Establishment of 3D hepatocyte genotoxicity testing assay / 中国药理学通报

Aim To establish the 3D hepatocyte model by selecting the humanized hepatocyte HepG2 cells and 3D cell culture methods, and to establish the 3D hepatocyte cytokinesis-block micronucleus cytome(CBMN-cyt)assay and 3D hepatocyte comet assay by using chemicals of different mode of action.Methods In this study, a scaffold-free culture method was used to successfully establish a 3D HepG2 hepatocyte spheroid model.The appearance of the sphere, the survival rate of cells inside the sphere, the gene expression of phase I and II metabolic enzymes, and the expression of liver-specific biomarkers were selected as the observation indicators to obtain the best culture conditions for the 3D hepatocyte model.The 3D hepatocyte model was combined with in vitro micronucleomics test and in vitro comet test to explore its applicability for genotoxicity test.Results The best culture conditions for the 3D hepatocyte model was 5×103 cells/20 μL /drop inoculation, cultivating for seven days.A 3D hepatocyte CBMN-cyt assay was established using mitomycin C(MMC), a micronucleus positive compound, and the results showed that it could successfully detect the genotoxicity and cytotoxicity of MMC.Compared with the CBMN-cyt results of 2D hepatocyte model, 3D hepatocyte model had higher sensitivity in detecting MN and Nbud.The 3D hepatocyte comet assay methods were established using the known in vivo and in vitro comet assay positive compound methyl methanesulfonate(MMS), and the results showed that MMS could significantly increase the tail DNA% of 3D hepatocytes with low cytotoxicity.The sensitivity of 3D hepatocyte model to MMS genotoxicity detection was higher than that of 2D cells.Conclusions The 3D hepatocyte model established in this study is easy to use and low in cost, and shows good sensitivity and specificity in the in vitro micronucleus test and comet test, suggesting that the 3D hepatocyte genotoxicity test method is used in early drug genotoxicity screening.It has good application prospects in additional experimental research.

Effects of tRNA and tRNA-derived Fragments on Skeletal Muscle Development / 中国生物化学与分子生物学报

Skeletal muscle is an important tissue of human and livestock. The study of the muscle development is of great significance for treating muscle diseases and improving livestock meat quality. The process of muscle development is controlled by several myogenic transcription factors and signaling pathways. In addition, recent findings established that several noncoding RNAs play a critical role in the regulation of muscle development such as long non-coding RNA (lncRNA), microRNA (miRNA) and circu- lar RNA (circRNA), etc. The detailed mechanism of muscle development is not well understood. Transfer RNAs (tRNAs) are fundamental components in the translation machinery as an adaptor molecule, and tRNA pool could be differentially exploited to modulate expression of mRNAs. In addition, tRNA can be cleaved into tRNA-derived fragments (tRFs) by a variety of ribonucleases (RNases) upon various stress conditions. Unlike the post-transcriptional regulation of lncRNA and miRNA on muscle development, tRNA has been implicated in various aspects of muscle development. Mitochondria play a central role in a plethora of processes related to the maintenance of muscle cellular homeostasis and genomic integrity. Mitochondrial tRNA(mt-tRNA) gene mutations lead to multiple myopathy because human mitochondrial genome is extremely small. The regulation of tRF is similar to miRNAs in regards to the related physiological processes, but are more conservative than miRNA. It is generally believed that tRF has strong tissue specificity, disease specificity and temporal specificity. Some skeletal muscle-specific tRFs could act posttranscriptionally via RNAi or targeting related genes. However, the tRF-sequencing analysis and functional mechanism of tRF are rarely studied in skeletal muscles. The myopathy caused by mitochondrial tRNA gene mutations are particularly complex, which are one of the challenges to diagnose, treat, or prevent diseases. Compared with other noncoding RNAs, the structural complexity of tRF also brings great challenges to data mining and analysis. In this review, we summarize the formation and function of tRNA and tRF especially in muscle development, which will deepen our understandings of related myopathy, and provide new ideas and directions for the investigation of skeletal muscle.

Analysis on the rule of clinical acupoint selection in treatment of puerperal insufficient lactation with acupuncture and moxibustion / 中国针灸

The rule and characteristics of clinical acupoint selection were explored in treatment of puerperal insufficient lactation with acupuncture and moxibustion. The clinical articles on the treatment of puerperal insufficient lactation with acupuncture and moxibustion were retrieved from the databases of CNKI, VIP and Wanfang from the date of establishment to June 1, 2019. The articles were screened in accordance with the inclusion and exclusion criteria. The prescriptions of acupuncture and moxibustion were extracted and normalized. Using Microsoft Excel 2016 software, the use frequency, meridians involved and acupoint distributions were analyzed. Using SPSS Statistics 21.0 software, the cluster and factor analyses were conducted. Totally, 102 articles were included,108 acupoint prescriptions were extracted, 65 acupoitns were designed and the total use frequency was 654 times. The top three acupoints used in treatment of puerperal insufficient lactation were Danzhong (CV 17), Rugen (ST 18) and Shaoze (SI 1). The most frequently involved meridians were the stomach meridian, the conception vessel, the small intestine meridian and the liver meridian. The acupoints were mainly distributed in the chest and four extremities. It was showed in cluster analysis that Rugen (ST 18), Shaoze (SI 1), Zusanli (ST 36) and Danzhong (CV 17) represent 3 clusters and a total of 7 common factors were extracted. The acupoint selection is based on syndrome differentiation in treatment of puerperal insufficient lactation with acupuncture and moxibustion, of which, the local acupoints are predominated and the distal acupoints are combined.

Optimization of Formulation of Co-loaded Docetaxel and Gambogic Acid Albumin Nanoparticles and Evaluation of Its Quality / 中国实验方剂学杂志

Objective: The formulation of co-loaded docetaxel(DTX) and gambogic acid(GA) albumin nanoparticles(DTX-GA-BSA NPs) was optimized by central composite design-response surface methodology to prepare DTX-GA-BSA NPs, and its quality was evaluated. The optimal synergistic ratio of DTX and GA was screened by coefficient of drug interaction(CDI). Method: NabTM method was used to prepare DTX-GA-BSA NPs with bovine serum albumin(BSA) as the carrier material. Design-Expert 8.0.6 software was used to design the experiment and process the data, overall desirability(OD) of particle size and polydispersity index(PDI), encapsulation rate were taken as indexes. The particle size and Zeta potential of the nanoparticles were measured. Individual and synergistic inhibitory effects of DTX and GA on the proliferation of MGC-803 and HGC-27 cells were determined by methyl thiazolyl tetrazolium(MTT) assay, respectively. Result: The optimum prescription of DTX-GA-BSA NPs was as follows:BSA concentration of 5 g·L-1, water-oil phase volume ratio of 1:17, drug-loading ratio(mass ration of drug to carrier) of 1:10.The average particle size of DTX-GA-BSA NPs was 135.8 nm and PDI was 0.09, Zeta potential was -21.4 mV. The deviation between the predicted value and the observed value of the model was small, the model had good predictability. For MGC-803 cell, when the concentrations of DTX and GA were 0.004, 0.12 μmol·L-1, respectively(mass ratio of DTX to GA was 1:23), the CDI value was the smallest and the synergistic proliferation inhibition was the most significant. For HGC-27 cell, when the concentrations of DTX and GA were 0.004, 1 μmol·L-1, respectively(mass ratio of DTX to GA was 1:195), the synergistic proliferation inhibition was the most significant. Conclusion: The optimized formulation of DTX-GA-BSA NPs is stable and reliable. The established mathematical model has good predictive ability and practicability. DTX combined with GA has synergistic effect on MGC-803 and HGC-27 cells without concentration dependence.

Optimization of preparation procedures for nano-sponges of flavonoids fromGlycyrrhizae Radix et Rhizoma by Box-Behnken response surface methodology based on CRITIC weighted evaluation / 中国中药杂志

In this paper, nano-sponges of flavonoids from Glycyrrhizae Radix et Rhizoma (LF-NSP) were prepared by agitation-freeze drying method. Box-benhnken design and response surface method based on the single factor experiment was used to optimize the preparation process, with the stirring temperature as well as stirring time and speed as the independent variables, while with drug loading, particle size and the generalized "normalized value" as the response values. In addition, the nano-sponges were characterized by scanning electron microscope (SEM), infraredspectroscopy (FT-IR) and differential scanning calorimetry (DSC), and its release in vitro was also investigated. The results showed that the optimum preparation conditions for glycyrrhizin nano-sponges were as follows：The proportion of main drug and auxiliary drug was 1:2; the proportion of crosslinking agent DPC and β-CD was 4:1; stirring temperature 45 °C for 4.8 h at 245 r·min⁻¹. The comprehensive score of LF-NSP prepared under these conditions was 94.78. FT-IR and DSC results indicated the formation of Glycyrrhiza flavonoids nano-sponges, and SEM showed that they were spherical particles in shape. In release experiment in vitro, the cumulative release of glycyrrhizin flavonoids nano-sponges for 240 min was 81.8%, while that of crude drug was only 31.5%. Nano-sponges can significantly improve the dissolution of flavonoids from Glycyrrhizae Radix et Rhizoma.

Preparation of nanosuspensions of flavonoids from Glycyrrhizae Radix et Rhizoma and the in vitro dissolution rate / 中成药

AIM To prepare nanosuspensions of flavonoids from Glycyrrhizae Radix et Rhizoma and to determine the in vitro dissolution rate.METHODS Precipitation-high pressure homogenization method was adopted in the preparation of nanosuspensions.With mean particle size and polydispersity index (PDI) as evaluation indices,concentrations of flavonoids,povidone K30 (PVP K30) and polyethylene glycol 400 (PEG 400) as influencing factors,central composite design-response surface method was applied to optimizing the preparation.For the freedried powder prepared by freeze-drying method,the optimal kind and ratio of lyoprotectant were screened.Then the in vitro dissolution rates of freeze-dried powder and physical mixture were compared.RESULTS The optimal conditions were determined to be 10.00 mg/mL for flavonoids' concentration,and 2.30 mg/mL for both PVP K30 and PEG 400 concentrations,the mean particle size and PDI were (172.3 ± 1.2) nm and 0.175 ± 0.004,respectively.The optimal lyoprotectant was 5％ mannitol-lactose (3 ∶ 2),the mean particle size and PDI after redissolution were (239.7 ±2.1) nm and 0.193 ±0.032,respectively.The in vitro dissolution rate of lyoprotectant reached 87.7％ within 60 min,which was much higher than that of physical mixture (less than 30％).CONCLUSION Nanosuspension can effectively improve the in vitro dissolution rate of flavonoids from Glycyrrhizae Radix et Rhizoma.

Development of therapeutic antibodies for gastric and colorectal cancers / 浙江大学学报·医学版

With the elucidation of structures and functions, antibodies are widely applied in the diagnosis and treatment of diseases. Today, therapeutic antibodies have played ever increasing roles in the treatment of cancers. In fact, there are over 20 monoclonal antibodies which have been approved by the U.S.Food and Drug Administration (FDA) for the therapeutic use in cancers. For the gastric and colorectal cancers, there are at least 9 antibodies have been approved for cancer therapy or for clinical trials. These antibody drugs target to tumor associate antigens and can destroy the cancer cells through several mechanisms such as antibody-dependent cell cytotoxicity, complement-dependent cytotoxicity, blockage of blood nutrition and crucial signaling pathways. With the progress in gene engineering technology, the diverse structures of antibodies can be created. In addition, the antibody-conjugates with radioisotopes, toxins and cytotoxins, are also designed for targeted therapy of gastric and colorectal cancers. In this article, we review the trends in the clinical development and application of antibody drugs for future research and development of the rapidly expanding therapeutic modality in gastric and colorectal cancers.