Use of Network Pharmacology and Experiment Validation to Uncover the Mechanism of Jianshen Lishui Prescription in the Treatment of Intracerebral Hemorrhage.

BACKGROUND: As a Chinese medicinal formula, the Jianshen Lishui prescription has been clinically proven to be effective in treating intracerebral hemorrhage (ICH). Yet, the mechanisms involved are unknown. METHODS: (1) Network pharmacology analysis: It involved the screening of active components in the Jianshen Lishui prescription, identification of potential targets for these components, and the screening of ICH-related targets. Common targets for both disease and drug were identified. Protein- protein interaction networks were constructed, followed by further screening of core targets. Gene Ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes( KEGG) pathway enrichment analysis were performed on these core targets. Finally, molecular docking verification was carried out using the active components and core targets. (2) Experimental verification: It was conducted using a rat model of intracerebral hemorrhage. This involved observing neurological deficit scores in the rats and measuring cerebral water content. The effects of Jianshen Lishui prescription on the neurological function, cerebral water content, and brain tissue core targets were observed through HE staining, Western blot and qPCR. RESULTS: (1) In this study, 29 common targets were obtained by intersecting 256 potential drug targets and 642 genes associated with ICH. 9 core targets were obtained by employing the protein- protein interaction (PPI) construction system to screen more specific targets. In addition, the findings revealed that the molecular mechanism of Jianshen Lishui prescription in treating ICH was mainly related to cancer signaling pathways and signal transduction pathways, based on the results of GO and KEGG enrichment analysis. Molecular docking results showed that the active constituent of Jianshen Lishui prescription mannitol has the highest binding activity with KRAS, luteolin, and Poria sterol with AR, INS1 and KRAS, cerebrosterol with GNB1, INS and ESR1, and sitosterol with AR, INS1 and KRAS. (2) Animal experiments verified that Jianshen Lishui prescription significantly alleviated encephaledema and improved nerve functions of the rat model of ICH. And INS1 expression levels were upregulated and the expression levels of AR, KRAS, PTGS2, and ESR1 were down-regulated by the prescription. CONCLUSION: Jianshen Lishui prescription protects the nerve function of ICH patients by inhibiting inflammation and reducing cerebral edema. This study provides more supportive evidences for the clinical use of traditional Chinese prescriptions in ICH treatment.

Moxibustion modulates working memory in patients with amnestic mild cognitive impairment: a functional magnetic resonance imaging study.

OBJECTIVE: To investigate the correlation between changes in brain activity associated with working memory and assessment scales of memory scores in amnestic mild cognitive impairment (aMCI) before and after moxibustion therapy. METHODS: aMCI patients were randomized into the moxibustion treatment (MT) group and the placebo moxibustion (PM) group. Each group received either moxibustion therapy or a placebo moxibustion for eight weeks. Neuropsychological performance and functional brain responses to a working memory task were assessed at baseline and at the end of treatment. Memory function was evaluated individually by the Rivermead behavioral memory test (RBMT), and working memory was assessed by the N-back task. RESULTS: Compared with the PM group, RBMT score changes were significant ( < 0.05). In the MT group, the accuracy of the N-back texts increased compared with those before the intervention. After moxibustion intervention, the right insula, postcentral gyrus, precentral gyrus, superior temporal gyrus, thalamus, lingual gyrus, calcarine sulcus, posterior cingulate gyrus, middle frontal gyrus and anterior frontal gyrus were significantly activated (= 0.01, Cluster-level Family-Wise Error = 0.05). Pearson correlation analysis showed that the insula, lingual gyrus and posterior cingulate gyrus were associated with changes in N-back score. Spearman correlation analysis showed that the postcentral gyrus, superior temporal gyrus, thalamus, lingual gyrus, and posterior cingulate gyrus were correlated with RBMT score changes. CONCLUSION: Moxibustion treatment improved memory in aMCI patients and was associated with the activation of the brain region of the insula, lingual gyrus, posterior cingulate gyrus, postcentral gyrus, superior temporal gyrus, and thalamus, which may be an important mechanism by which moxibustion improves the memory function.

Construction of a 10rolGLP-1-expressing glucose-lowing Saccharomyces cerevisiae by CRISPR/Cas9 technique / 生物工程学报

To develop a novel glucose-lowering biomedicine with potential benefits in the treatment of type 2 diabetes, we used the 10rolGLP-1 gene previously constructed in our laboratory and the CRISPR/Cas9 genome editing technique to create an engineered Saccharomyces cerevisiae strain. The gRNA expression vector pYES2-gRNA, the donor vector pNK1-L-PGK-10rolGLP-1-R and the Cas9 expression vector pGADT7-Cas9 were constructed and co-transformed into S. cerevisiae INVSc1 strain, with the PGK-10rolGLP-1 expressing unit specifically knocked in through homologous recombination. Finally, an S. cerevisiae strain highly expressing the 10rolGLP-1 with glucose-lowering activity was obtained. SDS-PAGE and Western blotting results confirmed that two recombinant strains of S. cerevisiae stably expressed the 10rolGLP-1 and exhibited the desired glucose-lowering property when orally administered to mice. Hypoglycemic experiment results showed that the recombinant hypoglycemic S. cerevisiae strain offered a highly hypoglycemic effect on the diabetic mouse model, and the blood glucose decline was adagio, which can avoid the dangerous consequences caused by rapid decline in blood glucose. Moreover, the body weight and other symptoms such as polyuria also improved significantly, indicating that the orally hypoglycemic S. cerevisiae strain that we constructed may develop into an effective, safe, economic, practical and ideal functional food for type 2 diabetes mellitus treatment.

Fetal exposure of Aristolochic Acid I undermines ovarian reserve by disturbing primordial folliculogenesis.

The primordial follicle pool established in early life determines the ovarian reserve in the female reproductive lifespan. Premature exhaustion of primordial follicles contributes to primary ovarian insufficiency (POI), that is dependent by the initial size of the primordial follicle pool and by the rate of its activation and depletion. AAI, a powerful nephrotoxin with carcinogenic potential, is present in the Aristolochiaceae species, which can release AAI into soil as a persistent pollutant. In order to assess the potential risk of Aristolochic Acid I (AAI) exposure on mammalian oogenesis, we uncovered its adverse effect on primordial folliculogenesis in the neonatal mouse ovary and its effect on female fertility in adulthood. Pregnant mice were orally administrated with doses of AAI without hepatic or renal toxicity during late-gestation. Ovaries from offspring of administered female displayed gross aberrations during primordial folliculogenesis. Also, unenclosed oocytes in germ-cell cysts showed increased DNA damage. Furthermore, several key factors, including NANOS3, SOX9, KLF4, that govern early gonad's differentiation were abnormally expressed in the exposed ovary, while the follicle formation was partially restored by knockdown of Nanos3 or sox9. In adulthood, these aberrations evolved into a significant reduction in offspring number and impaired ovarian reserve. Together, our results show that AAI influences primordial folliculogenesis and, importantly, affected female fertility. This study shows that administration of drugs herbs or consumption of vegetables that contain AAs during pregnancy may adversely influence the fertility of offspring.

Sedentary behavior among children and adolescents with intellectual disabilities in Jinan City / 中国学校卫生

Objective@#To understand the sedentary behavior level of children and adolescents with intellectual disabilities in Jinan City, and to provide a reference basis for developing health behavior intervention strategies.@*Methods@#By used the method of cluster random sampling,the Children s Leisure Activities Study Survey was used to investigate the sedentary behavior level of 285 children and adolescents with intellectual disabilities aged 6-18 years from 7 special education schools in Jinan City.@*Results@#The sedentary behavior time during the whole week among children and adolescents with intellectual disabilities in Jinan City was 394.46 min/d, including 378.00 min/d on weekdays(Monday to Friday) and 388.80 min/d on weekends (Saturday and Sunday), the difference was statistically significant ( Z =-2.19, P <0.05). 80.4%(229) of children and adolescents with intellectual disabilities had sedentary behavior time of more than 2 h/d. The sedentary behavior time per day during the whole week among children and adolescents with intellectual disabilities was negatively correlated with the amount of time spent in moderate vigorous physical activity among them ( r =-0.16, P <0.05).@*Conclusion@#Excessive sedentary behavior has become a growing public health concern among children and adolescents with intellectual disabilities,which warrants targeted healthy behavior intervention.

Evaluation of right ventricular-pulmonary artery coupling in patients with acute pulmonary embolism by the ratio of tricuspid annular plane systolic excursion and tricuspid regurgitation pressure gradient / 中华超声影像学杂志

Objective:To evaluate the feasibility of a new ultrasonic parameter to assess right ventricular-pulmonary artery (RV-PA) coupling in patients with acute pulmonary embolism (APE).Methods:A retrospective analysis was performed in 140 patients with APE diagnosed by computed tomography pulmonary angiography (CTPA) in the Second Affiliated Hospital of Harbin Medical University from August 2017 to June 2020. According to the tricuspid annular plane systolic excursion/pulmonary arterial systolic pressure (TAPSE/PASP) ratio cutoff value 0.40 mm/mmHg reported by the European Society of Cardiology in 2020, the patients were divided into the coupling group ( n=99) and the uncoupling group ( n=41). The conventional ultrasonic parameters of the 2 groups were measured, and then several ultrasonic parameter ratios were obtained. The new ultrasonic parameter, which can replace the TAPSE/PASP ratio, was screened out by Spearman correlation analysis, and ROC curve was plotted to calculate the diagnostic efficacy of this parameter. Results:①Compared with the coupling group, patients in the uncoupling group were older and more likely to be accompanied by dyspnea and venous thrombosis in the lower extremities (all P<0.05), but there was no significant difference in other general data(all P>0.05); ②Compared with the coupling group, tricuspid regurgitation velocity (TRV), tricuspid regurgitation pressure gradient(TRPG), PASP, right ventricle end-diastolic transverse diameter(RVTD), inferior vena cava(IVC) diameter and the ratio of early diastolic tricuspid inflow to tricuspid lateral annular velocity(E/e′), in the uncoupling group increased significantly (all P<0.05), and TAPSE, peak systolic velocity of tricuspid annulus(s′), TAPSE/PASP ratio, TAPSE/TRPG ratio, TAPSE/RVTD ratio and s′/TRPG ratio decreased significantly (all P<0.05); ③The TAPSE/TRPG ratio was highly correlated with TAPSE/PASP ratio ( rs=0.970, P<0.001); The TAPSE/TRPG ratio was still highly correlated with TAPSE/PASP ratio in the uncoupling and coupling groups ( rs=0.966, 0.922; all P<0.001). ④ROC analysis showed that the area under curve for TAPSE/TRPG in diagnosing RV-PA coupling was 0.992. At the cutoff of TAPSE/TRPG <0.625 mm/mmHg for indicating RV-PA coupling, the sensitivity and specificity were 97.6% and 92.9%, respectively. Conclusions:TAPSE/TRPG ratio can be used as a new ultrasonic parameter to reflect RV-PA coupling, which is helpful for clinical identification of APE patients with high risk and poor prognosis.

Effects of early applying basic fibroblast growth factor on corneal haze after surface ablation surgery and its mechanism / 中华实验眼科杂志

Objective:To investigate the effects of early applying of basic fibroblast growth factor (bFGF) on corneal haze formation after surface ablation surgery in rabbits.Methods:The right eyes of 60 healthy New Zealand white rabbits received photorefractive keratectomy (PRK) and were randomized into PRK+ normal saline group, PRK+ bFGF group and simple PRK group, with 20 rabbits in each group.Normal saline solution and bFGF were topically administered according to grouping, respectively, 3 times per day, 1 drop for each time until the sacrifice of the animals, and no drug was used in the PRK group.Another 8 normal rabbits were served as blank control group.The corneal healing response and haze formation were evaluated by anterior segment photography and anterior segment optical coherence tomography (AS-OCT) and graded based on Fantes criteria.Corneal histopathology was examined by hematoxylin-eosin staining.Immunohistochemistry was used to detect the expression of transforming growth factor-β 1(TGF-β 1), α-smooth muscle actin (α-SMA) and matrix metalloproteinase-2 (MMP-2) in cornea.This study protocol was approved by the Experimental Animal Ethic Committee of Affiliated Hospital of Binzhou Medical University (20180209-03). The use and care of the animals complied with the Statement of ARVO. Results:The corneal epithelium was completely healed in 3-4 days following surgery and there was not significantly different in healing time among the three groups.( F=0.57, P=0.57). The haze grading was significantly different among different groups at different time points ( Fgroup=41.736, P<0.01; Ftime=129.445, P<0.01) and showed the highest score in the PRK+ bFGF group on the 28th day after operation.On the 7th day after surgery, AS-OCT image showed that the surface reflection of corneal epithelium was continuous and smooth and corneal epithelium was not tightly attached to the superficial stromal layer; the reflection of the superficial stromal layer was enhanced in all the operation groups.The proliferation of corneal epithelial cells and superficial stromal layer in the operation area were seen under the optical microscope, and the arrangement of collagen fibers in the stromal layer was disordered with the most obvious changes in the PRK+ bFGF group in comparison with the PRK+ normal saline group and the simple PRK group, and these findings became worse on postoperative 28 days.The corneal epithelial surface reflection in the blank control group was continuous and smooth.Immunohistochemistry showed that a few MMP-2 positive cells were seen in the blank control group.TGF-β 1, α-SMA and MMP-2 proteins were positively expressed in the corneas 7 days after surgery in the three groups, and their expressions were the most obvious in the PRK+ bFGF in comparison with the PRK+ normal saline group and the PRK group and were enhanced 28 days after operation, showing statistically differences (all at P<0.05). Conclusions:Early application of bFGF following surface ablation surgery promotes the proliferation of corneal epithelial cells and irregular arrangement of collagen in the superficial stromal layer, which is associated with the expressions of haze-related factors TGF-β 1, α-SMA and MMP-2 in corneas.

Dvelopment of a DNA-based microarray for detection of human adenovirus serotypes / 军事医学

Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,quick and accurate detection of serotypes of human adenovirus (HAdV ),namely,HAdV3,HAdV7,HAdV11,HAdV14 and HAdV55.Methods Based on the specific gene sequences in the conserved region of adenovirus from GenBank, oligonucleotide primers and probes were designed and synthesized to prepare the oligonucleotide microarray.The specific genomic sequences were amplified by multiplex PCR method.The multiplex PCR amplification products were hybridized with the specific probes of microarray that was scanned after washing and chemiluminescenceb before the result was analyzed.After optimization of the multiple PCR system,hybridization reactions and conditions of chemiluminescence,the specificity,sensitivity and reproducibility of the chip were evaluated.Results The microarray displayed high sensitivity, specificity and reproducibility.The minimum detection limit of plasmidg DNA was 3 ×103 copies/reaction.The microarray detection results of 38 clinical samples were approximately consistent with those using the direct sequencing method(37 /38).Conclusion A chemiluminescence imaging DNA microarray method for quick,sensitive and specific detection of five serotypes of HAdV is established,which can provide a new means for detecting serotypes of HAdV.

Development of a DNA-based microarray for detection of nine pathogens causing rash and fever illness / 军事医学

Objective To develop a chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine rash-and fever-causing pathogens,namely,Measles virus,Rubella virus,Enterovirus type 71, Varicella zoster virus,Dengue fever virus,Human small FDNA virus B19,Coxsackie virus type A16,A-βStreptococcus pyogenes (hemolytic streptococcus)and Salmonella typhi.Methods Primers and probes were designed based on the specific sequence in the conserved region of genomes of the nine pathogens.The nucleic acids of the nine pathogens were amplified and labelled by multiplex PCR method.The multiplex PCR amplification products were hybridized with specific probes of microarray that was scanned after washing and chemiluminescence coloration to identify the nine pathogens.After the optimization of the multiplex PCR system,hybridization and chemiluminescence imaging,the specificity,sensitivity and reproducibility of the chip were evaluated.The serial diluted nucleic acid of Enterovirus type 71 was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Results Nine specific primers and eleven specific probes were selected.The microarray demonstrated high sensitivity and specificity.The minimum detection limit of plasmid DNA and in vitro transcribed RNAs was 3 ×103 copies per reaction.The detection sensitivity of this microarray was 10 percent of that by the real-time PCR method.The rate of sensitivity and specificity of clinical sample detection was 95% and 85.7% respectively,and the rate of accuracy was 93.2%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous,fast and accurate detection of nine pathogens that cause rash and fever illnesses is established successfully,which can serve as a new high throuthput screening method for clinical diagnosis and epidemiological investigation of rash and fever illnesses.

DNA microarray for simultaneous screening and detection of seven rickettsia / 军事医学

Objective To develop a chemiluminescence ( CL ) imaging DNA microarray method for simultaneous detection of seven rickettsiae.Methods Primers and probes were designed based on the specific sequence of seven rickettsia genomes.The probes were immobilized on the aldehyde modified glass surface to prepare DNA microarray for rickettsiae.The nucleic acids of the selected rickettsiae were amplified and labelled by multiplex PCR method, and then hybridized with microarray that was scanned after washing and chemiluminescence coloration, before the results were analyzed.Facilitated by the optimization of the multiplex PCR system, hybridization, and chemiluminescence imagination, we evaluated the specificity,sensitivity and reproducibility of the chip.The serial diluted nucleic acid of Rickettsia mooseri was detected using microarray and real-time PCR approach to compare the sensitivity of these two methods.Double-blind simulated samples were prepared to further evaluate the accuracy of the detection methods.Results One universal primer, four specific primers, one universal probe, and nine specific probes were selected.This DNA microarray demonstrated high specificity and sensitivity.The detection sensitivity of plasmid DNA and double-blind simulated sample DNA was 1.5 ×102 -3 ×103 copies per reaction and 103 -104 copies/μl.The detection results of real-time PCR method was consistent with the microarray, and the microarray possessed 10 fold lower detection sensitivities than the real-time PCR method.The coincidence rate of double-blind simulated sample detection was 100%.Conclusion A chemiluminescence imaging DNA microarray method for simultaneous detection of seven rickettsiae is established successfully,which can serve as a new high throuthput detection method for clinical diagnosis and epidemiological investigation of rickettsiae.

Gene chip technique in detection of vancomycin-resistant Enterococcus gene / 军事医学

Objective To develop a detection method based on the technology of gene chips which can quickly distinguish genes of Enterococcus faecalis, E.faecium and vancomycin resistance.Methods Based on the specific gene ( ddl) sequences of two types of Enterococcus from GenBank, oligonucleotide probes which could detect and distinguish special genes and drug resistance genes ( vanA,vanB) of Enterococcus were designed and compounded.Then,the probes were dotted to modified slide.The target DNA fragments of vancomycin-resistant Enterococcus ( VRE) were labeled with biotin by multiple PCR amplification, and then hybridized with oligonucleotide probes on slide.The results were analyzed by portable imager.The multiple PCR system, hybridization reaction and condition of the chemiluminescence method were optimized before the specificity, sensitivity and reproducibility of the chip were evaluated.Results One universal primer, four specific primers, one universal probe and four specific probes were selected.This gene chip was demonstrated of high specificity and repeatability.The detection sensitivity was 103 CFU/ml.The gene chip detection results of 10 clinical samples were basically consistent with the drug sensitivity test ( 8/10 ) .Conclusion A gene chip technique for the detection of VRE is established successfully.It is possible to distinguish the type of VRE and detect the genetic phenotypes of drug resistance by gene chip technique.