2% in high, moderate and low differentiated tumors, respectively, P = 0.046), higher T stage (10.9% in T1, and 89.1% in T2 and T3 tumor samples, P < 0.001) and TNM stage (10.9% in stage I, and 89.1% in stages II and III tumor samples, P < 0.001). The rate of lymph node metastasis was significantly higher in the SENP1 over-expression group (76.4%) than that in the SENP1 low expression group (33.3%, P < 0.001). Sixty three patients received postoperative chemotherapy, including 34 with SENP1 over-expression and 29 with SENP1 low expression. Among the 34 patients with SENP1 over-expression, 22 (64.7%) patients developed recurrence or metastasis, significantly higher than those in the low expression group 27.6% (8/29) (P = 0.005). Multivariate Cox regression analysis showed that lymph node metastasis (P = 0.015), TNM stage (P = 0.001), and SENP1 expression level (P = 0.002) were independent prognostic factors for the survival of NSCLC patients.

CONCLUSIONSSENP1 may be a promising predictor of survival, a predictive factor of chemo-sensitivity for NSCLC patients, and potentially a desirable drug target for lung carcinoma target therapy.

Antineoplastic Agents ; therapeutic use ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; metabolism ; Cell Line, Tumor ; Cysteine Endopeptidases ; Endopeptidases ; genetics ; metabolism ; Female ; Humans ; Immunohistochemistry ; In Vitro Techniques ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; Male ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDElevated fibrinogen (Fg) level is a known risk factor for ischemic stroke. There are few clinical trials on oral fibrinogen-depleting therapies for secondary ischemic stroke prevention. We aimed to assess the effects of one-year therapy with oral lumbrokinase enteric-coated capsules on secondary ischemic stroke prevention.

METHODSThis is a multicenter, randomized, parallel group and controlled study that began treatment in hospitalized patients with ischemic stroke and continued for 12 months. Patients were randomized to either the control group that received the standard stroke treatment or the fibrinogen-depleting group that received the standard stroke treatment plus enteric-coated lumbrokinase capsules. The NIH Stroke Scale scores (NIHSSs) and plasma Fg level were recorded. The carotid artery intima-media thickness (IMT) and status of plaques were examined through carotid ultrasound examination. Primary outcomes included all-cause mortality, any event of recurrent ischemic stroke/transient ischemic attack (TIA), hemorrhagic stroke, myocardial infarction and angina, and other noncerebral ischemia or hemorrhage. Kaplan-Meier survival analysis and the Long-rank test were used to compare total vascular end point incidence between the two groups. Comparison of median values between two groups was done by the Student t test, one-way analysis of variance (ANOVA), or non-parametric rank sum test.

RESULTSA total of 310 patients were enrolled, 192 patients in the treatment group and 118 patients in the control group. Compared to the control group, the treatment group showed favorable outcomes in the Fg level, carotid IMT, the detection rate of vulnerable plaques, the volume of carotid plaques, NIHSS scores, and incidence of total vascular (6.78% and 2.08%, respectively) and cerebral vascular events (5.93% and 1.04%, respectively) (P < 0.05). In the treatment group, the volume of carotid plaques was significantly related to the carotid IMT, the plaque diameter, width and number (P = 0.000, 0.000, 0.000, 0.022; F = 13.51, 2.52, 11.33, -3.29, but there was a weak correlation with the Fg level (P = 0.056). After 1-year therapy, the incidence of overall vascular end points was reduced by 4.7%.

CONCLUSIONLong-term oral fibrinogen-depleting therapy may be beneficial for secondary ischemic stroke prevention.

Administration, Oral ; Aged ; Carotid Intima-Media Thickness ; Endopeptidases ; administration & dosage ; therapeutic use ; Female ; Fibrinogen ; metabolism ; Humans ; Male ; Middle Aged ; Secondary Prevention ; Stroke ; prevention & control

Alzheimer's disease (AD) is a complex neurodegenerative disorder which seriously causes the dementia in elderly people and afflicts millions of people worldwide. Drug discovery for Alzheimer's disease therapy has been a hot research area and a big challenge, in which development of acetylcholinesterase (AChE) inhibitors design was the most active and some AChE inhibitors are commercially available for AD medication already. However, practical using of commercial AChE inhibitors showed their limited usefulness and related adverse effects. Thus, it is extremely urgent to find novel AChE inhibitors with higher potency and less adverse effects. Based on the accurate crystallographic studies about AChE, strategies for multi-binding site AChE inhibitors have been formed, followed by design of the multi-target directed ligands. In this review, the structures and binding modes of commercial AChE inhibitors were briefly discussed, together with the development of AChE inhibitor design for AD therapy: from multi-binding site inhibitors to multi-target directed ligands.
Acetylcholinesterase ; chemistry ; metabolism ; Alzheimer Disease ; drug therapy ; Amyloid Precursor Protein Secretases ; antagonists & inhibitors ; Amyloid beta-Peptides ; metabolism ; Animals ; Aspartic Acid Endopeptidases ; antagonists & inhibitors ; Binding Sites ; Butyrylcholinesterase ; chemistry ; metabolism ; Cholinesterase Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; therapeutic use ; Drug Design ; Humans ; Ligands ; Monoamine Oxidase Inhibitors ; chemical synthesis ; chemistry ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; Structure-Activity Relationship

PURPOSE: The purpose of this study was to determine the pharmacogenetic effects of complement factor H (CFH) Y402H, LOC387715 and high-temperature requirement factor A1 (HTRA1) genotypes on the treatment of exudative age-related macular degeneration (AMD) by intravitreal bevacizumab injection in a Korean population. METHODS: Seventy-five patients diagnosed with exudative AMD were treated with intravitreal bevacizumab (2.5 mg) monotherapy. All patients received three initial intravitreal bevacizumab injections every four weeks and were then treated "as needed" based on clinical findings, optical coherence tomography and fluorescein angiography during the 12 month follow-up period after the third injection. RESULTS: The difference in visual acuity improvement among the three genotypes of LOC387715 were statistically significant at six months post-treatment (logarithm of the minimum angle of resolution; TT, 0.346; GT, 0.264; GG, 0.188; p = 0.037). Among the LOC387715 genotypes, the number of additional injections was lower in patients who had the risk T allele (GG, 2.143; GT, 2.000; TT, 1.575; p = 0.064). There was no significant difference between visual acuity and central macular thickness change in the CFH Y402H polymorphism group during the 12 month follow-up period. However, the TC group of CFH Y402H required more additional bevacizumab injections than the TT group (TT, 1.517; TC, 3.363; p = 0.020). CONCLUSIONS: This study demonstrated that different LOC387715/HTRA1 genotypes resulted in different bevacizumab treatment responses on exudative AMD. Patients with the risk allele had an improved treatment response and less need for additional injections. However, patients with the CFH Y402H risk allele needed more additional injections of bevacizumab in order to improve visual acuity. This study illustrates how pharmacogenetic factors may help determine treatment modality and dosing. This could ultimately provide basic data for 'personalized medicine' in AMD.
Aged ; Alleles ; Angiogenesis Inhibitors/administration & dosage/therapeutic use ; Antibodies, Monoclonal, Humanized/*administration & dosage/therapeutic use ; DNA/*genetics ; Female ; Follow-Up Studies ; Genotype ; Humans ; Intravitreal Injections ; Macular Degeneration/drug therapy/epidemiology/*genetics ; Male ; Pharmacogenetics/*methods ; *Polymorphism, Genetic ; Retrospective Studies ; Serine Endopeptidases/*genetics/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors ; Visual Acuity

OBJECTIVETo observe the effects of combination of Lumbrukinase Capsule (LC) and Probucol Tablet (PT) in treating cerebral infarction (CI) patients' unstable atheromatous plaque of the carotid artery.

METHODS150 patients were randomly assigned to the PT group and the LC group, 75 cases in each. Patients in the PT group took 0.5 g PT each time, twice daily. On the basis of PT, patients in the LC group also took 600 thousand U LC, thrice daily. The treatment course was 12 months for all. The serum levels of TC, TG, HDL-C, LDL-C, fibrinogen (FIB), and changes of the carotid atherosclerotic plaque were measured before treatment, 6 and 12 months after treatment. Meanwhile, adverse events were recorded.

RESULTSThe serum levels of TC, TG, and LDL-C were all lower 6 months after treatment than before treatment in the two groups, showing statistical significance (P < 0.05). The serum level of HDL-C was higher 6 months after treatment than before treatment in the two groups, showing no statistical significance (P > 0.05). When compared with before treatment in the same group, the serum level of FIB significantly decreased after treatment. Besides, there was statistical difference between the two groups (P < 0.05). There was no statistical difference in the serum levels of blood lipids or FIB between 12-month treatment and 6-month treatment in the same group (P > 0.05). The plaque effective rate in the LC group was superior to that of the PT group, showing statistical significance (P < 0.01). During the treatment period, the occurrence of cerebrovascular event was lower in the LC group than in the PT group (P < 0.05). Partial patients in the two groups had gastric discomfort.

CONCLUSIONSThe combination of LC and PT could prevent and treat arteriosclerosis, stabilize the plaque, effectively lower the occurrence of ischemic events. Its clinical application did not increase the risk of hemorrhage. It was safe and effective, worthy of spreading. It was necessary to further observe whether combination of LC and PT could increase side effects of the digestive tract.

Adult ; Aged ; Animals ; Biological Products ; therapeutic use ; Carotid Artery Diseases ; drug therapy ; Cerebral Infarction ; drug therapy ; pathology ; Female ; Humans ; Male ; Materia Medica ; therapeutic use ; Middle Aged ; Oligochaeta ; enzymology ; Plaque, Atherosclerotic ; drug therapy ; Probucol ; therapeutic use ; Serine Endopeptidases ; therapeutic use ; Treatment Outcome