Objective To explore prescription medication rules and potential mechanism of traditional Chinese medicine(TCM)in the treatment of alcoholic liver disease(ALD)based on the technology of data mining and network pharmacology.Methods The prescriptions related to the treatment of ALD were retrieved in Chinese National Knowledge Infrastructure,Wanfang,Chinese Biomedical Literature and VIP databases.After the data were collated according to the filter criteria,IBM SPSS Statistics 27.0 and IBM SPSS Modeler 18 software were used to analyze the prescription rules and association rules.Then,the medication rules of TCM in the treatment of ALD were summarized,and the core drug combinations were obtained.Active ingredients in the core drug combinations for ALD and their targets were screened by network pharmacology.GO and KEGG analysis were performed on the main targets,and molecular docking technique was used to verify the binding ability of active ingredients to main targets.Results A total of 143 prescription for ALD were screened,involving 222 Chinese medicine,among which 28 high-frequency Chinese medicine were used with a frequency≥25 times.Eight core drug combinations were obtained by associations rule analysis.It has been found that there are 215 intersection targets between"Poria-Atractylodis macrocephalae Rhizoma-Hearba Artemisiae Scopariae"and ALD,including six core targets of AKT1,TNF,VEGFA,IL-1β,SRC,EGFR.One hundred and sixty-eight of signaling pathways are involved,including cancer pathways,PI3K/AKT signaling pathways,chemical carcinogenesis-reactive oxygen species,lipid and atherosclerosis,etc.Molecular docking results showed that the main active components including cerevisterol,genkwanin and demethoxycapillarisin had good binding ability to AKT1.Conclusion The main active ingredients in"Poria-Atractylodis macrocephalae Rhizoma-Hearba Artemisiae Scopariae"can participate in the regulation of key signaling pathways such as PI3K/AKT by acting on key target proteins(AKT1,TNF,and VEGFA).Subsequently,they play a role in inhibiting inflammatory response and apoptosis,slowing down liver fibrosis,and promoting hepatocyte repair.This study provides data support and theoretical guidance for the study of TCM in the treatment of ALD.

The paper reports a case of coenzyme Q10 deficiency nephrotic syndrome associated with coenzyme Q2 gene mutation and reviews the literature on this topic. The patient presented with hematuria, proteinuria, and a diminution of vision as clinical manifestations. But the proteinuria was not relieved after sufficient doses of glucocorticoids for over 2 months. The patient′s birth history was unremarkable, and his parents were both healthy and not consanguineous. Whole exome sequencing revealed that the patient had a mutation of coenzyme Q2 gene at c.973A>G(p.T325A) and c.517C>T(p.R173C). Combined with renal biopsy pathology, the patient was diagnosed with hereditary nephropathy and started the supplements of coenzyme Q10 after stopping glucocorticoid treatments immediately. After 5 weeks of therapy, the patient′s 24-h urine protein quantification decreased from 6.01 g to 1.53 g.

Objective:Through the investigation of the pathogenicity of COL4A4 heterozygous splicing mutations and the genotype-phenotype correlation in autosomal dominant Alport syndrome (ADAS), to better understand the impact of COL4A4 heterozygous splicing mutations on ADAS. Methods:The study was a case series analysis. Patients from 5 ADAS families with COL4A4 heterozygous splicing mutations detected by whole exome sequencing were recruited by three hospitals. In vivo transcriptional analysis and/or in vitro minigene splicing assay were conducted to determine the splicing patterns and assess the pathogenicity of COL4A4 heterozygous splicing mutations. Results:In the five ADAS pedigrees carrying COL4A4 heterozygous splicing mutations, four novel ADAS splicing patterns were described. In pedigree 1-4, most patients presented with continuous hematuria or/and microalbuminuria. Otherwise,the proband in pedigree 4 presented with macroalbuminuria and the proband in pedigree 1 had progressed to chronic kidney disease stage 2 at the age of 70 years old. In pedigree 5, all patients developed end-stage renal disease between 28 and 41 years old. c.735+3A>G detected in pedigree 1 and pedigree 2 and c.694-1G>C detected in pedigree 3 both led to exon 12 skipping in COL4A4, resulting in 42 nucleotides in-frame deletion (c.694_735del). c.2056+3A>G detected in pedigree 4 led to COL4A4 exon 26 skipping, which caused in-frame deletion of 69 nucleotides (c.1988_2056del). c.2716+5G>T detected in pedigree 5 led to a 360 nucleotides large in-frame deletion, including 100 bp sequence at the 3'end of exon 29,the whole sequence of exon 30 and 89 bp sequence at the 5'end of exon 31 (c.2446_2805del). Conclusions:Renal prognosis differs significantly for patients with small in-frame deletions versus large in-frame deletion splicing abnormalities. Determination of the pathogenicity and the splicing patterns of COL4A4 heterozygous splicing mutations using in vivo and in vitro transcriptional analysis may provide renal prognostic information.

Objective:To observe the effects of pyrroloquinoline quinine (PQQ) on the mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells (BMSCs) under oxidative stress, and to explore its mechanism.Methods:BMSCs of rats were cultured in vitro with Dulbecco′s minimum essential medium/F12 medium containing fetal bovine serum in the volume fraction of 10% (hereinafter referred to as normal medium). The rat BMSCs of third to fifth passages in logarithmic growth phase were selected for the following experiments. (1) The cells were divided into normal control group, normal control+ PQQ group, hydrogen peroxide (H 2O 2) alone group, and H 2O 2+ PQQ group. The cells in normal control group were cultured in normal medium for 24 hours; the cells in normal control+ PQQ group were cultured in normal medium containing 100 μmol/L PQQ for 24 hours; the cells in H 2O 2 alone group were cultured in normal medium containing 200 μmol/L H 2O 2 for 24 hours; the cells in H 2O 2+ PQQ group were pre-incubated with normal medium containing 100 μmol/L PQQ for 2 hours, and then with H 2O 2 added to the concentration of 200 μmol/L and cultured for 24 hours. The cell morphology of each group was observed under the inverted phase contrast microscope, and the cell survival rate was detected by cell count kit 8 method. (2) Five batches of cells were collected, and the cells of each batch were divided into normal control group, H 2O 2 alone group, and H 2O 2+ PQQ group. The cells in each group received the same treatment as that in the corresponding group of experiment (1). After 24 hours of culture, one batch of cells was collected for apoptosis detection by flow cytometry, and the apoptosis rate was calculated. One batch of cells was subjected to mitochondrial membrane potential assay and JC-1 fluorescent staining observation using the JC-1 mitochondrial membrane potential detection kit and the inverted phase contrast fluorescence microscope, respectively. One batch of cells was collected for mitochondrial morphology observation under the transmission electron microscope. One batch of cells was subjected to catalase (CAT) and superoxide dismutase (SOD) activity assay by CAT activity assay kit and SOD activity assay kit, respectively. One batch of cells was subjected to Western blotting for determination of protein level of Epac1, adenine monophosphate activated protein kinase (AMPK), phosphorylated AMPK, cysteinyl aspartate-specific proteinase 3 (caspase-3), and cleaved caspase-3, and the phosphorylation level of AMPK and cleaved caspase-3/caspase-3 ratio were calculated. Six replicates were measured in each group for each index except for morphological observation. Data were statistically analyzed with one-way analysis of variance and independent sample equal variance t test. Results:(1) After 24 hours of culture, compared with those in normal control group (the cell survival rate was set to 100.0%), there was an increase in cell vacuole and a decrease in cell number in H 2O 2 alone group, and the cell survival rate was significantly reduced to (74.3±2.9)% ( t=6.39, P<0.01). Compared with those in H 2O 2 alone group, the cell morphology of H 2O 2+ PQQ group was significantly improved, and the cell survival rate was significantly increased to (116.9±4.2)% ( t=6.92, P<0.01); the cell survival rate in normal control+ PQQ group was (101.2±1.1)%, close to that of control group ( t=1.06, P>0.05). (2) After 24 hours of culture, compared with (13.6±1.0)% in normal control group, the apoptosis rate of cells in H 2O 2 alone group was significantly increased to (37.1±2.0)% ( t=10.57, P<0.01). Compared with that in H 2O 2 alone group, the apoptosis rate of cells in H 2O 2+ PQQ group was significantly declined to (17.0±0.7)% ( t=9.49, P<0.01). (3) After 24 hours of culture, compared with those in normal control group, the mitochondrial membrane potential of cells in H 2O 2 alone group was depolarized, the JC-1 fluorescent dye mainly existed in the cytoplasm in the form of monomer, which emitted green fluorescence, and a significant decrease in mitochondrial membrane potential was shown ( t=4.18, P<0.01). Compared with those in H 2O 2 alone group, the mitochondrial membrane potential of cells in H 2O 2+ PQQ group was increased to normal level ( t=4.43, P<0.01), and the JC-1 fluorescent dye accumulated in mitochondria following the polarized mitochondrial membrane potential and emitted red fluorescence. (4) After 24 hours of culture, compared with that in normal control group, the mitochondrial structure of cells in H 2O 2 alone group was disordered, with disappeared mitochondrial cristae and decreased mitochondrial matrix density. Compared with that in H 2O 2 alone group, the mitochondrial structure of cells in H 2O 2+ PQQ group was regular and intact, with clearly visible mitochondrial cristae and increased mitochondrial matrix density. (5) After 24 hours of culture, compared with those in normal control group, the CAT activity of cells in H 2O 2 alone group was significantly increased ( t=4.54, P<0.05), and the SOD activity was significantly decreased ( t=3.93, P<0.05). Compared with those in H 2O 2 alone group, the CAT activity of cells in H 2O 2+ PQQ group was obviously increased ( t=8.65, P<0.01), while there was no significant change in the SOD activity ( t=0.72, P>0.05). (6) After 24 hours of culture, compared with those in normal control group, the protein expression of Epac1 of cells in H 2O 2 alone group was significantly decreased ( t=4.67, P<0.01), while the AMPK phosphorylation level and the cleaved caspase-3/caspase-3 ratio were significantly increased ( t=7.88, 3.62, P<0.01). Compared with those in H 2O 2 alone group, the protein expression of Epac1 and the AMPK phosphorylation level of cells in H 2O 2+ PQQ group were both significantly increased ( t=4.34, 16.37, P<0.01), while the cleaved caspase-3/caspase-3 ratio was significantly declined ( t=3.17, P<0.05). Conclusions:Pretreatment with PQQ can improve the mitochondrial function, reduce cell apoptosis rate, and enhance cell survival rate of rat BMSCs under oxidative stress, which may be related to the up-regulation of Epac1 protein expression, activation of AMPK signaling pathway, and down-regulation of cleaved caspase-3 protein level.

Objective: To investigate the effects of hypoxia on the phenotype transformation of human dermal fibroblasts to myofibroblasts and the mechanism. Methods: The third passage of healthy adult human dermal fibroblasts in logarithmic phase were cultured in DMEM medium containing 10% fetal bovine serum for the following five experiments. (1) In experiments 1, 2, and 3, cells were divided into normoxia group and hypoxia group according to the random number table, with 10 dishes in each group. Cells of normoxia group were cultured in incubator containing 21% oxygen, while those of hypoxia group with 1% oxygen. At post culture hour (PCH) 0 and 48, 5 dishes of cells were collected from each group, respectively. mRNA expressions of markers of myofibroblasts including alpha smooth muscle actin (α-SMA), type Ⅰ collagen, and type Ⅲ collagen of cells were determined with real time fluorescent quantitative reverse transcription polymerase chain reaction in experiment 1. Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting in experiment 2. The protein expression of nuclear factor-kappa B (NF-κB) of cells was determined with Western blotting in experiment 3. (2) In experiment 4, cells were divided into normoxia group, hypoxia group, and hypoxia+ pyrrolidine dithiocarbamate (PDTC) group according to the random number table, with 5 dishes in each group. Cells in the former two groups were treated the same as those in experiment 1. Cells in hypoxia+ PDTC group were treated the same as those in hypoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, the protein expression of NF-κB of cells was determined with Western blotting. (3) In experiment 5, cells were divided into normoxia group, hypoxia group, hypoxia+ PDTC group, and normoxia+ PDTC group according to the random number table, with 5 dishes in each group. Cells in the former three groups were treated the same as those in experiment 4. Cells in normoxia+ PDTC group were treated the same as those in normoxia group plus adding 4 mL PDTC with a final molarity of 10 μmol/L in the culture medium. At PCH 48, protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen of cells were determined with Western blotting. Data were processed with analysis of variance of factorial design, one-way analysis of variance, and LSD-t test. Results: (1) Compared with those of normoxia group at corresponding time point, mRNA expressions and protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen and the protein expression of NF-κB in fibroblasts of hypoxia group were not changed obviously at PCH 0 (with t values from -1.21 to 2.04, P values above 0.05), while mRNA expressions and protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen and the protein expression of NF-κB significantly increased at PCH 48 (with t values from -12.57 to -3.44, P values below 0.01). (2) At PCH 48, the protein expression of NF-κB in fibroblasts of hypoxia group was 0.83±0.12, significantly higher than that of normoxia group (0.17±0.06, t=-16.96, P<0.001). The protein expression of NF-κB in fibroblasts of hypoxia+ PDTC group was 0.31±0.08, significantly lower than that of hypoxia group (t=12.73, P<0.001). (3) At PCH 48, protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in fibroblasts of hypoxia group were 0.73±0.09, 1.25±0.10, and 1.16±0.07, respectively, significantly higher than those of normoxia group (0.14±0.06, 0.87±0.08, and 0.77±0.13, respectively, with t values from 9.24 to 11.24, P values below 0.001). The protein expression of α-SMA in fibroblasts of normoxia+ PDTC group was 0.24±0.07, significantly higher than that of normoxia group (t=4.22, P<0.01). Protein expressions of type Ⅰ collagen and type Ⅲ collagen in fibroblasts of normoxia+ PDTC group were 0.25±0.06 and 0.32±0.11, respectively, significantly lower than those of normoxia group (with t values respectively -4.31 and -3.88, P values below 0.01). Protein expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in fibroblasts of hypoxia+ PDTC group were 0.09±0.08, 0.38±0.12, and 0.47±0.08, respectively, significantly lower than those of hypoxia group (with t values from 11.78 to 22.98, P values below 0.001). Conclusions Hypoxia can significantly up-regulate the expressions of α-SMA, type Ⅰ collagen, and type Ⅲ collagen in human dermal fibroblasts, which may promote the phenotype transformation of fibroblasts to myofibroblasts, and this is likely to be associated with the activation of NF-κB signal pathway.

In vitro fertilization and embryo transfer (IVF- ET) technology is now widely used in the world in infertility patients. Psychological intervention can help the IVF- ET patients and their spouses reduce the negative emotions and improve pregnancy outcomes. This review provides a summary of the negative emotions in IVF- ET couples and the psychological intervention on them, so as to provide reference to establish a systematic and standardized clinical psychological nursing mode.

Objective To investigate the dynamic characteristics of the pacemaker current of canine sino-atrial node cells and compare them with the wild type mHCN2 pacemaker current overexpressed in neonatal rat myocardial cells.Methods Fresh canine sino-atrial node cells were enzymatically isolated in a calcium-free solution containing collagenase and elastase,and the funny current was recorded and compared with the mHCN2 current overexpressed in cultured neonatal rat myocardial cells under the same experimental conditions.Results The canine sinus node cells were elongated,spindle-shaped or polygonal,with well-defined boundaries,and showed spontaneous beating.The elicited pacemaker current was an inward current and its rise in amplitude quickened as the hyperpolarization potential increased.At V ＝-75 mV,the canine sinus atrial node pacemaker current was (-2.1±0.3) pA/pF and had the same activation kinetics as those of the mHCN2 channel current overexpressed in neonatal rat myocardial cells [τact:(728±137) ms vs.(530±65) ms,P＞0.05].Conclusions Within the physiological range,the pacemaker current in canine sino-atrial node cells and the wild type mHCN2 pacemaker current over expressed in neonatal rat myocardial cells have similar activation kinetics.

OBJECTIVETo discuss the authorized patent of compound traditional Chinese medicines with different efficacy in 2010, in order to provide reference for R&D of relevant compounds and patent protection.

METHODLiteratures for patents of compound traditional Chinese medicines were searched to screen relevant data and create a sample space. The samples were classified by hierarchical cluster procedures and iterative partitioning procedures using "authorized percentage" and "authorized time interval" as variable quantities. The comprehensive results generated by the two clustering methods were used to draw a conclusion.

RESULTThe samples were classified into four groups by clustering methods, each has significant difference in authorized patents' number and authorized time interval with others.

CONCLUSIONAmong compounds showing therapeutic advantage of traditional Chinese medicines, patents with short authorization period and in less number can be given most attention for patent application. Those with longer authorization period and in less number can be given more attention. While those with shorter authorization period and in large number can also be given attention for information guidance for traditional Chinese medicine science and technology and commercialization of patent achievements.

Chemistry, Pharmaceutical ; legislation & jurisprudence ; standards ; statistics & numerical data ; China ; Cluster Analysis ; Drug Therapy ; Drugs, Chinese Herbal ; chemistry ; standards ; therapeutic use ; Humans ; Medicine, Chinese Traditional ; standards ; Patents as Topic ; legislation & jurisprudence ; statistics & numerical data

OBJECTIVETo study current regional development and optimization schemes of patents of traditional Chinese medicine compounds.

METHODSimple statistics and cluster analysis were adopted for calculating application quantity, maintenance quantity and quantity of scientific research papers related to patents of traditional Chinese medicine compounds in different regions. On that basis, cluster analysis was used for studying current development patents of traditional Chinese medicine compounds in different regions.

RESULTThe 34 regions, including Chinese mainland, Hong Kong, Macau and Taiwan, were divided into four groups by cluster analysis according to the difference in quantities of patents and research papers. The first and third region are better, the second region is medium, while the forth region is not ideal.

CONCLUSIONDifferent regions shall adopt suitable development schemes for the development of traditional Chinese medicine compounds according to their actual situations. Reasonable regional alliance is helpful for inter-regional win-win and co-flourishing.

Chemistry, Pharmaceutical ; legislation & jurisprudence ; standards ; China ; Cluster Analysis ; Drug Therapy ; Drugs, Chinese Herbal ; chemistry ; standards ; therapeutic use ; Humans ; Medicine, Chinese Traditional ; standards ; Patents as Topic ; legislation & jurisprudence

Current patent protection of traditional Chinese medicine (TCM) compounds is far from being satisfactory with increasing research and development achievements. As patent protection of traditional Chinese medicine compounds is closely related with many fields such as research and development of new TCM drugs, industrial development and TCM internationalization, the development of research and harmonious development of TCM compounds and their patent protection is bound to have a far-reaching influence on domestic and even international societies.
Chemistry, Pharmaceutical ; economics ; legislation & jurisprudence ; China ; Drugs, Chinese Herbal ; chemistry ; economics ; Humans ; Medicine, Chinese Traditional ; economics ; Patents as Topic