Current Biotechnology

Advance on MicroRNA Involved in Nutrient Element Stress in Plant
Dan CAO, Linlong MA, Yanli LIU, Lili WANG, Xiaofang JIN
2022, 12(6): 801-805. DOI: 10.19586/j.2095-2341.2022.0140
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Nutrient elements participate in various physiological and metabolic processes of plants in different ways， such as photosynthesis， respiration， energy metabolism and signal transduction， and are important material basis for plant growing development， yield and quality. MicroRNAs （miRNAs） are a kind of endogenous （non-coding） single-stranded RNAs， which play important roles in response to nutrient element stress in plants. This paper summarized the research progress of miRNAs in regulating nutrient element stress， especially phosphorus， sulfur， nitrogen and potassium， and prospected the future research of miRNAs in this field of plant nutrient stress. It was hoped to provide new ideas for molecular breeding using miRNAs for efficient use of nutrient elements.

Research Progress of CRISPR‍⁃‍Cas9 Technology on the Production of Plant Secondary Metabolites
Yang YANG, Fenglin WANG, De LIU, Yuanyuan LUO, Jianhua ZHU
2022, 12(6): 806-816. DOI: 10.19586/j.2095-2341.2022.0056
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Gene editing technology can carry out target gene insertion， knockout and replacement. Gene editing technology based on CRISPR-Cas9 was the third generation of gene editing technology after zinc finger nuclease and transcription activator-like effector nuclease. In recent years， CRISPR-Cas9 has been applied in multi-aspects as a research hotspot， such as medicine， pharmacy， botany， zoology and microbiology. However， its application in the field of plant secondary metabolites is still in the exploratory period. The development history of gene editing technologies， the principle of CRISPR-Cas9 and the application of technologies were briefly introduced. More importantly， the application of CRISPR-Cas9 technology on plant secondary metabolites production were summarized. CRISPR-Cas9 system can be used to knock out， mutate and insert the plant genome at fixed points， so as to improve the content of plant secondary metabolites， improve crop quality and plant resistance. This technology has shown an increasingly important role in the edition of key enzyme genes involved in the biosynthesis of plant secondary metabolites.

Research Progress on Improving Aroma of Tobacco Leaf with Bio-enzymes and Microorganism Technology
Jie HAO, Qiang JI, Liqun LI, Chao ZHENG, Na WU, Han WU, Xuanwen LI, Zhikang SUN
2022, 12(6): 817-824. DOI: 10.19586/j.2095-2341.2022.0070
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Biological enzyme and microorganism technology play significant roles in tobacco fermentation. The utilization of bio-enzymes and microorganisms to improve the quality and aroma of tobacco has become the focus of tobacco industry. Treating tobacco with bio-enzyme preparation can degrade biomolecules such as protein， pectin and cellulose in tobacco to proteins， pectins， cellulose and other biological macromolecules in tobacco， thus improving the quality and aroma of tobacco. Using microorganisms to ferment tobacco can adjust and improve the proportion of chemical components and increase the formation of aroma substances in tobacco. The application and progress of the technology of bio-enzyme and microbial technology during tobacco flavor production and fermentation， and the mechanism of tobacco fermentation and aroma enhancement techonolgy， were reviewed，especially expatiated on the microbes and enzymes improve tobacco aroma， improving the quality of tobacco leaves， and degradation of protein， starch， pectin， cellulose and other macromolecules substances in tobacco leaves. The problems in practical application of the microbes and enzymes were analysed， so as to provide a theoretical basis for the use of bio-enzymes and microorganism technology to improve tobacco aroma in the future.

Progress of Bimolecular Fluorescence Complementation Technology in Animal Viruses Research
Shuijuan ZHOU, Zhongjian GUO
2022, 12(6): 825-836. DOI: 10.19586/j.2095-2341.2022.0048
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There are a series of protein-protein interactions in the process of virus infection. Therefore， understanding the interactions between the viruses and the hosts are of great significance for the in-depth studies of virology. Currently， there are many techniques used in protein interactions research， among which the bimolecular fluorescence complementation （BiFC）is widely used due to its ability to visualize interactions in living cells. This paper introduced the principle， development and advantages of BiFC technology. The application of BiFC technology in animal viruses and antiviral drugs were also summarized， and the principle of some new bimolecular fluorescence systems were further elaborated， so as to provide new ideas for the studies of virus pathogenic mechanism and the development of antiviral drugs.

Advances in Measuring Techniques and Standardization of Glycated Hemoglobin A_1c (HbA_1c)
Jingjing LI, Haibo ZHAO, Rui ZHAI, Liang LIANG, Peilei FAN, Yujia ZHAO
2022, 12(6): 837-846. DOI: 10.19586/j.2095-2341.2022.0094
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Diabetes is not only a worldwide disease but also a serious public health problem. Glycated hemoglobin A_1c （HbA_1c） has been defined by the World Health Organization （WHO） as the diagnosis criteria for diabetes， which has important clinical significance in the diagnosis， monitoring， and treatment of diabetes. In recent years， much research on HbA_1c laboratory measuring techniques and standardization have been carried out at all over the world， forming a series of test methods and standard systems， which have achieved fruitful results. This paper mainly introduced the representative HbA_1c laboratory testing techniques and the international and domestic HbA_1c standardization process， and future analyzes and prospects for the existing problems， which was expected to be helpful for the selection of clinical laboratory measurement.

Research Progress on the Relationship Between Hydrogen and Intestinal Flora
Zijia LIU, Xue JIANG, Yang YI, Meng WANG, Cheng MA, Yifei SONG, Fei XIE
2022, 12(6): 847-852. DOI: 10.19586/j.2095-2341.2022.0060
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Hydrogen has been proved to have significant medical effects on a variety of diseases， however， the molecular mechanism of its effect have not been fully elucidated. Intestinal flora has been regarded as an important "organ" of human body， which is closely related to human health. Studies have shown that there are a large number of bacteria capable of hydrogen metabolism in human intestinal flora， and the changes of these bacteria may be closely related to the occurrence and development of a variety of diseases. In addition， the study also found that exogenous hydrogen intervention may improve inflammatory bowel disease， fatty liver disease and other diseases through the remodeling of intestinal flora. This paper reviewed the hydrogen metabolism of intestinal microflora and its relationship with the occurrence and development of diseases， as well as the related research on the effect of exogenous hydrogen intervention on disease progression by regulating intestinal microflora， hoping to provide help for researchers who are committed to studying the medical effects of hydrogen from the perspective of intestinal flora.

Progress on the Mechanism of Soybean Protein Derived Peptides Regulating Glucose and Lipid Metabolism
Yanyi LI, Na LYU, Jinli CHEN, Xiao LI, Weiting ZHANG, Hongxia ZHANG
2022, 12(6): 853-860. DOI: 10.19586/j.2095-2341.2021.0161
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Soybean protein derived peptides is a peptide mixture obtained by separating and refining soybean protein through acid or enzymatic hydrolysis. It is an important source of plant protein derived peptides. Soybean protein derived peptides can inhibit intestinal cholesterol absorption， interact with cholesterol， regulate cholesterol metabolism related genes （used to reduce cholesterol）， triglyceride metabolism related genes， dipeptidyl peptidase Ⅳ and glucose metabolism related genes， it plays an important role in reducing cholesterol， triglycerides， anti-obesity， inhibiting fatty acid synthase and anti-diabetes and other metabolic regulation. This paper reviewed the physiological functions of soybean protein derived peptides in improving lipid and sugar metabolism in vitro and in vivo， and forecasted the prospects of soybean derived peptides， which was expected to provide reference for the research and application of soybean proteinderived peptides.

Advances on the Mechanism of Obesity-induced Skeletal Muscle Atrophy
Jingya GUO, Ping ZHANG, Yuhan ZHAO, Mengjie LI, Kunlun HUANG, Tao TONG
2022, 12(6): 861-868. DOI: 10.19586/j.2095-2341.2022.0081
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Skeletal muscle is the main storage and metabolic reservoir of amino acids and proteins in the body， and its normal function and metabolic processes are affected by a variety of pathological factors. Skeletal muscle atrophy occurs in a state of severe imbalance in skeletal muscle homeostasis， which imposes a heavy burden on patients' lives and social and medical care. In recent years， obesity-induced skeletal muscle atrophy is increasingly becoming a serious public health challenge due to the proliferation of obesity in the world. Obesity-induced skeletal muscle atrophy involves changes in various signaling molecules or pathways， such as ubiquitin protease system， autophagy-lysosomal system， insulin/IGF1-PI3K-Akt， myostatin， interleukin-6， tumor necrosis factor， etc. After these signal molecules or pathways are activated or inhibited in the state of obesity， they can jointly affect the balance of protein synthesis/catabolism and further cause skeletal muscle atrophy. Based on the above-mentioned signaling molecules or pathways， this study systematically summarized and discussed the mechanism underlying obesity-induced skeletal muscle atrophy， hoping to provide theoretical references for exploring preventive or therapeutic targets of obesity-induced muscle atrophy， and to extend the development and utilization of natural phytochemicals.

Research Progress on the Regulatory Mechanism of Ferroptosis in Spinal Cord Injury
Kaiyan DU, Chenxu QI, Jingyu CAO, Meng CHEN, Jing GAO, Chengmei LIU
2022, 12(6): 869-874. DOI: 10.19586/j.2095-2341.2022.0050
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Ferroptosis is an iron-dependent， programmed cell death characterized by accumulation of intracellular lipid reactive oxygen species. It is widely existed in tumour， cancer， acute kidney injury and other diseases. Spinal cord injury （SCI） is a severe traumatic nervous system disease with high morbidity， mortality and disability. At present， the exact mechanism and efficient treatment of spinal cord injury are still being explored， which is also an urgent problem to be solved in the worldwide. Studies have shown that the regulation of programmed nerve cell death after spinal cord injury is the focus of treatment of SCI. However， the molecular mechanism of ferroptosis involved in spinal cord injury is not well understood. The paper collected and sorted out the domestic and foreign literature on ferroptosis after spinal cord injury in recent years， and reviewed the regulatory mechanism and research progress of ferroptosis involved in spinal cord injury， in order to bring new ideas for the treatment of spinal cord injury.

Mechanism and Research Progress of SIRT6 in Ischemia Reperfusion Injury
Jing CHENG, Guo ZHANG, Wenfei ZHANG, Liquan WU, Zhibiao CHEN
2022, 12(6): 875-879. DOI: 10.19586/j.2095-2341.2022.0082
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The restoration of blood supply after ischemic injury will lead to ischemia/reperfusion （IR） injury， which could lead to further aggravation of tissue injury. IR injury is accompanied by a series of mechanisms， including glutamate excitotoxicity， calcium overload， oxidative stress， inflammation and apoptosis， resulting in cell death. IR injury processes are regulated by the Sirtuins family. In the Sirtuins family， SIRT6， specifically located in the nucleus， can promote resistance to DNA damage and oxidative stress， inhibit genomic instability， and play a role in metabolic homeostasis， and SIRT6 is highly expressed in the brain. However， there are few studies on SIRT6 in brain IR injury， so combined with the latest research progress at home and abroad， we made a retrospective summary and analysis of the role of SIRT6 in IR injury， hoping to provide some reference basis for domestic and foreign scholars on the study of SIRT6 in stroke.

EMSA Experiments with Fluorescently Labeled Probes to Analyze the Binding Sites of Opaque2 Protein with the ZmGRAS11 Promoter
Jiameng ZHU, Haiyang JIANG, Rumei CHEN, Xiaojin ZHOU
2022, 12(6): 880-887. DOI: 10.19586/j.2095-2341.2022.0084
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The electrophoretic mobility shift assay （EMSA） is an experimental technique for studying the binding between proteins and nucleic acids. Since the establishment of the EMSA technology， radioactive isotope and biotin labeling are the main techniques for tracing nucleic acid probes， but these two traditional labeling techniques have some disadvantages， such as poor stability of radioactive probe and complex detection steps of biotin. Although fluorescently labeled probes have been used in EMSA recently， the systematic workflow has not been reported. This study optimized and systematically summarized the technical procedure of fluorescently labeled EMSA. The 6-carboxy-fluorescence （FAM） labeled ZmGRAS11 promoter probe was applied to detect its binding to Opaque2， and the appropriate protein to probe ratio was 8∶1. Several mutated GCN4 motif sequences were applied to determine the binding site between Opaque2 and ZmGRAS11 promoter， and the results showed that the core motif， TGAC， may play an essential role in the recognition by Opaque2. This study provided data surport for further exploring the mechanism of the Opaque2-ZmGRAS11 transcriptional regulatory module in maize kernel development.

Study on the Function of Cassava Sugar Transporter Mesweet18 by VIGS
Jingjing XUE, Feifei AN, Xiuqin LUO, Jie CAI
2022, 12(6): 888-893. DOI: 10.19586/j.2095-2341.2022.0075
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Sugar will eventually be exported transporter （SWEET） is a key protein for plants， which plays an important role in plant sugar transport， reproduction and developnent， stress resistance， and interaction with pathogens. In this study， we used potted plantings of cassava SC9 as materials， designed the target gene region of Mesweet18 silencing in cassava， used virus-induced gene silencing （VIGS） technology to infect. qRT-PCR results showed that the expression of Mesweet18 in silencing plant were 46.80%，30.23%，21.12%， respectively， which were significantly down-regulated than the control. The results of chlorophyll and soluble sugar content in leaves showed that chlorophyll a， b and total content were decreased， sucrose and fructose contents increased significantly， while glucose content decreased slightly， which compared with the control. The study on molecular function of Mesweet18 in cassava laid a foundation for the molecular mechanism of sugar transporter SWEET in cassava.

Effect of Salinity Rising of Inland Water on Physiological Characteristics of Chlorella vulgaris
Kaituo ZHAO, Zhilong LI, Da HE
2022, 12(6): 894-899. DOI: 10.19586/j.2095-2341.2022.0066
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With the change of global climate， the problem of salinity slowly rising of inland water has a remarkable effect on the phytoplankton. To investigate the effect on physiological property of freshwater phytoplankton under the increasing salty environment， Chlorella vulgaris was taken as the research object and cultured at raw water and salinity（1， 3， 5 g·L^-1）. The effect of low salt level on growth and multiplication of Chlorella vulgaris was studied and its physiological response such as antioxidant enzymatic activity and photosynthesis were analyzed. The results showed that， with the rising of salinity， the growth of Chlorella vulgaris slowed down， the generating rate of chlorophyll-a decreased， its membrane structure was badly damaged， and the photosynthetic activity markedly decreased. All the results showed that high salinity （>3 g·L^-1） very likely damaged cells physiological structure and enzyme system of algae cells so that inhibited its growth and multiplication. The effects of salinity changes on planktonic algae were studied from the perspective of cell proliferation and physiological characteristics， which was expected to provide reference for protecting the biodiversity of inland water.

Isolation, Identification and Biological Characterization of Vibrio parahaemolyticus CHY5-M1M Phage
Liping LIU, Honghua WANG, Feng WANG, Peng DING, Zihao SUN, Zhe WANG, Ying QIAO, Zhumei HOU
2022, 12(6): 900-905. DOI: 10.19586/j.2095-2341.2022.0069
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Vibrio parahaemolyticus disease brings huge losses to the aquaculture industry. With the banning of antibiotics， people have started to explore novel therapeutic methods regarding the disease in aquatic animals， such as phage therapy. In this paper， 15 samples of seafood culture water and sewerage effluent were collected from Chengyang wholesale aquatic products market in Qingdao， and 12 strains of Vibrio parahaemolyticus phage were obtained by isolation and purification using the spot-drop method with Vibrio parahaemolyticus as the host bacterium. The biological characteristics of CHY5-M1M， such as the optimal infection complex， one-step growth curve， stability of temperature and pH and sensitivity to UV light， were studied using the double-layer plate method. The results showed that， the potency of CHY5-M1M phage was 8.65×10¹³ CFU·mL^-1； the potency was highest at 100 infection replicates； the latency period of one-step growth curve was 20 min， the lysis time was 100 min， the plateau was reached after 140 min； the potency of phage was highest at 40 ℃， the activity started to decrease above 60 ℃； the pH adaptation range of phage was 5~11. The phage concentration decreased significantly with the increase of UV irradiation time； the genome length was 43 193 bp， 44 genes， no virulence factor gene and antibiotic resistance gene. The results of this study indicated that phage CHY5-M1M has good application prospects in the development of novel anti-vibrio drugs for the prevention and treatment of severe vibriosis in marine animals， and could be an alternative antibacterial product that can be further investigated.

The Role of COPII Coat Protein SEC24A in Mammalian Macroautophagy
Yunpeng ZHAO, Haolin ZHANG, Qianqian XIONG, Yuting LI, Juan WANG
2022, 12(6): 906-914. DOI: 10.19586/j.2095-2341.2022.0053
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Autophagy is an important and conserved intracellular degradation process in which cellular contents are degraded by the formation of bilayered autophagosomes that encapsulate cellular contents. During starvation-induced stress， COPII vesicles are considered to be the membrane source of autophagosomes. The aim of this study was to explore the role of COPII coat protein SEC24A in mammalian macroautophagy pathway. SEC24A was knocked down by siRNA interference technology. Autophagy was induced by starvation in EBSS for 2 h. Western blot and immunofluorescence experiments were used to detect the levels of autophagy substrate protein p62 and autophagy marker protein LC3-II to determine whether SEC24A is involved in autophagy. The numbers of autophagosomes and autolysosomes were detected by RFP-GFP-LC3 tandem fluorescence assay. Proteinase K sensitivity assay was used to verify that autophagy defects occurred before or after autophagosome closure. Immunofluorescence experiments were used to detect the effect of knockdown of SEC24A on ATG complexes in the autophagy pathway to identify the site where SEC24A regulates the autophagy pathway. Co-immunoprecipitation assay verified the interaction between SEC24A and autophagy-related protein ATG9A. Under starvation conditions， knockdown of SEC24A resulted in accumulation of p62 and decreased LC3-II protein levels. RFP-GFP-LC3 tandem fluorescence experiments showed that the number of autophagosomes and autolysosomes decreased in SEC24A-depleted cells. Protease K protection assay showed reduced protection of p62 and GFP-LC3 in SEC24A-depleted cells. Immunofluorescence experiments showed that the punctate structures of ATG14L and ATG16L1 were reduced after knockdown of SEC24A， indicating that SEC24 acted on the upstream of ATG14L and ATG16L1. Co-immunoprecipitation assay showed that SEC24A interacted with ATG9A. This study not only deepened the understanding of the autophagosome formation process and molecular mechanism， but also provided information for a comprehensive interpretation of the important role of COPII vesicles and their coat proteins in autophagy.

Cloning and Functional Identification of ectABC Gene Cluster from Vibrio neocaledonicus CGJ02-2
Lin TAN, Ni Win Htet NWE, Chang CHEN, Zhiqiang PAN
2022, 12(6): 915-921. DOI: 10.19586/j.2095-2341.2022.0049
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This study aimed to clone and functionally characterize ectABC gene cluster so as to lay a foundation for the production of ectoine. The ectABC gene cluster was cloned from Vibrio neocaledonicus CGJ02-2 by PCR and ligated to expression vector pBAD， which was transformed into Escherichia coli BW25113 and expressed with L-arabinose induction. The recombinant protein was detected by SDS-PAGE and authenticated by LC-MS. The whole cell biocatalysis was used to synthesize ectoine， and high resolution mass spectrum was employed to identify the production of ectoine， the catalysis conditions were optimized regarding to aspartate concentration， KCl concentration， temperature and pH. Results showed the size of ectABC gene cluster from the genome of Vibrio neocaledonicus CGJ02-2 was 2 235 bp. Three recombinant proteins were detected in the expression product by SDS-PAGE， and LC-MS analysis indicated that the molecular weight of the three recombinant proteins is consistent with the theoretical molecular weight of ectA， ectB， ectC， respectively. High resolution tandem mass spectrum identification found that ectoine was produced in the supernatant of whole cell catalysis. The optimal catalysis conditions obtained as follows： aspartate concentration 100 mmol·L^-1， KCl concentration 100 mmol·L^-1， temperature 30 ℃， pH 7 with a yield of 1.11 mg·mL^-1. In this study， the gene cluster ectABC for ectoine biosynthesis was cloned from genus Vibrio， and it was heterologously expressed with biosynthesis of ectoine in low saline environment， which has laid foundation for the scale production of ectione.

Discussion on the Construction Technology of Mouse Model with Mumanized Immune
Bei FAN, Yuanyuan WANG, Yunxia ZHAO, Kui JIANG, Bin WANG
2022, 12(6): 922-928. DOI: 10.19586/j.2095-2341.2022.0129
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To improve the construction success rate of the mouse model with humanized immune system and provide preclinical animal model for further research on tumor immunotherapy drugs， the methods and techniques of the mouse model with humanized immune system construction were explored. The mouse model with humanized immune system was established by injecting HuPB-MNC cells with different numbers and five donors into tail vein of severely immunodeficient mice NCG. The body weight and mortality were recorded twice a week； on the 7^th， 14^th， 21^st， 28^th， 35^th day of the experiment， blood were collected from orbital venous plexus， and the human CD45⁺T cells reconstruction level in NCG mice peripheral blood was detected by flow cytometry. The level of CD45⁺T cells in peripheral blood of NCG mice increased gradually with the time after injection， and reached more than 25% at about 3~4 weeks. The results showed that， the construction of the mouse model with humanized immune system should consider the construction method， the strain， sex and feeding of immunodeficient mice， the donor， number and cell state of immune cells. Strategies such as overcoming the differences by adopting multiple donors， and maximizing the window period by optimizing tumor model and immune cell donor combined inoculation plan， which can improve the success rate of model construction and application conversion rate， further improve the construction method of the mouse model with humanized immune system in laboratory， and provide a more favorable drug development tool for tumor immunotherapy.

Study on Strain Optimization of Coix Seed Multi Lactobacillus Fermentation Broth and its Inhibition on Melanin Production
Xinliang MAO, Hui ZHANG, Wenjie CHEN, Yanwu CHEN, Honghong REN, Guiyuan YU, Yadong HUANG, Qi XIANG, Xiaomin LI
2022, 12(6): 929-936. DOI: 10.19586/j.2095-2341.2022.0137
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With coix seed as the fermentation substrate， the better lactic acid bacteria that can improve the activity in vitro of the fermentation liquid were screened， and the inhibitory effect of the dominant lactic acid bacteria coix seed fermentation broth on the melanin production of fish embryos was explored and analyzed. By comparing and analyzing the chemical indexes such as reducing sugar， total phenol， free amino acid， protein， total acid， lactic acid content， and hydroxyl radical scavenging ability and tyrosinase activity inhibition rate of coix seed fermentation broth of three kinds of single lactic acid bacteria Lactococcus lactis， Streptococcus thermophilus and Lactobacillus bulgaricus and three kinds of compound lactic acid bacteria of fermented coix seed with single or mixd Lactobacillus， the better fermentation strains were determined. High throughput sequencing was used to explore the microbial community structure and succession. The inhibitory effect of fermentation broth on melanin production was studied by zebrafish model. It was screened and determined that the compound fermentation of Lactococcus lactis， Streptococcus thermophilus and Lactobacillus bulgaricus had more advantages than that of single Lactobacillus. Lactococcus lactis and Streptococcusthermophilus were the dominant bacteria in the early stage of fermentation， and Lactobacillus bulgaricus was the dominant bacteria in the middle and late stage of fermentation. After fermentation with compound Lactobacillus， the hydroxyl radical scavenging rate and tyrosinase activity inhibition rate of fermented coix seed increased by 20.82% and 87.26%， respectively. The results of zebrafish model experiment showed that fermented coix seed could significantly reduce the distribution of melanin on zebrafish surface and when the content was 2%， the inhibition rate of melanin production could reach 59.45%. The research results can provide scientific data for the development of coix seed multi Lactobacillus fermentation broth as a new functional raw material with skin whitening properties， and promote the progress of coix seed industry.

Inhibitory Effect of Metformin Hydrochloride on Glioblastoma Cells
Mengting SUN, Pengxiang ZHAO, Yang YI, Meng WANG, Fei XIE, Yao Mawulikplimi ADZAVON, Mengyu LIU
2022, 12(6): 937-945. DOI: 10.19586/j.2095-2341.2022.0062
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The aim of this study was to explore the effect of metformin hydrochloride on the proliferation of different glioblastoma U87 cells， GL261 cells and C6 cells， a mouse GBM cell line GL261， a rat GBM cell line C6 and a human GBM cell line U87MG were selected and treated with metformin hydrochloride. The cell proliferative activity was detected by CCK-8 method； cell apoptosis level was detected by cell real-time fluorescence detection； the clonal formation ability of GBM cells was detected by plate cloning experiment； the intracellular ATP level was detected by CCK-L method； Akt and its phosphorylation were detected by Western blot. Results showed that， compared with the control group， metformin hydrochloride significantly inhibited the proliferative activity of GBM cells and affected cell morphology with the increase of the concentration. Compared with the control group， at the same concentration， metformin hydrochloride increased the apoptotic level of GBM cells， inhibited the clonal formation ability of GBM cells， and reduced the production of ATP in GBM cells. After 24 h of metformin hydrochloride treatment， the p-Akt expression in GBM cells was significantly down-regulated， but there was no significant change in Akt. The results showed that metformin hydrochloride could inhibit the proliferation and clonal formation of a variety of GBM cells and reduce the intracellular ATP level in vitro， which might be related to the phosphorylation of Akt. The results was expected to provide a theoretical basis for further research on the mechanism of metformin hydrochloride against glioblastoma in vitro.

Effects of Pregnancy Dexamethasone Exposure on Hippocampal Neuron Proliferation and Synaptic Plasticity of Rat Offspring
Jiaxin QIN, Baozhen YAO
2022, 12(6): 946-952. DOI: 10.19586/j.2095-2341.2022.0026
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Dexamethasone is a glucocorticoid drug with various pharmacological effects such as anti-inflammatory and immunosuppressive effects. It is widely used in multiple departments to treat multiple autoimmune diseases. Dexamethasone is often used during pregnancy to promote lung maturation in preterm infants and prevent fetal respiratory distress syndrome. Current epidemiological and experimental studies have shown that dexamethasone exposure during pregnancy could lead to an increased risk of cartilage and kidney damage in the offspring. This study investigated the effects of prenatal dexamethasone exposure （PDE） on the proliferation of fetal hippocampal neurons and the formation of synaptic plasticity in the fetal hippocampus. Wistar rats were injected subcutaneously with dexamethasone （0.2 mg·kg^-1·d^-1） at mid-to-late gestation， and the control group was treated with isodose 0.9% sodium chloride solution. The hippocampus of GD20 offspring was collected and examined for morphology and functions related to hippocampal neuron proliferation and synaptic plasticity formation by real-time fluorescence quantitative PCR and Western blot. We further examined adaptor protein containing pH domain， PTB domain， and leucine zipper motif 1 （APPL1）. Restlts showed that， compared with the blank control group， the specific indexes of Ki67， proliferating cell nuclear antigen （PCNA）， synaptic fusion protein （syntaxin）， and 25 kD synaptic-associated protein （SNAP25） in PDE fetal hippocampus were significantly reduced， suggesting that PDE inhibited neural proliferation， synaptic development， and other functions in the fetal hippocampus. Western blot showed significant downregulation of postsynaptic dense protein 95 （PSD95） in the PDE group， confirming that PDE could lead to impaired synaptic plasticity in the offspring hippocampus. APPL1 results showed that APPL1 expression was down-regulated in the hippocampus of PDE offspring， so it was hypothesized that PDE might affect hippocampal synaptic plasticity in offspring by regulating APPL1 expression. The study suggested that PDE is developmentally toxic to the hippocampus and can reduce hippocampal neurons' proliferation and impaired synaptic plasticity. This study provided the experimental and theoretical basis for guiding the rational use of drugs during pregnancy and effectively assessing the risk of hippocampal developmental toxicity in fetuses.

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