Construction and expression of circular RNA in vitro / 中国生物制品学杂志

@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.

Construction of luciferase-expressing pancreatic cancer cell lines and evaluation of their application in detecting cytotoxicity efficacy of immune cells / 中华微生物学和免疫学杂志

Objective:To construct pancreatic cancer cell lines expressing luciferase and mesothelin (MSLN), and evaluate the feasibility of using them as target cells in analyzing the cytotoxicity activity of immune cells.Methods:Lentiviral vectors expressing luciferase and MSLN genes were constructed, and pancreatic cancer cell lines were infected after lentivirus packaging. Single-cell clones were obtained by limited dilution following antibiotic screening, and the stable expression of the target genes were verified. These cells were used as target cells to detect the cytotoxicity of immune cells by real-time cell analysis (RTCA) and luciferase activity. Besides, these luciferase-expressing cells were transplanted into B-NDG mice to establish the animal models of pancreatic cancer, and in vivo optical imaging technology was used to detect the expression of luciferase and monitor the tumor growth in mice. The cytotoxicity of chimeric antigen receptor T (CAR-T) cells was verified in these animal models. Results:Three pancreatic cancer cell lines, panc-1-luc, panc-1-luc-MSLN and capan-2-luc, that could stably express luciferase and MSLN genes were successfully constructed. The expression of the reporter gene in these cells were high, and positively correlated with the number of cells. There were 95.6% of panc-1-luc-MSLN cells expressing MSLN. MSLN-CAR-T cells had specific killing effect on MSLN-positive panc-1-luc-MSLN cells and capan-2-luc cells, with the minimum killing rates of (70.00±18.19)% and (57.00±5.29)%, respectively. But they had no cytotoxicity to MSLN-negative panc-1-luc cells. RTCA results showed that MSLN-CAR-T cells were able to lyse all three pancreatic cancer cell lines, and the minimum killing rates were (56.33±7.64)%, (93.00±2.65)% and (26.33±28.15)%, respectively. The killing of target cells by NK-92MI cells was not depended on MSLN expression. The cytotoxicity in the mice models of pancreatic cancer was consistent with the results in vitro. The in vivo and in vitro test results suggested that the expression of luciferase by target cells could reflect the cytotoxicity of immune cells. Conclusions:This study establishes three pancreatic cancer cell lines stably expressing luciferase, which can be used to evaluate the cytotoxicity of immunotherapy products targeting tumor cells in vitro and in vivo.

Establishment and verification of a drug screening system for cardiac fibrosis based on FAP gene promoter / 中国药理学与毒理学杂志

OBJECTIVE Based on fibroblast activation protein(FAP)gene promoter as the response element,to develop a new dual luciferase reporting system for the screening of drugs related to myocardial fibrosis.METHODS The promoter fragment of mouse FAP gene was synthesized in vitro and cloned into plasmid psiCHECK2 to replace HSV-TK promoter,and then a new recombinant plasmid psiCHECK2-FAP was obtained.After the recombinant plasmid psiCHECK2-FAP was digested by restriction endonucliase Hind Ⅲ,the product digested was identified by agar-gel electrophoresis and sequencing.After psiCHECK2-FAP was transient transfected into mouse cardiac fibroblasts(MCFs),and continued cultured for 24 h,and MCFs were treated with Ransforming growth factor β1(TGF-β1,5 μg·L-1)or angiotensinⅡ(Ang Ⅱ,1 μmol·L-1)or palmitic acid(PA,100 μmol·L-1)for 0,12,24,48 h,respectively,the double luciferase reporter gene assay was used to detect luciferase activity;After psiCHECK2-FAP was transient transfected into MCFs,the cells were pretreated with Dapa(1 μmol·L-1)for 1 h,and supplemented with TGF-β1(5 μg·L-1)or AngⅡ(1 μmol·L-1)or PA(100 μmol·L-1),continued treatment for 24 h,the double luciferase reporter gene assay was used to detect luciferase activity,and the expression of collagenⅠand collagen Ⅲ protein was detected with Western blotting.RESULTS The recombinant plasmid psiCHECK2-FAP was digested into two fragments by Hind Ⅲ with the expected strip size,and the sequencing results were completely consistent with the theoretical sequence;Compared with control group,the collagenⅠand collagen Ⅲ protein expression were significantly increased by TGF-β1 or Ang Ⅱ or PA in MCFs(P<0.05,P<0.01).However,compared with TGF-β1 or Ang Ⅱ or PA group,the intervention of Dapa significantly alleviated the promoter activity of FAP gene and the expression of collagenⅠand collagen Ⅲ protein(P<0.05,P<0.01);Compared with control group,luciferase activity was significantly increased by TGF-β1 or Ang Ⅱ or PA(P<0.05,P<0.01),reaching the peak at 24 h.Compared with TGF-β1 or AngⅡ or PA group,the intervention of Dapa significantly decreased luciferase activity(P<0.05,P<0.01).CONCLUSSION Based on the promoter of FAP gene as the response element,a noval dual luciferase reporter gene system was established and showed a good sensitivity to the promyocardial fibrosis factor in MCFs,which can provide strategies for the development of antimyocar-dial fibrosis drugs.

Prognostic significance of microRNA-20a-5p levels which promotes proliferation and invasion by targeting cyclin G2 in small cell lung cancer

MicroRNA-20a-5p (miR-20a-5p) has been shown to function as a tumor promoter factor in several cancers. However, its role in small cell lung cancer (SCLC) remains unclear. In this study, we have made an attempt to measure the tumor tissue levels of miR-20a-5p in patients with SCLC using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). The biological function of miR-20a-5p in SCLC cells was investigated in vitro and in vivo studies, including cell proliferation, migration assays and tumorigenicity in nude mice. Meanwhile?we conducted the luciferase reporter assay to verify the biological relationship between miR-20a-5p and CCNG2. The expression of miR-20a-5p was significantly upregulated in human SCLC compared to that in normal tissues. Kaplan-Meier analysis indicated that patients with high expression of miR-20a-5p are closely related with the shorter survival of SCLC. Further, multivariate analysis showed that miR-20a-5p was an independent prognostic factor. Increasing miR-20a-5p expression promotes the proliferation, migration and invasion of the NCI-H446 cells in vitro and in vivo. Dual-luciferase reporter gene assay demonstrated that miR-20a-5p directly targets CCNG2. These findings suggest that miR-20a-5p levels might be a novel diagnostic and prognostic marker of SCLC. Inhibiting miR-20a-5p could be a promising therapeutic strategy for SCLC.

Development and optimization of a cell screening system for farnesoid X receptor agonist / 生物工程学报

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

Advance on detection method and application based on bioluminescence of luciferase / 中国药科大学学报

@#Bioluminescence is a widespread phenomenon in nature, and luminescent organisms can be found both on land and in the ocean. Among them, luciferase based bioluminescence systems have been widely studied, inspiring the exploration of genetic and epigenetic aspects and the development of a series of related assays for in vivo and in vitro studies. This paper summarizes the recent developments of luciferase based bioluminescence assays in terms of bioluminescence systems, types of luciferases, and the development and application of luciferase bioluminescence assays.

Construction of recombinant influenza virus expressing Gaussia luciferase and analysis of its in vitro biological characteristics / 中华微生物学和免疫学杂志

Objective:To construct recombinant influenza viruses expressing Gaussia luciferase (Gluc) with different influenza virus backbones and analyze their growth characteristics, genetic stability, ability to express Gluc and in vitro anti-influenza drug activity. Methods:The C-terminal of PR8NA was modified by inserting the porcine teschovirus-2A autocleavage peptide (P2A) and the Gluc-coding gene. Recombinant viruses, PR8NAGluc/PR8 and PR8NAGluc/WSN, were rescued using the eight-plasmid system of influenza virus reverse genetics, with seven plasmids derived from A/Puerto Rico/8/34(PR8) (H1N1) and A/WSN/1933 (WSN) H1N1. The genetic stability of the recombinant viruses was verified by RT-PCR. The fluorescence activity and the growth kinetics of the two recombinant viruses were compared. The correlation between the fluorescence activity of PR8NAGluc/WSN and median tissue culture infective dose (TCID 50), and the anti-drug activity of PR8NAGluc/WSN against oseltamivir, favipiravir, and Lianhua Qingwen in vitro were also analyzed. Results:The Gluc-expressing recombinant viruses constructed using PR8 and WSN backbones were successfully rescued by reverse genetics. Compared with the PR8 backbone, the WSN backbone significantly improved the fluorescence activity of Gluc. Moreover, the PR8NAGluc/WSN virus expressed stably in embryonated egg, and its replication kinetics was slightly lower than that of wild type. The fluorescence activity of PR8NAGluc/WSN virus had a good correlation with its TCID 50. The PR8NAGluc/WSN virus was sensitive to oseltamivir, favipiravir and Lianhua Qingwen. Conclusions:The recombinant virus with a WSN backbone exhibited higher fluorescence expression intensity as compared with the recombinant virus with a PR8 backbone. This study provided reference for high-throughput screening of anti-influenza drugs and the development of influenza virus vector vaccines.

Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer / 中山大学学报(医学科学版)

ObjectiveTo construct a human ovarian cancer cell line SKOV3 （SK-Luc-EGFP） stably co-expressing luciferase （Luc） and enhanced green fluorescent protein （EGFP） and to explore its application in ovarian cancer research both in vitro and in vivo. MethodsThe recombinant plasmid pCDH-Luc-T2A-EGFP-Puro was constructed by introducing a Luc-T2A-EGFP fusion gene fragment amplified by Overlap PCR into plasmid vector. The three-plasmid lentivirus packaging system was transfected into HEK 293T cells and the viral supernatant was harvested to infect SKOV3 cells. SK-Luc-EGFP cell line with the highest fluorescence intensity of EGFP was obtained by puromycin selection and flow cytometry assessment， and the Luc expression of the cell line was subsequently validated by in vitro bioluminescent assay. SK-Luc-EGFP cells were further explored for the following applications： distinguishing SK-Luc-EGFP cells from non-tumor cells in ascites by flow cytometry and confocal microscopy； visualizing adhesion of SK-Luc-EGFP cells to mesothelial cells or omentum by fluorescence microscopy； monitoring process of SK-Luc-EGFP tumorigenesis by in vivo bioluminescence imaging. ResultsA recombinant lentiviral expression plasmid pCDH-Luc-T2A-EGFP-Puro was constructed and packaged into lentiviral particles that were then transfected into SKOV3 cells to generate SK-Luc-EGFP cell line. The purity of SK-Luc-EGFP cells based on EGFP expression was 100% as validated by fluorescence microscopy and flow cytometry； SK-Luc-EGFP cells could be visually distinguished from non-tumor cells in ascitic fluid by flow cytometry and confocal imaging. Moreover， Luc expression in SK-Luc-EGFP cells was verified by in vitro bioluminescence assay， and a linear relationship with a correlation coefficient of 0.997 9 was found between cell number and the bioluminescent signal. Adhesion of SK-Luc-EGFP cells to mesothelial cells was directly observed by fluorescence imaging in in vitro adhesion assay； peritoneal adhesion of SK-Luc-EGFP cells to omentum was also observed after intraperitoneal （i.p.） injection of SK-Luc-EGFP cells in nude mice； in the peritoneal metastasis mouse model established by i.p. injection of SK-Luc-EGFP cells， monitoring of tumorigenesis process was achieved by in vivo bioluminescence imaging. ConclusionSK-Luc-EGFP cell line is a useful tool for investigating ovarian cancer in vitro and in vivo.

Baicalin modulates let-7i-3p/PI3K/Akt/NF-KB signaling axis to reduce NLRP3 inflammasome activation in fibroblast like synoviocytes of rheumatoid arthritis / 中国药理学通报

Aim To study the effect of baicalin on the activation of NLRP3 inflammasomes in human fibroblast like synoviocytes of rheumatoid arthritis ( HFLS-RA) and its mechanism. Methods To confirm that baicalin alleviated the activation of NLRP3 inflammasome in HFLS-RA, immunofluorescence was used to observe the expression of NLRP3 before and after baicalin treatment. Western blot was used to detect the protein expression of p-PI3K, p-Akt, NF-κB p65, NL-RP3, ASC and caspase-1 after baicalin treatment for 48 h, and ELISA was employed to detect the contents of IL-1 and IL-18 in the supernatents. In order to explore the mechanism of baicalin alleviating the activation of NLRP3 inflammasome, double luciferin and Westen blot analysis were applied to verify the corresponding relationship between let-7i-3p and PIK3CA. RT-qPCR was utilized to determine the expression of let-7i-3p and PI3K before and after baicalin intervention. let-7i-3p interference was used to verify whether baicalin mitigated the activation of enhanced NLRP3 inflammasomes. Results Baicalin (50, 100 mg · L

A cell model for high-throughput screening GLP-1 receptor agonists / 中国药理学通报

Aim To establish a high-throughput screening cell model for GLP-1 receptor agonists. Methods A pEGFP-GLP-1R-3 C recombinant plasmid was constructed and transfected into HEK293T cells. The cells were screened with G418 and flow cytometry. The established stable cell line was named HEK293TGLP-lR-3C-eGFP cell line. The expression level of GLP-1 R-3C-eGFP protein was confirmed by Western blotting and laser confocal microscopy. Then cyclic adenosine monophosphate (cAMP) response element reporter gene was transfected into the HEK293T-GLP-lR-3C-eGFP cells. The luminescence values were detected by One-Step Luciferase Reporter Gene Assay Kit after stimulation with different concentrations of GLP-1 peptide. The luminescence values reflected the cellular cAMP level, which was verified using the cAMP kit (E L I S A). Results HEK293T-GLP-lR-3C-eGFP cell line was successfully constructed. The relative light unit change trend after stimulation with different concentrations of GLP-1 was similar to that of the cellular cAMP level change trend. The value of Z' in this experiment was 0.52. Conclusions A recombinant HEK293T cell line is established, which can be used for high-throughput screening of GLP-1 receptor agonists.

Reporter gene assay for detection of biological activity of human growth hormone / 中国生物制品学杂志

@#Objective To develop and verify a reporter gene assay(RGA) for the detection of biological activity of human growth hormone(hGH).Methods The biological activity of hGH was evaluated by the expression of luciferase(Luc)activated by hGH binding to hGH receptor(hGHR) on HEK293/GH-Luc cell membrane.The developed detection conditions were as follows:the initial concentration of sample was 1 μg/mL;the cell inoculation amount was(2.45～2.66) × 10~4 cells/well;the sample was of 3-fold serial dilution,with a total of 8 dilutions and the incubation time was 18～24 h.The relative biological activity of the sample was calculated by measuring Luc intensity and comparing it with the national standard by four-parameter fitting.The developed method was verified for specificity,repeatability,intermediate precision,relative accuracy,linear range and durability.Results The excipient components in product and the serum components in culture medium showed no effect on the activity detection results;The geometric coefficient of variation(GCV) of relative titer of one batch of samples in six repeated detections was 6.794%,much lower than 20%.The relative titer GCV detected by two experimenters in different batches of samples at different times were both lower than 20%;The relative deviations of the relative titer determination values of samples at different concentrations were within ±12%,the slope of linear regression equation was 0.982,the linear range was 0.6～1.6 μg/mL,and the coefficient of determination(R~2) was 0.997;The GCV of three batches of stock solutions and one batch of finished products were 4.758%,4.430%,7.294% and 2.771% respectively under the conditions of different cell generation,cell density and sampling location,all of which were less than 20%.Conclusion The developed RGA showed good specificity,repeatability,intermediate precision,relative accuracy,linear range and durability,which met the application requirements and was expected to replace the traditional in vivo biological activity detection methods for the activity evaluation and quality control of hGH.

Evaluation of stability of Luc2P reporter cell lines by duplex digital PCR / 中国生物制品学杂志

@#Objective To develop a duplex digital PCR(dPCR) for evaluation of the stability of luciferase(Luc) gene in Luc2P reporter cell lines(CHO-K1,Hacat,HEK293 and UT7).Methods Genomic DNAs of Luc2P reporter cell lines were extracted,a duplex dPCR was developed to determine the copies of reference gene RPP30 and the target gene Luc,and the relative copy number of Luc(copies Luc/copy RPP30) was employed as the indicator for the stability evaluation of Luc gene;The developed method was verified for the specificity,precision,linearity,accuracy and durability,and analyzed for the applicability,according to the related requirements in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).Results All the original cells without reporter gene transfection were negative,while all the four reporter cell lines were positive,and the negative and positive regions in dPCR results were clearly distinguished;The relative standard deviation(RSD) of the eight repeated detections of the same genomic DNA sample and the six independent extractions of genomic DNA sample of the same cell were all less than 10%,and the linear fitting R~2 values were more than 0.99 for both Luc and RPP30.The spike recoveries of five groups of samples detected by the developed method were between 50% and 100%,and the results of chip-type dPCR and droplet-type dPCR were consistent.This method distinguished the relative copy number of Luc in different cell clones,and the results of detecting the relative copy number of Luc in three passages(P8,P12 and P31) were highly consistent.Conclusion The developed duplex dPCR method has good specificity,precision,linearity,accuracy,durability and applicability,and might be used to evaluate the stability of Luc gene in Luc2P reporter cell lines.

miR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene / 生物工程学报

This study aimed to explore the effect of miR-23b-3p on the differentiation of goat intramuscular preadipocytes, and to confirm whether miR-23b-3p plays its roles via targeting the PDE4B gene. Based on the pre-transcriptome sequencing data obtained previously, the miR-23b-3p, which was differentially expressed in goat intramuscular adipocytes before and after differentiation, was used as an entry point. real-time quantitative-polymerase chain reaction (qPCR) was used to detect the expression pattern of miR-23b-3p during the differentiation of goat intramuscular preadipocytes. The effects of miR-23b-3p on adipose differentiation and adipose differentiation marker genes were determined at the morphological and molecular levels. The downstream target genes of miR-23b-3p were determined using bioinformatics prediction as well as dual luciferase reporter assay to clarify the targeting relationship between miR-23b-3p and the predicted target genes. The results indicated that overexpression of miR-23b-3p reduced lipid droplet accumulation in goat intramuscular adipocytes, significantly down-regulated the expression levels of adipogenic marker genes AP2, C/EBPα, FASN, and LPL (P < 0.01). In addition, the expressions of C/EBPβ, DGAT2, GLUT4 and PPARγ were significantly downregulated (P < 0.05). After interfering with the expression of miR-23b-3p, lipid droplet accumulation was increased in goat intramuscular adipocytes. The expression levels of ACC, ATGL, AP2, DGAT2, GLUT4, FASN and SREBP1 were extremely significantly up-regulated (P < 0.01), and the expression levels of C/EBPβ, LPL and PPARγ were significantly up-regulated (P < 0.05). It was predicted that PDE4B might be a target gene of miR-23b-3p. The mRNA expression level of PDE4B was significantly decreased after overexpression of miR-23b-3p (P < 0.01), and the interference with miR-23b-3p significantly increased the mRNA level of PDE4B (P < 0.05). The dual luciferase reporter assay indicated that miR-23b-3p had a targeting relationship with PDE4B gene. MiR-23b-3p regulates the differentiation of goat intramuscular preadipocytes by targeting the PDE4B gene.

Establishment of mouse models of lymphoma with dual-labeled EBV-immortalized lymphoblastoid cell lines by intravenous versus subcutaneous injection / 中国肿瘤临床

Objective:To establish a green fluorescent protein(GFP)and firefly luciferase(Luc)double-labeled Epstein-Barr virus(EBV)infec-ted B lymphoblastoid cell lines(B-LCL)and apply them to mouse models,then compare the advantages and disadvantages of models inocu-lated by intravenous(IV)or subcutaneous(SC).Methods:B lymphoblastoid cell lines double-tagged with GFP/Luc(B-LCL-GL)were con-structed through lentivirus transduction,puromycin intervention.Subcutaneous xenograft and hematogenous metastasis models were re-spectively established by subcutaneous or intravenous injection of B-LCL-GL cells at three concentrations in(NOD)/Prkdcscid/IL-2Rγnull(NPG)mice for in vivo bioluminescence imaging.Results:In the B-LCL-GL group,the ratio of the GFP-positive cell population was 92.5%,and the average luminescence intensity was as high as 4.80E+08 Photons/s,which was considerably higher than that of untreated B-LCLs.In the hematogenous metastasis models,tumor bioluminescence was initially located in the peritoneal area and then spread throughout the en-tire body between 7 and 28 days.In the subcutaneous xenograft models,strong central and weak peripheral tumor-related biolumines-cence signal was detected on day 7 in the three groups,which then spread throughout the body on day 28 in the high-dose group.Taken to-gether,there was no significant difference in tumor progression between the two routes of administration when using the same dose of B-LCL-GL cells.However,the survival analysis indicated that the IV injection group,in which all the mice ultimately died,had a shorter time frame for testing than that of the SC injection group,in which the mice survived until day 100 in the low-dose and medium-dose groups,thus allowing for long-term testing.Conclusions:GFP and Luc dual-positive B-LCLs were successfully established to generate hematogenous metastasis and subcutaneous xenograft models,which allow the monitoring of the location and size of lymphomas in vivo.It provide plat-form for the study of tumor characteristics and selecting anti-tumor drugs.

Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays

SARS-CoV-2 main protease (Mpro) is one of the most extensively exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based FlipGFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery.

Activating Effect of Effective Components in Medicinal and Edible Substances on Human Pregnane X Receptor and Cytotoxicity Screening / 中国实验方剂学杂志

ObjectiveTo explore the activating effects of ten important effective components in seven medicinal and edible substances on human pregnane X receptor （PXR）， including Glycyrrhizae Radix et Rhizoma （liquiritin and glycyrrhizic acid）， Houttuyniae Herba （quercetin and houttuyfonate）， Prunellae Spica （rosmarinic acid）， Cassiae Semen （aurantio-obtusin）， Poria （pachymic acid）， Lilii Bulbus （Lilium brownii saponin and colchicine）， and Lycii Fructus （Lycium barbarum polysaccharide） and screen potentially toxic components. MethodCell counting kit-8 （CCK-8） assay was used to investigate the cytotoxic effect of liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， pachymic acid， aurantio-obtusin， and colchicine （10， 20， and 50 μmol·L-1）， and L. brownii saponin and L. barbarum polysaccharide （10， 20， and 50 mg·L-1） on normal human hepatocyte cell line （L02）. The release of lactate dehydrogenase （LDH） in L02 cells after drug treatments was detected by the biochemical analyzer. The apoptosis induced by ten effective components was explored by Hoechst 33342 staining. The secreted luciferase reporter system was used to co-transfect the PXR expression vector and reporter gene vector containing cytochrome P450 3A4 （CYP3A4） transcriptional regulatory region into L02 cells， with 10 μmol·L-1 rifampicin （RIF） as a positive control. After treated with liquiritin， glycyrrhizic acid， quercetin， houttuyfonate， rosmarinic acid， aurantio-obtusin， pachymic acid， and colchicine （5， 10， and 20 μmol·L-1） and L. brownii saponin and L. barbarum polysaccharide （5， 10， and 20 mg·L-1） for 24 h， the cells were tested for secreted luciferase activity. ResultCompared with the control group， colchicine， L. brownii saponin， and quercetin decreased the cell viability （P<0.05， P<0.01）. Compared with the control group， quercetin， rosmarinic acid， glycyrrhizic acid， colchicine， aurantio-obtusin， and pachymic acid increased the release rate of LDH in L02 cells （P<0.05， P<0.01）. The proportion of hyperchromatic nuclei increased gradually after rosmarinic acid， liquiritin， and L. barbarum polysaccharide treatments as compared with the control group （P<0.05， P<0.01）. In terms of co-transfection of pcDNA3.1-PXR and pGLuc-CYP3A4 into L02 cells， compared with the control group， aurantio-obtusin and pachymic acid showed activating effects on PXR （P<0.05）， whereas liquiritin and glycyrrhizic acid showed inhibitory effects （P<0.05）. ConclusionThe findings suggest that when medicinal and edible substances are taken for a long time， attention should be paid to their influence on drug-metabolizing enzymes and possible interactions， so as to improve their safety.

Mechanism of miR-381 on macrophage infiltration in polymyositis by targeting stromal cell derived actor-1 / 解剖学报

Objective To explore the mechanism of miR-381 on the infiltration of polymyositis (PM) macrophages by targeting stromal cell derived factor-1 (SDF-1). Methods PM model mouse was constructed by rabbit myosin (1.5 mg), mycobacterium tuberculosis (5 mg) and pertussis toxin (500 ng). The 30 PM model mice were divided into control group and PM+miR-381 group (n = 15/group). During the same period, 15 healthy mice were used as a control group. Mice in the PM+miR-381 group were injected with miR-381 agomir (300 μg) intraperitoneally for 2 weeks. Serum creatine kinase (s-CK), interleukin (IL)-1β and IL-6 levels in serum of each group of mice, and the pathological changes of muscle tissue were detected and compared. The macrophage marker protein F4/80 was detected by immunohistochemical staining to assess the infiltration of macrophages. The expression levels of miR-381 and SDF-1 mRNA and protein in muscle tissues of each group were detected. The target relationship between miR-381 and SDF-1 was verified by dual luciferase report. Mouse macrophages were divided into miR-381 NC group and miR-381 mimic group. The SDF-1 mRNA and protein levels in each group were detected by Real-time PCR and Western blotting. Transwell was used to detect the level of cell migration to evaluate the infiltration capacity. Results The above indicators of the three groups were significantly different (P<0.05). The level of miR-381 in the muscle tissue of the PM group was significantly lower than that of the control group, s-CK, IL-1β, IL-6, histological score, macrophage infiltration, and SDF-1 mRNA and protein expression levels were significantly higher than those of the control group (P<0.05). The level of miR-381 in the muscle tissue of the PM+ miR-381 group was significantly higher than that of the PM group, s-CK, IL-1β, IL-6, histological score, macrophage infiltration, and SDF-1 mRNA and protein expression levels were significantly lower than those in the PM group (P<0.05). The dual luciferase report result indicated that miR-381 could target binding to SDF-1. The expression levels of SDF-1 mRNA and protein in macrophages in the miR-381 mimic group were significantly lower than those in the miR-381 NC group (P<0.05). The number of migrating cells in the miR-381 mimic group was significantly lower than that in the miR-381 NC group (P<0.05). Conclusion Increasing the level of miR-381 can inhibit the inflammatory infiltration ability of macrophages by targeting the expression of SDF-1, thereby alleviating PM.

A small molecule UPR modulator for diabetes identified by high throughput screening

Unfolded protein response (UPR) is a stress response that is specific to the endoplasmic reticulum (ER). UPR is activated upon accumulation of unfolded (or misfolded) proteins in the ER's lumen to restore protein folding capacity by increasing the synthesis of chaperones. In addition, UPR also enhances degradation of unfolded proteins and reduces global protein synthesis to alleviate additional accumulation of unfolded proteins in the ER. Herein, we describe a cell-based ultra-high throughput screening (uHTS) campaign that identifies a small molecule that can modulate UPR and ER stress in cellular and

Construction and efficacy evaluation of Rb luciferase reporter gene detection system / 西安交通大学学报(医学版)

【Objective】 To construct the Rb luciferase reporter gene assay system and detect the activation ability of Rb gene for screening the targeted drugs. 【Methods】 The synthetic Rb gene sequence was annealed to form a double-stranded DNA structure and then inserted into the polyclonal site of pGL6-TA. The junction product was transformed into E.coli DH5α competent cells for expanded culture, and the constructed pGL6-Rb-Luc plasmid and pGL6-TA plasmid were transfected into HEK293 cells. The monoclonal cell line HEK293-Rb-Luc with stable expression was screened by G418, and the activation and inhibition of Rb in HEK293-Rb-Luc were tested by serum and CDK4/6 inhibitor Palbociclib. 【Results】 The sequence of Rb reaction elements in pGL6-Rb-Luc was completely correct. The recovery of serum culture significantly increased the luciferase activity in HEK293-Rb-Luc (P<0.001). Compared with 0 nmol/L, 25, 50, 75 and 100 nmol/L, CDK4/6 inhibitor Palbociclib made the inhibition rate of Rb activity rise to 6.90%, 40.23%, 50.57% and 52.07%, respectively (P<0.05). 【Conclusion】 The Rb luciferase reporter gene detection system HEK293-Rb-Luc was successfully constructed, which can effectively detect the activation level of Rb.

MicroRNA-141-3p expression in serum of patients with cerebral hemorrhage and its mechanism / 解剖学报

Objective To analyze the expression level of microRNA-141-3p (miR-141-3) in patients with intracerebral hemorrhage (ICH), and explore the effect and mechanism of miR-141-3p on cerebral hemorrhage injury in rats. Methods Forty patients with ICH and 40 healthy controls in total were enrolled in this study. The expression of miR- 141-3p in peripheral blood serum was determined by the Real-time PCR method. The target relationship between miR-141- 3p and NOD-like receptor 3 (NLRP3) 3′ UTR was confirmed by dual luciferase reporter assay. miR-141-3p agonist and agonist NC were injected into rats via the lateral ventricle, respectively. On day 7 after treatment, the neurological function score was evaluated, and then all rats were killed to obtain brain tissue. Brain water content was examined by the dried and wet mass. HE staining was conducted to observe the pathological changes of cerebral tissue. The mRNA expressions of NLRP3 and miR-141-3p were detected by Real-time PCR. The protein expression of interleukin (IL)-lβ, IL-6 and IL-18 were detected by Western blotting analysis. Results The expression of miR-141-3p in serum of ICH patients was significantly down-regulated compared to healthy controls and negatively correlated with the severity of edema around the hematoma [(0.068±0.038) vs (0.520±0.028), t = 15.93, P<0.001; r =-0.8948, -0.9434 to-0.8087, P<0.001 ]. The result of luciferase reporter assay showed that miR-141-3p was related to the regulation of NLRP3 gene expression. The relative expression levels of miR-141-3p in agonist group were significantly higher than those in the agonist NC group (P< 0.001), while the expression levels of NLRP3, IL-lβ, IL-6 and IL-18 were significantly lower than those in the agonist NC group (P< 0.001). Compared with the agonist NC group, the cerebral water content reduced significantly (P< 0.001), and the neurological function score was significantly improved on the day 7 after treatment in agonist group (P< 0.001). The result of HE staining showed that injection of miR-141-3p in ICH rats could reduced the severity of edema around the hematoma. Conclusion MiR-141-3p alleviates ICH-induced inflammatory injury in rat possibly by modulating miR-141-3p.