1.Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system
LIU Xin-yi ; AN ni ; ZHANG Qing ; WANG Hong ; KONG Xiang-yu ; WANG Ming-yue ; PANG Li-li ; DUAN Zhao-jun
Chinese Journal of Biologicals 2023;36(2):145-150+157
Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.
2. Discussion on the Pathogenesis of Central Precocious Puberty
Na NI ; Xiang-Yang KONG ; Na NI ; Hen SU
Chinese Journal of Biochemistry and Molecular Biology 2021;37(6):727-732
Precocious puberty (PP) is a common childhood sexual dysplasia. Central precocious puberty (CPP) is a disease in which the hypothalamic-pituitary-gonadal (HPG) axis is activated in early age, leading to the early release of gonadotrophin releasing hormone, the early development of gonads, and the early onset of puberty. The occurrence of puberty is regulated by gene and environment. Current clinical studies have found that gain-of-function mutations in the KISS1 gene, and loss-of-function mutations in KISS1R (also named GPR54), MKRN3 and DLK1 genes are all important single-gene causes of central precocious puberty. KISS1 is a tumor metastasis suppressor gene. KISS1R codes a G protein-coupled receptor which with its ligand, kisspeptin, forms an excitatory neuroregulator system for GnRH secretion. They play a role in the upstream of the HPG axis. MKRN3 is a maternal-imprinted gene. DLK1 is a gene that regulates the growth of cell. They play a role in the downstream of HPG axis. Recently, the incidence of central precocious puberty has become higher and higher, which is related to the excessive exposure of environmental endocrine disruptors (EEDs) due to the continuous development of social economy. A number of investigations have found that children’s exposure to EEDs is significantly related to the incidence of precocious puberty. In humans, these EEDs also affect the metabolism of gut microbes. This paper aims to review the current studies on the single-gene pathogenesis, epigenetics, gut microbiota and environmental factors of central precocious puberty, so as to provide help for the treatment and prevention of this disease.
3.Network Pharmacology Study of Astragaloside Ⅳ in Treatment of Ischemic Stroke
Ming-zhu QI ; Ni-xue ZHANG ; Xiao-hui SU ; Xiang-ying KONG
Chinese Journal of Experimental Traditional Medical Formulae 2021;27(3):163-170
Objective:To study the mechanism of astragaloside Ⅳ in the treatment of ischemic stroke by means of network pharmacology. Method:The targets of astragaloside Ⅳ were predicted using Swiss Target Prediction platform, and the targets of ischemic stroke were retrieved using GeneCards, Therapeutic Target Database (TTD), Traditional Chinese Medicine Integrated Database (TCMID) and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) databases. The potential targets of astragaloside Ⅳ acting on ischemic stroke were obtained by the intersection of the targets of astragaloside Ⅳ and ischemic stroke. STRING platform was used to build protein-protein interaction (PPI) network, and eigenvalues were calculated through network topology analysis to screen core targets. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the related targets in DAVID database. Finally, molecular docking verification was conducted to further clarify the core targets of astragaloside Ⅳ acting on ischemic stroke. Result:The 44 common targets were obtained after the intersection of the targets of astragaloside Ⅳ and ischemic stroke. PPI network topology analysis showed that RAC-alpha serine/threonine-protein kinase (Akt1), renin (REN), epidermal growth factor receptor (EGFR), vascular endothlial growth factor A (VEGFA) and neuronal proto-oncogene tyrosine-protein kinase (SRC) were the core targets of astragaloside Ⅳ in the treatment of ischemic stroke. Enrichment analysis results of KEGG pathway showed that the pathways of astragaloside Ⅳ acting on ischemic stroke involved the neuroactive ligand-receptor interaction pathway, cGMP-PKG signaling pathway, calcium signaling pathway, Rap1 signaling pathway, PI3K/Akt signaling pathway, etc. Conclusion:Astragaloside Ⅳ may promote angiogenesis and inhibit platelet activity by acting on Akt1, REN, EGFR, VEGFA, SRC, thus improving cerebral blood flow. It can also inhibit the apoptosis of ischemic brain tissue cells and inflammation to reduce the damage of nerve function, and finally treat ischemic stroke. This study provides ideas and guidance for further exploring the mechanism of astragaloside Ⅳ in the treatment of ischemic stroke.
4.Research Progress of Role of Autophagy in Rheumatoid Arthritis and Traditional Chinese Medicine Intervention
Ni-xue ZHANG ; Xiao-hui SU ; Ya-ge TIAN ; Na LIN ; Xiang-ying KONG
Chinese Journal of Experimental Traditional Medical Formulae 2021;27(6):169-175
For rheumatoid arthritis, glucocorticoids or immunosuppressive agents are currently used in clinical treatment, but long-term use of these drugs has large side effect on humans, and immunosuppressive agents are expensive. To a certain extent, its wide application is limited. The treatment of rheumatoid arthritis with traditional Chinese medicine(TCM) has a long history and little toxic and side effect, but its specific mechanism of action needs further exploration. The process of autophagy is an active biological process in which cells themselves are stimulated by the outside world through intracellular signal transduction to maintain a stable internal environment. Its abnormality is involved in the occurrence of many diseases. At present, studies have shown that abnormal autophagy is closely related to the occurrence and development of rheumatoid arthritis, which can interfere with the pathological changes of RA pannus formation, synovial inflammation and bone destruction and affect the disease process. In recent years, many studies have found that traditional Chinese medicine and its active ingredients can affect the pathological development of rheumatoid arthritis by regulating autophagy. This article investigates the relevant literature on the intervention of rheumatoid arthritis by regulating autophagy through using TCM, with a view to providing new ideas for basic research, new drug development and clinical treatment of rheumatoid arthritis.
5.Application of tendon-derived stem cells and bone marrow-derived mesenchymal stem cells for tendon injury repair in rat model.
Xiangpeng KONG ; Ming NI ; Guoqiang ZHANG ; Wei CHAI ; Xiang LI ; Yucong LI ; Yan WANG
Journal of Zhejiang University. Medical sciences 2016;45(2):112-119
OBJECTIVETo evaluate the application of tendon-derived stem cells (TDSC) and bone marrow-derived mesenchymal stem cells (BMSC) for patellar tendon injury repair in rat model.
METHODSTDSCs and BMSCs were isolated from patellar tendons or bone marrow of healthy SD rats. The patellar tendon injury model was induced in 60 SD rats, then the animals were divided into 3 groups with 20 in each group: rats in TDSC group received transplantation of TDSC with fibrin glue in defected patellar tendon, rats in BMSC group received BMSC with fibrin glue for transplantation and those in control group received fibrin glue only. The gross morphology, histology and biomechanics of the patellar tendon were examined at 1, 2, 4, 6 and 8 weeks after the treatment.
RESULTSGross observation showed that the tendon defects in TDSC group and BMSC group almost disappeared in week 8, while the boundary of tendon defects in control group was still visible. Histology examination showed that the neo-tendon formation in TDSC group and BMSC group was observed at week 8, while there was no neo-tendon formation in control group. Biomechanics study showed that the ultimate stress and Young Modulus, relative ultimate stress and relative Young Modulus increased with the time going in all groups(all P<0.05); the ultimate stress and Young Modulus, relative ultimate stress and relative Young Modulus of TDSC and BMSC groups were significantly higher than those in control group at week 4, 6 and 8(all P<0.05). There was no difference in ultimate stress and Young Modulus between TDSC group and BMSC group(P>0.05), however, the relative Young Modulus of TDSC group was significantly higher than that in BMSC group at week 8(P<0.05).
CONCLUSIONAllogeneic TDSC and BMSC transplantation facilitates the repair of tendon injury and improves the biomechanics of tendon. TDSC is more suitable for in vivo tendon regeneration than BMSC.
Animals ; Bone Marrow ; Elastic Modulus ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Regeneration ; Tendon Injuries ; therapy ; Tendons ; cytology ; Wound Healing
6.Advances in taste masking technology based on preparation technology
Lei ZHAO ; Xiang-an-ni KONG ; Chang LI ; Chun-meng SUN ; Jia-sheng TU
Acta Pharmaceutica Sinica 2023;57(11):3185-3190
Good medicine tastes bitter, but it is often difficult to swallow because the drug is bitter and astringent, so that the compliance of patients with medication is poor. However, the use of taste masking technology can better improve this situation. Appropriate and effective taste masking technology can improve the drug compliance of patients, especially children, it can also improve the curative effect and the clinical value of drugs. Herein, we summarize the latest research progress of taste masking technology, and summarize the traditional taste masking technology from the aspects of action mechanisms and application scopes. Finally, the novel and efficient taste masking technologies were presented.
7.Preparation modification strategies for clinical treatment drugs of Parkinson's disease
Meng-jiao HE ; Yi-fang XIAO ; Xiang-an-ni KONG ; Zhi-hao LIU ; Xiao-guang WANG ; Hao FENG ; Jia-sheng TU ; Qian CHEN ; Chun-meng SUN
Acta Pharmaceutica Sinica 2024;58(3):574-580
Parkinson's disease (PD) is a chronic neurodegenerative disease. At present, levodopa and other drugs are mainly used for dopamine supplementation therapy. However, the absorption of levodopa in the gastrointestinal tract is unstable and its half-life is short, and long-term use of levodopa will lead to the end-of-dose deterioration, dyskinesia, the "ON-OFF" phenomenon and other symptoms. Therefore, new preparations need to be developed to improve drug efficacy, reduce side effects or improve compliance of patients. Based on the above clinical needs, this review briefly introduced the preparation modification strategies for the treatment of PD through case analysis, in order to provide references for the research and development of related preparations.
8.Analysis of Serum Metabonomics in Patients with Multiple Myeloma Based on Liquid Chromatography-Mass Spectrometry.
Xiao-Meng XU ; Xiang-Tu KONG ; Hui YU ; Xiao-Li CHEN ; Peng-Jun JIANG ; Hai-Wen NI
Journal of Experimental Hematology 2021;29(2):520-524
OBJECTIVE:
To observe the changes of serum metabolites in patients with multiple myeloma (MM) by metabonomics, and explore the potential biomarkers for diagnosis, prognosis, and progression of MM.
METHODS:
Serum samples were collected from 26 patients with MM and 50 healthy controls. The data detected by liquid chromatography-mass spectrometry (LC-MS) was input into SIMCA-14.0 software for multivariate statistical analysis. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze the changes of metabolites.
RESULTS:
The metabolic change of uric acid and trans-vaccenic acid in serum samples of MM patients was 9.39 times and 2.77 times of these in healthy people, respectively, which were significantly higher than those of healthy people, and the difference was statistically significant(P<0.01).
CONCLUSION
Uric acid and trans-vaccenic acid are expected to be important metabolic indicators for the diagnosis, prognosis, and efficacy evaluation of MM, thus providing some clues for the pathogenesis of MM.
Biomarkers
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Chromatography, Liquid
;
Discriminant Analysis
;
Humans
;
Mass Spectrometry
;
Metabolomics
;
Multiple Myeloma
9.Taste masking pharmaceutical excipients and their applications
Xiang-an-ni KONG ; Lei ZHAO ; Wen-zhen ZHAN ; Yu-xuan LI ; Chang LI ; Jia-sheng TU ; Chun-meng SUN
Acta Pharmaceutica Sinica 2023;57(11):3179-3184
The taste of drugs has an important impact on the compliance of patients, but most of the active drug ingredients have an uncomfortable taste, especially traditional Chinese medicine. Through a variety of pharmaceutical excipients with taste masking properties combined with corresponding technologies can improve the taste of drugs and the characteristics of other dosage forms, so as to improve patient compliance. Here, we mainly summarize the auxiliary materials used for taste masking, explain the mechanism of taste masking from the point of view of excipients and introduces related uses, so as to provide reference for further research on taste masking of pediatric preparations.
10.Effect of Celastrol Based on IRAK4/ERK/p38 Signaling Pathway on Proliferation and Apoptosis of Multiple Myeloma Cells.
Xiao-Meng XU ; Di KANG ; Xin-Yu ZHU ; Xiang-Tu KONG ; Hui YU ; Xiao-Li CHEN ; Peng-Jun JIANG ; Hai-Wen NI
Journal of Experimental Hematology 2022;30(1):175-182
OBJECTIVE:
To investigate the effect of celastrol on the proliferation and apoptosis of human multiple myeloma (MM) cell lines, reveal the relationship between IRAK4/ERK/p38 signaling pathway and celastrol regulating the proliferation and apoptosis of H929 and ARP-1 cells, and explore whether celastrol combined with bortezomib has synergistic effect.
METHODS:
CCK-8 method was used to detect the viability of MM cell lines H929 and ARP-1 treated by different concentrations of celastrol, bortezomib, and their combination, and the synergistic effect was determined by Kim's formula. The apoptosis rate of H929 cells and necrosis rate of ARP-1 were detected by Annexin V/PI method. The expression of key proteins and apoptosis proteins in IRAK4/ERK/p38 signaling pathway were detected by Western blot.
RESULTS:
Celastrol could significantly inhibit the proliferation of H929 and ARP-1 cells (r=0.9018, r=0.9244) and induce apoptosis in a time-dependent manner. Compared with the control group, celastrol could significantly up-regulate the expression of PARP and cleaved caspase-3 while down-regulate the expression of p-IRAK4, p-ERK, and p-p38 in H929 and ARP-1 cells. Celastrol and bortezomib alone inhibited the proliferation of H929 and ARP-1 cells. Compared with celastrol and bortezomib alone, their combination had lower cell survival rate and higher apoptosis rate (P<0.05).
CONCLUSION
Celastrol can inhibit the proliferation and promote the apoptosis of H929 and ARP-1 cells, which may be related to inhibiting the phosphorylation of IRAK4 and blocking the activation of IRAK4/ERK/p38 signaling pathway. Celastrol combined with bortezomib has synergistic effect, which can more effectively inhibit the proliferation and induce apoptosis of H929 and ARP-1 cells.
Apoptosis
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Bortezomib/pharmacology*
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Cell Line, Tumor
;
Cell Proliferation
;
Humans
;
Interleukin-1 Receptor-Associated Kinases
;
Multiple Myeloma
;
Pentacyclic Triterpenes
;
Signal Transduction