Current Biotechnology

Strengthening Innovation in Synthetic Biotechnology and Leading the Leapfrog Development of Modern Agriculture
Min LIN, Bin YAO
2022, 12(3): 321-324. DOI: 10.19586/j.2095-2341.2022.0052
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Synthetic biotechnology emerging in the early 21^st century is regarded as one of the disruptive technologies affecting the future of the world. The application of synthetic biotechnology in agriculture will provide revolutionary solutions to the problems of global agricultural production， foster new forms of biological economy such as agricultural carbon economy and ammonia economy， and lead the development of new drivers of growth and new forms of business， such as cellular agriculture， low-carbon agriculture and smart agriculture. In this review， we briefly summarized the development status and trend of synthetic biotechnology in the world. The development strategy and overall goal of agricultural synthetic biotechnology innovation in China were discussed.

Research Progress on PRNs Gene Family
Jinrui FAN, Lei WANG, Junjie ZOU
2022, 12(3): 325-331. DOI: 10.19586/j.2095-2341.2021.190
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Pirins （PRNs， PIRs） are gene family containing cupin domain and belong to the functionally diversity cupin superfamily. The N-terminal domain of PRN contains a metal-ion binding site， which is highly conserved in prokaryotes and eukaryotes. Human hPRN could participate in cancer development as transcriptional regulator and redox sensor， suggesting that hPRN is a potential therapeutic target. In plants， there are several members in PRNs gene family， whereas their biological functions remain largely unknown. This paper outlined the basic structure and biochemical characteristic of PRNs， and reviewed the biological research progresses in human， microorganism and plants， in order to provide clues to clarify the PRNs molecular mechanism of crop such as rice， and provide target genes for the genetic improvement of crops production or adapation by gene editing and other molecular breeding techniques.

Biomedical Research Progress of Hydrogen‍⁃‍rich Water and Hydrogen‍⁃rich Saline： Animal Experiment
Meng WANG, Yang YI, Mengting SUN, Zijia LIU, Xue JIANG, Chen MA, Yifei SONG, Fei XIE
2022, 12(3): 332-343. DOI: 10.19586/j.2095-2341.2022.0028
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Since the discovery that hydrogen inhalation could effectively protect against cerebral ischemia-reperfusion injury in 2007， the biological effects of hydrogen have been discovered successively. Hydrogen-rich water or hydrogen-rich saline as the main hydrogen intervention method， which has been widely used in preclinical medicine and clinical research and has been proven to have preventive and therapeutic effects on a variety of diseases. In the past， most of the studies on hydrogen-rich water or hydrogen-rich saline were aimed to introduction of its medical effects. This paper introduced the changes of hydrogen concentration in the body after the intervention of hydrogen-rich water or hydrogen-rich saline， their effects on normal physiological functions， and the protective effects on diseases and the impact on gut microbiota. We also focused on the hydrogen concentration of hydrogen-rich water or hydrogen-rich saline， the time point of intervention， the duration of intervention， and the dose of each intervention in different animal experiment， which amied to provide theoretical basis for the basic research of hydrogen molecules.

Biomedical Research Progress of Hydrogen⁃rich Water and Hydrogen⁃rich Saline: Clinical Trial
Yang YI, Pengxiang ZHAO, Mengyu LIU, Mawulikplimi Adzavon YAO, Fei XIE, Xuemei MA
2022, 12(3): 344-351. DOI: 10.19586/j.2095-2341.2021.0180
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Hydrogen molecular has been verified to have a wide range of biological effects， and previous studies showed that hydrogen has a significant therapeutic effect on many diseases. In recent years， there are many reports about the clinical application of hydrogen molecules at home and abroad， which provided a new way to treat diseases. Drinking hydrogen-rich water or injecting hydrogen-rich saline are the commonly used methods for hydrogen intake. It has attracted extensive attention because of its simple operation and high safety. In this paper， the clinical research progress of hydrogen-rich water and hydrogen-rich saline in metabolic diseases， nervous system diseases， inflammatory diseases， tumors， skin diseases， and exercise fatigue were summerized， aiming to provide references for clinical application and mechanism research of hydrogen-rich water and hydrogen-rich saline.

Research Progress of Nucleic Acid⁃based Self⁃assembling Nanocarriers
Keren CHEN, Weishen WANG, Longjiao ZHU, Yangzi ZHANG, Xiaoyun HE, Kunlun HUANG, Wentao XU
2022, 12(3): 352-357. DOI: 10.19586/j.2095-2341.2021.0160
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As the rapid development of nucleic acid nanotechnology， nucleic acid-based self-assembling nanocarriers have become a research focus in the field of drug delivery. This article reviewed the application of nucleic acid-based self-assembling nanocarriers in drug delivery， discussed different nucleic acid-based self-assembling strategies， and expounded a variety of targeted delivery and controlled drug release methods. In addition， this article also summarized the application of nucleic acid-based self-assembling nanocarrier in the delivery of protein drugs， nucleic acid drug， small molecule drugs and nanodrugs. At the same time， the challenges and future development trends in this field were summarized and prospected， in order to provide references for the development of drug delivery fields and new drug systems.

Development and Application of Monoclonal Antibody-based Drug
Yiqi YANG, Zhigao ZHANG, Xiaolong YOU, Jing ZHANG, Guanfeng LIN, Yingsong WU
2022, 12(3): 358-365. DOI: 10.19586/j.2095-2341.2021.0173
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Monoclonal antibody-based drug at first were mouse-derived antibodies， with the problem of high immunogenicity and short serum half-lives. After the development of several decades， monoclonal antibody-based drug developed from murine antibodies to chimeric antibodies， humanised antibodies and full human antibodies. By recombining antibody fragments， modifying sites and conjugating drugs， researchers have developed different kinds of monoclonal antibody-based drug， which included antibody fusion protein， antibody-drug conjugates， bispecific antibodies and antibody fragments. All of them played important roles in the treatment of malignant tumour， autoimmune diseases and infectious diseases. This article summarized the humanization course of monoclonal antibody-based drug， the types of antibodies' construction and characteristics， the application of monoclonal antibody-based drug in the treatment of coronavirus disease 2019（COVID-19）， and offered a brief future perspective for monoclonal antibody-based drug. The paper was expected to provide references for the development of antibody-based drugs.

Progress on the Role of SHH Signaling Pathway in Hippocampus Neuroplasticity and Related Nervous System Diseases
Lulu XIE, Baozhen YAO
2022, 12(3): 366-372. DOI: 10.19586/j.2095-2341.2021.153
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Sonic hedgehog （SHH） is a secreted protein that controls the formation of neural progenitors， neurons and glial cells during development. Studies found that hippocampus is a very important brain area of studing and memory. SHH plays an important role in the formation and plasticity of hippocampal neuronal circuits and mediates hippocampal neurogenesis and synaptic plasticity. Activation of SHH receptors in dendrites of hippocampal neurons is involved in transneuronal signaling pathways that accelerate axon growth and enhance glutamate release from presynaptic terminals. Impaired SHH signaling pathway transduction can lead to central nervous system injury and related diseases （such as autism， depression and neurodegenerative diseases）. Therefore， SHH signaling pathway transduction controlling， such as using SHH pathway inhibitors or agonists， may be helpful in the treatment of related diseases. This paper reviewed the hippocampal neuroplasticity of SHH signaling pathway and its effects on the development of central nervous system and related diseases， in order to lay a theoretical basis for elucidating the mechanism of SHH signal transduction impairment leading to hippocampus nervous impairment and central nervous system impairment and related diseases.

Research Progress of Body Fluid Markers of Osteoarthritis Synovial Lesions
Jiawei YAO, Bo QIU
2022, 12(3): 373-378. DOI: 10.19586/j.2095-2341.2021.092
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Osteoarthritis （OA） is a disease characterized by progressive articular cartilage degradation， osteophyte formation， and subchondral bone sclerosis. In recent years， studies have found that the inflammation of synovium is also an important pathological mechanism in the pathogenesis of osteoarthritis. The serum and synovial fluid of patients with osteoarthritis contain a large number of metabolites of synovial cells and chondrocytes， such as collagen degradation products， C-reactive protein degradation products， hyaluronic acid， cytokines， and glycoproteins. These metabolites of synovial cells or chondrocytes are closely related to osteoarthritis， and they play important roles in the occurrence and development of synovial diseases of osteoarthritis， which can reflect the degree of inflammation of joint synovium and the progress of osteoarthritis in patients with osteoarthritis to a certain extent. The synovial lesions of early osteoarthritis are difficult to be identified and diagnosed in medical imaging examinations. Therefore， finding sensitive and specific biomarkers related to osteoarthritis synovial lesions is helpful for osteoarthritis synovium early diagnosis of inflammation and evaluation of its progress. This paper summarized the research progress of the relationship between metabolites of synovial cells or chondrocytes above and osteoarthritis synovial lesions， which was expected to provide a theoretical basis for the development of corresponding biochemical detection methods and drugs in the future.

Functional Analysis of ELONGATED HYPOCOTYL5 Regulating Branching in Arabidopsis thaliana
Qin ZHOU, Yurong XIE
2022, 12(3): 379-386. DOI: 10.19586/j.2095-2341.2021.0102
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Light is one of the most important external environmental factors that affects plant branching， however， whether ELONGATED HYPOCOTYL5 （HY5） regulating plant branching is still unknown. In this study， HY5 transgenic overexpression plants were created and commercial T-DNA insertion mutant homozygous plants were obtained. By comparing the number of branches of wild type （WT）， overexpression plant （HY5-OE） and mutant （hy5?215）， the results showed that the overexpression plants had much more rosette branches， while the mutants had less branches compared with wild type plants. The transcriptional levels of BRC1 （BRANCHED1）， a key negative regulatory factor of Arabidopsis thaliana branches were further compared. The results also showed that BRC1 transcription was significantly down-regulated in overexpressed plants and up-regulated in mutants. The results suggested that HY5 promotes Arabidopsis thaliana branching by inhibiting the transcription of BRC1. These results of this research provided a theoretical basis for elucidating the biological functions of HY5 regulatory branches.

Expressional and Functional Characterization of the Ferric Uptake Regulator Fur in Pseudomonas stutzeri A1501
Yichao LIU, Chao LU, Yuhua ZHAN, Xiubin KE, Wei LU, Yongliang YAN
2022, 12(3): 387-395. DOI: 10.19586/j.2095-2341.2022.0061
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Iron is one of the inevitable metal elements for cell physiological metabolisms， and an important co-factor for a variety of oxidoreductases and peroxidases. In Gram-negative bacteria， the intracellular iron transport， storage and utilization are mainly regulated by the ferric uptake regulator （Fur）. A fur homologous gene was identified in the genome of Pseudomonas stutzeri A1501 which is a root-associated nitrogen-fixing bacterium， but its function was still unclear. In this study， the biological information， especially the sequence difference of both the fur genes and the Fur proteins among A1501 and other typical bacteria were compared. The expression characteristics of the fur gene and the Fur protein in A1501 were explored via the application of both real-time quantitative PCR （qRT-PCR） and Western blotting. The results showed that both the Fur protein and the fur gene were highly expressed under iron-limited conditions but the expression inhibited under iron-exceed conditions. The fur knockout mutant and functional complementary strains were constructed. It was observed that deletion of fur affected the carbon source metabolic spectrum of A1501 and also caused higher sensitivity to hydrogen peroxide stress. Meanwhile， the level of intracellular iron in the fur mutant was 1.3 times of that in wild type. Further qRT-PCR analysis indicated that Fur regulated the expression of the intracellular iron transport system and superoxide dismutase genes. This study provided a theoretical basis for the analysis of the intracellular iron homeostatic regulation and rhizosphere environment adaptation of rhizosphere microorganisms.

Prokaryotic Expression of Outer Membrane Ferritin Receptor pvuA Gene of Vibrio parahaemolyticus and Optimization of Induction Conditions for Product
Lei LIU, Xinyue ZHOU, Zhen ZHU, Ye CAO, Wenbin WANG
2022, 12(3): 396-404. DOI: 10.19586/j.2095-2341.2021.0152
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To obtain the recombinant plasmid of ferric vibrioferrin receptor pvuA and improve the expression yield of pvuA in Escherichia coli BL21， optimize the expression conditions， and lay a foundation for its immunogenicity study， the ferric vibrioferrin receptor gene pvuA was amplified from the genomic DNA of Vibrio parahaemolyticus. The recombinant plasmid pET-28a （+） - ferriferrin receptor was constructed， transformed into E. coli BL21， and induced by isopropyl β-D-thiogalactoside （IPTG）. Based on the single factor experiment， the effects of independent variables and their interaction on the production yield of ferric vibrioferrin receptor were studied by response surface methodology. The independent variables were initial cell concentration， induction time and induction temperature， inducer concentration， and the response value was the yield of bacterial protein. The induction conditions were optimized by the combination of Design Expert and response surface analysis. The ferric vibrioferrin receptor was expressed as inclusion bodies. The optimal expressioninitial cell concentration was OD₆₀₀=0.6， temperature was 37 ℃， induction time was 10 h， IPTG concentration was 1.0 mmol·L^-1. At this time， the protein content of inclusion bodies precipitation was the highest （11.00 mg·mL^-1）. The E. coli recombinant plasmid of ferric vibrioferrin receptor pvuA was constructed， the yield of purified protein was improved by optimizing the expression conditions， which laid a foundation for the preparation and application of polyclonal antibody against Vibrio spp..

Development of A Fluorescent Probe for CRP Detection Based on Ligand Peptides
Siya WANG, Yuhao HAN, Zhenbao LI, Tingting ZHANG, Jiaxin LIANG, Huifeng LIN, Shuhua ZHANG
2022, 12(3): 405-410. DOI: 10.19586/j.2095-2341.2021.0179
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C-reactive protein （CRP） is an effective marker of inflammation in body. Early detection is the key to estimate inflammation related diseases， therefore， the development of new CRP detection preparations is of great significance. The specific affinity ligand of CRP was screened by using phage surface display techniques， and target ligands synthesized by solid?phase peptide synthesis technology. The fluorescent probe for CRP was successfully obtained by biomarker， high performance liquid chromatography （HPLC） and mass spectrometry （MS）. After three rounds of screening， ELISA detection， recombinant phage sequencing and sequence alignment， a target ligand peptide was obtained： S-P-H-N-R-S-N-L-V-Q-E-L； by solid phase peptide synthesis and biomarker technology， a fluorescent probe for CRP was obtained： FITC?（Acp）?S?P?H?N?R?S?N?L?V?Q?E?L. This study provided a new preparation to detect CRP effectively.

The Mechanisms of PKM2 Promoting Tumor Progression
Caihong WANG, Jianfei CHEN, Jiujun JU, Xuejie ZHU, Caixia WANG
2022, 12(3): 411-418. DOI: 10.19586/j.2095-2341.2021.0181
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To investigate the expression of pyruvate kinase M2 （PKM2） in different tumor tissue and its relationship with the clinical prognosis of cancer patients， and to explore the effects of PKM2 on tumor cell proliferation and migration and its mechanism of action， TCGA database and immunoblotting were used to analyze the expression of PKM2 in 33 tumors. The relationship between PKM2 and the prognosis of different tumor patients was explored. PKM2 was overexpressed in lung cancer cell lines， and the effect of PKM2 on lung cancer cell proliferation and migration were explored by CCK8 and Transwell assay. The effect of PKM2 overexpression on heat shock protein 90 alpha （Hsp90α） secretion in different tumor cells was detected by immunoblotting. Key proteins in the EMT process were detected after PKM2 knockdown. PKM2 was found highly expressed in 15 tumors， including breast cancer and lung cancer， and the high expression of PKM2 in 9 tumors was significantly correlated with the prognosis of tumors. After the expression of PKM2， the proliferation and migration ability of lung cancer cells was significantly enhanced. Overexpression of PKM2 could significantly increase Hsp90α secretion in breast cancer and lung cancer. Knocking down PKM2 could inhibit the expression of N-cadherin and Vimentin and increase the expression of E-cadherin. Results showed that， PKM2 was highly expressed in a variety of tumors and was significantly correlated with tumor prognosis. PKM2 could promote tumor progression by affecting the secretion of Hsp90α and the levels of epithelial-mesenchymal transition （EMT） related proteins. PKM2 may be a potential broad-spectrum tumor marker and therapeutic target.

Study on the Role of Long Non⁃coding RNA LINC00885 in Human Esophageal Cancer Cells
Shan XUE, Duo TANG, Zijie ZHAO, Qihao HONG, Qian WANG, Zijia LIU, Zhixiang ZHOU
2022, 12(3): 419-426. DOI: 10.19586/j.2095-2341.2021.0195
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To investigate the effect of long non-coding RNA LINC00885 on the proliferation， migration and invasion of human esophageal cancer cell EC109 in this study. The LINC00885 gene overexpression plasmid （pcDNA3.1-LINC00885） and shRNA knockdown plasmid （pLKO.1-LINC00885） were constructed. Colony formation experiment was used to detect the proliferation ability of EC109 cells； the scratch healing experiment was used to detect the lateral migration ability of EC109 cells； the Transwell chamber experiment was used to detect the longitudinal migration ability and invasion ability of EC109 cells； the flow cytometry experiment was used to detect the regulation of LINC00885 on the cell cycle； real-time quantitative PCR method was used to detect the LINC00885 mRNA transcription level； Western blot was used to detect the expression levels of BMP7 and epithelial-to-mesenchymal transition （EMT） pathway related proteins （VIMENTIN， β-catenim and ZO-1）. In EC109 cells overexpressing LINC00885， the proliferation， migration and invasion capabilities of the cells were significantly enhanced， and the expression of BMP7 protein was increased. While LINC00885 down-expression significantly inhibited the proliferation， migration and invasion capabilities of EC109 cells， and BMP7 protein expression was decreased. In addition， overexpressing LINC00885 in EC109 cells， the expression levels of EMT pathway proteins VIMENTIN and β-catenim increased significantly， while the expression levels of ZO-1 decreased significantly. By exploring the effect of LINC00885 on the proliferation and migration of esophageal cancer cells， this paper aimed to verify the function of Linc00885 in esophageal cancer， in order to lay a theoretical foundation for clinical treatment of esophageal cancer.

Expression and Biological Function of Long Noncoding RNA ZFAS1 in Colorectal Cancer
Lina WU, Xiaozhong PENG, Chongmei LU
2022, 12(3): 427-435. DOI: 10.19586/j.2095-2341.2022.0035
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To explore the expression and biological function of long noncoding RNA （lncRNA） zinc finger nuclear transcription factor antisense RNA 1 （ZFAS1） in colorectal cancer， the expression level of ZFAS1 in colorectal cancer were detected by real-time PCR， and the relationship between its expression level and clinicopathological features was analyzed. The effect of ZFAS1 on the growth and migration of colorectal cancer cells was detected by cell function experiment. By knockdown of ZFAS1， the expression of its potential target gene zinc finger nuclear transcription factor 1 （ZNFX1） in colorectal cancer cell lines were delected. The effect of ZNFX1 on the growth and migration of colon cancer cells was detected by cell function experiment. The results showed that， ZFAS1 was highly expressed in colorectal cancer， and knockdown of ZFAS1 could inhibit the growth and migration of colorectal cancer cells. After knockdown of ZFAS1， ZNFX1 expression was up-regulated. ZNFX1 was lowly expressed in colorectal cancer cell lines. Overexpression of ZNFX1 could inhibit the growth and migration of colorectal cancer cells. The results of this study laid a theoretical foundation for clarifying the mechanism of ZFAS1 in colorectal cancer.

Study on the Protective Effect of Anthocyanins to Liver and Kidney Damage Induced by Lead
Na CHEN, Yunlei ZHANG, Bing LI, Yanan WEI, Yunfei CAO
2022, 12(3): 436-445. DOI: 10.19586/j.2095-2341.2021.0174
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To investigate the protective effect of anthocyanins （ACNs） on liver and kidney injury induced by lead in rats during critical development period， the lead-induced rat were employed. After administration with different doses of ACNs solution and dithioglysuccinic acid （DMSA） for 3 consecutive weeks， lead level， histopathology， biochemical criterions were analyzed at 24 hours after the last administration. The ability of ACNs to repair oxidative damage of liver and kidney in lead-induced developing period rats was evaluated comprehensively by integrated biomarker responses （IBR）. The results showed that lead accumulated in the liver and kidney of rats， causing pathological and oxidative damage to the organs. However， ACNs could promote the discharge of lead from the body， improve the histopathological structure， enchance the level of serum transaminase， creatinine and other biochemical indicators. ACNs could also increased the activity of antioxidant enzymes in the liver and kidney， reduce the content of reductive substances， and then improve the oxidative damage induced by lead. IBR analysis showed that ACNs could effectively improve the oxidative damage of rat organs induced by lead. The results showed that ACNs supplement can effectively antagonize the oxidative damage caused by lead， suggesting a protective effect of ACNs on the liver and kidney.

Effects of Ginsenoside Rg1 on BDNF-TrkB Signaling Pathways in Rats with Alzheimer′s Disease
Xiaoguang CHEN, Xiaofeng PAN, Fan WANG, Songbin PAN
2022, 12(3): 446-451. DOI: 10.19586/j.2095-2341.2021.0183
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The aim of the study is to explore the effects of ginsenoside Rg1 on brain-derived neurotrophic factor/tyrosine kinase receptor B （BDNF-TrkB） signaling pathways in rats with Alzheimer's disease （AD）. A total of 75 SD rats were enrolled and randomly divided into blank control group， model group， low-dose Rg1 group， medium-dose Rg1 group and high-dose Rg1 group， with 15 rats in each group. The brain tissues in each group were collected to prepare brain slices. Except blank control group， Aβ1-42 reagent was added into each group to prepare AD models. The low-dose Rg1 group， medium-dose Rg1 group and high-dose Rg group were treated with 60， 120 and 240 μmol·L^-1 Rg1， respectively. The pathological damage of brain tissues was observed by HE staining. The apoptosis of brain tissues was detected by TUNEL staining. The levels of acetylcholine （Ach） and 5-hydroxytryptamine （5-HT）， and activity of acetylcholinesterase （TChE） in brain slices were detected by colorimetry. The expressions of cleaved cysteine proteinase-3 （Cleaved Caspase-3）， B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax） and BDNF-TrkB signaling pathway related proteins in brain slices were detected by Western blot. Compared with blank control group， the number of brain tissues apoptosis， levels of Cleaved Caspase-3， Bax/Bcl-2 and TChE were significantly increased in model group， and 5-HT， Ach， BDNF and TrkB were significantly decreased in model group （P<0.05）. Compared with model group， the number of brain tissues apoptosis， levels of Cleaved Caspase-3， Bax/Bcl-2 and TChE were significantly decreased in low-dose group， medium-dose group and high-dose Rg1 group， and 5-HT， Ach， BDNF and TrkB were significantly increased in low-dose group， medium-dose group and high-dose Rg1 group （P<0.05）， which showing concentration-dependence. Ginsenoside Rg1 can effectively protect the brain tissue of AD rats and inhibit neuron apoptosis， which the mechanism of action may be related to activating BDNF-TrkB signaling pathways. By analyzing the protective mechanism of ginsenoside Rg1 on AD rat model， the paper was expected to lay a theoretical foundation for the use of ginsenoside Rg1 in the treatment of AD in this study.

Effects of Estrogen on the Expression of Cx43 Protein in Renal Tubular Epithelial Cells of SD Rats with Ischemia⁃reperfusion
Yibo MA, Yuechen CHANG, Ketao MA, Li LI, Qinzhang WANG, Yinglong LI
2022, 12(3): 452-459. DOI: 10.19586/j.2095-2341.2022.0023
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To explore the effect of estrogen （estrogen， E₂） on the expression of Cx43 protein in renal tubular epithelial cells of SD rats with ischemia-reperfusion （I/R）， 32 healthy female SD rats were selected as experimental materials. All female SD rats were first subjected to ovariectomized （OVX） treatment， the 32 female OVX SD rats were randomly divided into OVX group， OVX+sham operation group， OVX+ I/R group， OVX+I/R+E₂ group， with 8 animals in each group. After 2 weeks of ovariectomy， the OVX group received no treatment， the OVX+Sham group received sham operation， the OVX+I/R group received ischemia-reperfusion treatment， and the OVX+I/R+ E₂ group was treated with estrogen for 3 days before ischemia-reperfusion treatment. By comparing the levels of serum creatinine， blood urea nitrogen， renal HE staining and Paller score of SD rats in each group， the degree of renal tissue damage was evaluated， and the expression position and level of Cx43 protein in renal tubular epithelial cells of SD rats in each group were analyzed by immunohistochemistry and Western blotting. Compared with the OVX group， the serum BUN and SCr levels in the OVX+I/R group were significantly increased （P<0.05）. Compared with the OVX+I/R group， the serum BUN and SCr levels in the OVX+I/R+E₂ group were significantly decreased （P<0.05）. HE staining showed that， compared with the OVX group， the renal tubules in the OVX+I/R group were significantly enlarged， some cells were fragmented and necrotic， and the Paller score was increased （P<0.05）. Compared with the OVX+I/R group， the renal tubules in the OVX+I/R+E₂ group were slightly expanded， the cell structure was more complete， and the Paller score was decreased （P<0.05）. Compared with OVX group and OVX+Sham group， the positive expression in renal tubular epithelial cells was significantly increased （P<0.05）， and the expression of Cx43 protein in OVX+I/R+E₂ group was lower than that in OVX+I/R group （P<0.05）. The results of Western blotting showed that， compared with the OVX group， the expression of Cx43 protein in the renal tubular epithelial cells in the OVX+I/R group was increased （P<0.05）， and the expression of Cx43 in the renal tubular epithelial cells was significantly up-regulated after kidney injury （P<0.05）. Compared with OVX+I/R group， after estrogen intervention in OVX+I/R+E₂ group， renal function damage was alleviated， and the protein expression of Cx43 in renal tubular epithelial cells was down-regulated （P<0.05）. The results of this study showed that， estrogen could alleviate renal injury caused by renal ischemia-reperfusion injury， which might be achieved by regulating the expression of Cx43 in renal tubular epithelial cells. The result of this paper provided new ideas for clinical treatment of renal ischemia-reperfusion injury.

Effects of Rapamycin on the Biological Behaviors of Podocytes and mTOR Signaling Pathway in Rats with Diabetic Nephropathy
Qiaomu HUANG, Yan HE
2022, 12(3): 460-466. DOI: 10.19586/j.2095-2341.2021.0169
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To explore the effects of rapamycin on the biological behaviors of podocytes and mammalian target of rapamycin （mTOR） signaling pathway in rats with diabetic nephropathy， streptozotocin was injected intraperitoneally to construct a rat model of diabetic nephropathy. The podocytes taken from normal rats were set as control group， and the podocytes taken from the model rats were enrolled as diabetic nephropathy model group （DN）. The podocytes taken from DN group were intervened with 2 mg·kg^-1 rapamycin and selected as rapamycin group （RAPA）. 3-（4， 5-dimethylthiahiazo-z-y1）-2， 5-diphenytetrazoliumromide （MTT） was used to detect podocyte proliferation level， and Transwell was used for detection of cell migration and invasion and flow cytometry for cell apoptosis level. Western blot was adopted to measure the protein expression levels of epithelial-mesenchymal transition markers （E-cadherin， N-cadherin， vimentin）， mTOR and ribosomal protein S6 kinase 1 （S6K1）. The results showed that， compared to the control group， the cell proliferation level was significantly inhibited， the cell migration and invasion were significantly increased， the apoptosis rate was significantly enhanced， the expression of epithelial-mesenchymal transition marker E-cadherin was significantly down-regulated while the expressions of N-cadherin and Vimentin were significantly up-regulated， the mTOR/S6K1 signaling pathways were significantly activated in the DN group（P<0.05）. Compared to the DN group， the cell proliferation level was significantly increased， the cell migration and invasion in the RAPA group were significantly reduced， the apoptosis rate was significantly reduced， the expression of E-cadherin was significantly up-regulated while the expressions of N-cadherin and Vimentin were significantly down-regulated， the protein expressions of mTOR and S6K1 were significantly inhibited in the RAPA group （P<0.05）. The results indicated that rapamycin could promote the proliferation of podocytes in vitro and inhibit the cell migration， invasion， apoptosis and epithelial-mesenchymal transition by inhibiting the mTOR signaling pathway， and it exerts a protective effect on podocytes in rats with diabetic nephropathy.

Dexamethasone-induced Hippocampal Axon Developmental Damage in Offspring Rats
Lulu XIE, Baozhen YAO
2022, 12(3): 467-472. DOI: 10.19586/j.2095-2341.2021.0193
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In order to explore the effect of dexamethasone on the hippocampal axons of offspring rats， a prenatal dexamethasone exposure （PDE） model was established. Wistar rats were subcutaneously injected with dexamethasone （0.2 mg·kg^-1·d^-1） in the second and third trimesters， some offspring were sacrificed during the gestational days of 20 （GD20） and at postnatal week of 12 （PW12）， and the hippocampal glucocorticoid receptors （GR） activation index and axon damage index were detected. GR was activated in the hippocampus of PDE offspring， and the expressions of GR， glucocorticoid-regulated kinase 1 （SGK1） and FK506 binding protein 5 （FKBP5） were significantly increased. Axon damage indicators including growth-associated protein-43 （GAP43）， semaphorin 3A （SEMA3A） and agrin expression were significantly increased. However， the hippocampal GR of PDE adult offspring rats was not significantly activated， and the expressions of axonal injury indicators GAP43， SEMA3A and AGRIN were significantly increased. The results comfirmed that PDE induces axonal developmental damage by activating fetal hippocampal GR， and axonal damage can persist after birth.

Establishment of Anti-glomerular Basement Membrane Nephritis Model Suitable for Immunotherapy in Rats
Cancan PENG, Huiming WANG
2022, 12(3): 473-478. DOI: 10.19586/j.2095-2341.2021.0191
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Linear epitope peptide P14（a3_127-148） was used as antigen to establish the rat model of anti-glomerular basement membrane （GBM） disease suitable for immunotherapy. Rats were immunized with P14 （a3_127-148） and Freund's adjuvant emulsion at three points on the rear foot pad. 24 hours urine and blood samples were collected per week before and after immunization. All rats were killed 7 weeks after immunization. After immunization， 24 h urinary protein， albumin/urine creatinine ratio （ACR）， serum creatinine and blood urea nitrogen in nephritis model group were significantly higher than those in control group （P<0.05）. The level of circulating anti-P14 （a3_127-148） IgG antibody was significantly higher than that of control group （P<0.001）. PAS staining showed segmental-cellular-like necrosis， rich in cellular crescents. Immunohistochemical staining showed obvious infiltration of neutrophils and macrophages in the glomerulus. Immunofluorescence detection showed strong linear deposition of IgG along GBM. The fracture and contraction of GBM were observed by electron microscope. No change was observed in the control group. No lung lesion was observed in all rats by HE staining. WKY rats were successfully immunized with P14 （a3_127-148） linear peptide to establish a rat model of anti-GBM disease， which would be helpful for the development of more specific immunotherapy.

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