Table of Content

25 December 2020, Volume 40 Issue 6

Previous Issue    Next Issue

Expression and Distribution of Molecules Regulating Protein Degradation Pathway in Nervous System of Mice

SONG Binbin, DONG Wenzhou, JIA Bingquan, ZHEN Ran, PENG Yu, YANG Xuan, YU Jia

2020, 40(6):  463-469.  DOI: 10.3969/j.issn.1674-5817.2020.06.001

Asbtract ( 326 )   PDF (1284KB) ( 377 )  

Figures and Tables | References | Related Articles | Metrics

Objective To investigate the expression and distribution of the molecules regulating protein degradation pathway in the nervous system of mice. Methods The eye, olfactory bulb, cortex, hippocampus, striatum, midbrain, cerebellum, brainstem, spinal cord, sciatic nerve and muscle of 6 weeks wild-type C57BL/6J mice were collected. Then the distribution and expression of key proteins in protein degradation pathway in different tissues, anatomical sites and subcellular structures were determined by Western blotting. Results Cathepsin D, lysosomal-associated membrane protein 2 (LAMP2), microtubule-associated protein 1A/1B-light chain 3 (LC3A/B), autophagy-related protein (Atg3), Ras-related GTP-binding protein7 (Rab7), Calpain and Calpastatin had different expression levels in the eye, brain, spinal cord, sciatic nerve and muscle. Compared to the spinal cord, the sciatic nerve showed decreased levels of ubiquitin, Cathepsin D, LAMP2, total LC3A/B, Atg3, Rab7 and full length of Calpain1 large subunit (P<0.05), and increased level of Calpain small subunit (P<0.05). Conclusion The ubiquitin-proteasome system and autophagy mainly are widespread in the cell under physiological conditions, which mainly occurs in the cell body of neurons; but the level of spliced Calpain small in axon is relatively high, which suggests that Calpain degradation pathway may play a major role in axon.



Neurological Dysfunction and Pathological Changes of Rats with

SUN Chengcheng, LIU Jiangang, LIU Meixia, LI Hao, LUO Zenggang

2020, 40(6):  470-476.  DOI: 10.3969/j.issn.1674-5817.2020.06.002

Asbtract ( 355 )   PDF (2516KB) ( 390 )  

Figures and Tables | References | Related Articles | Metrics

Objective To compare the changes of neurological dysfunction, gross/microscopic pathology and serum inflammatory index of brain tissues after bilateral common carotid artery ligation in different durations. Methods Permanent bilateral common carotid artery ligation was performed in SPF Wistar rats after anesthesia. The successful modeling rats were randomly divided into 3 groups with 10 in each group; while 10 rats in the sham-operated group were treated by only puncture without ligation. The neurological function of each group was tested 2 weeks, 4 weeks, and 6 weeks postoperatively, and then the blood was taken from the abdominal aorta of the anesthetized rats. ELISA method was used to detect the serum levels of interleukin-6 (IL-6), interleukin-1? (IL-1?) and tumor necrosis factor-? (TNF-?). The brain tissue was stained with triphenyltetrazolium chloride (TTC) to evaluate the range of cerebral ischemia. HE staining was used for morphological observation of the brain tissues. Results Compared with the sham-operated group, the neurological function scores of the rats at 2 weeks, 4 weeks, and 6 weeks after bilateral common carotid artery ligation were decreased significantly (P<0.05). Compared with the 2 weeks group, the neurological function scores of the 4 weeks and 6 weeks groups were decreased significantly (P<0.05). Compared with the sham-operated group, the level of IL-6 in the 2 weeks and 4 weeks groups was significantly increased (P<0.05). Compared with the sham-operated group, the ischemic range of brain tissues increased in the 2 weeks, 4 weeks, and 6 weeks groups. Pathological morphology also showed that the brain tissue morphology of rats in the 2 weeks, 4 weeks and 6 weeks groups had different degrees of damage and inflammatory infiltration. Conclusion After ligation of bilateral common arteries, the pathological changes of the brain tissues vary from ischemia to infarction, accompanied with neurobehavioral dysfunction, and the changes are different over time. Thus, the intervention time should be decided according to the mechanism of drug action.



Establishment of Eimeria stiedai Infected Rabbit Model and Nested PCR Assay

WEN Fuli

2020, 40(6):  477-482.  DOI: 10.3969/j.issn.1674-5817.2020.06.003

Asbtract ( 352 )   PDF (1407KB) ( 474 )  

Figures and Tables | References | Related Articles | Metrics

Objective To establish an infection model and nested PCR assay method for Eimeria stiedai (E.stiedai) in rabbit. Methods The established infection model of E.stiedai in rabbits was identified by clinical examination, microscopic observation, blood biochemistry and histopathology. By collecting oocysts, DNA of E.stiedai was extracted, and the specific primers were designed to establish nested PCR for detection of E.stiedai. Results Clinical necropsy: the abdominal circumference of the rabbit was obviously enlarged. The liver enlargement and a large number of white and light yellow nodules were found by biopsy, while the gallbladder and bile duct were swollen, and the bile was pale yellow. Microscopic examination: the size of the eggs was (31.72-38.43) ?m×(18.10-22.69) ?m. Blood biochemical examination: the content of globulin (GLOB) was increased, while the levels of creatinine (CREA) and alkaline phosphatase (ALKP) were decreased, and the rest of the detection indicators were in the reference range. Histopathological examination: a large number of pink E.stiedai eggs were seen in the liver tissue and the bile duct. Nested PCR: the lower limit of detection was 1 oocyst DNA sample and 1.14×10³ copies of plasmids, and the negative control and blank control showed no bands. The variability coefficient was less than 5%. Conclusion The E.stiedai infection model in rabbits can be successfully constructed, and the nested PCR assay can amplify the specific fragments of E.stiedai with high sensitivity and good repeatability.



Preliminary Study on the Influencing Factors of Mouse Embryo Transfer

GUI Fei, YANG Weiwei, SUN Xiaopin, GU Meier

2020, 40(6):  483-488.  DOI: 10.3969/j.issn.1674-5817.2020.06.004

Asbtract ( 536 )   PDF (477KB) ( 400 )  

Figures and Tables | References | Related Articles | Metrics

Objective To analyze the influences of mouse strain, embryo transfer method and transferred embryos' quantity on the results of embryo transfer, and to further optimize and improve the efficiency of embryo transfer. Methods The embryos from 277 gene modified strains were obtained by superovulation and in vitro fertilization (IVF), then the different numbers of embryos were transferred into the oviducts of pseudo-pregnant female mice through unilateral or bilateral embryo transplantation. The effects of strains with different IVF rates, number of transplanted embryos and transfer methods on the litter size and rate were analyzed and compared. Results When the strains with IVF rate of 0%-39%, the litter size (4.61±1.92) and the litter rate [(19.21±9.70)%were significantly lower than those in other groups (both P<0.01). The litter rate was significantly higher in 60%-69% IVF rate group than that in the 90%-100% IVF rate group (P<0.05). When the number of transferred embryos was fewer than 5, the recipient female mice got pregnant, but the litter size was less, with easy occurrence of the phenomenon of cannibalism. When the number of transferred embryos was more than 10, the litter size and rate of each group were both higher. The litter rate was (46.67±11.56)% when the number of transferred embryos was about 15, which was significantly higher than those of other groups (P<0.01), indicating that the embryo utilization rate was the highest. The average litter size was significantly the largest (9.67±1.53) with P<0.01 when the number of transferred embryos was around 25. The litter size (8.33±0.58) and the litter rate [(27.33±2.31)%were both decreased when the number of embryos transferred increased to 30, indicating that the bearing capacity of uterus almost reached the maximum. There was no significant difference in litter size or rate between unilateral and bilateral transplantation (P>0.05). Conclusion The average litter size and rate are lower when the IVF rate is lower than 40%. The embryo utilization rate is the highest, when the number of transferred embryos is about 15. The litter size is the largest when the number of transferred embryos is about 25, and the bearing capacity of uterus is almost the maximum. There is no significant difference in the litter size or rate between the unilateral and bilateral transplantation.

Effect and Mechanism of Paeoniflorin on Depression and Anxiety Behavior Induced by Bayk8644 in Rats

LI Ping, LI Yijie, XUE Ling, SONG Chunhong

2020, 40(6):  489-495.  DOI: 10.3969/j.issn.1674-5817.2020.06.005

Asbtract ( 415 )   PDF (1781KB) ( 626 )  

Figures and Tables | References | Related Articles | Metrics

Objective To explore the mechanism of paeoniflorin in improving depression and anxiety of rats through L-type calcium channel. Methods Forty-eight rats were divided into a normal group, a Bayk8644 (L-type Ca²⁺ channel agonist) group, and high dose (200 mg·kg^-1·d^-1), middle dose (100 mg·kg^-1·d^-1) and low dose (50 mg·kg^-1·d^-1) paeoniflorin groups, and a positive control nimodipine group (1 mg·kg^-1·d^-1). The normal group and the Bayk8644 group were given normal saline, and the other groups were given corresponding drugs. After 7 days of continuous administration, Bayk8644 (0.2 mg·kg^-1·d^-1) was injected intraperitoneally into all the groups except the normal group. The open field test (OFT) and elevated plus maze test (EPM) were used to test the depression and anxiety behavior of rats. The expression levels of calmodulin-dependent proteins kinase type Ⅱ (CaMK Ⅱ), brain-derived neurotrophic factor (BDNF) and phosphorylated CaMK Ⅱ (pho-CaMK Ⅱ) in each group were detected by Western blotting. Results Compared with the normal group, the total distance, the time spent in the central area, the distance in the central area, the number of vertical movement, the percentage of open arm time (OT%) and the percentage of open arm entry (OE%) of the Bayk8644 group increased significantly (P<0.05 or P<0.01), the expression levels of CaMKⅡ and pho-CaMK Ⅱ increased significantly (P<0.05 or P<0.01), and the expression level of BDNF decreased significantly (P<0.05). Compared with the Bayk8644 group, the total distance in the central area of the high dose paeoniflorin group increased significantly (P<0.05), the time spent in the central area of the middle and high dose paeoniflorin groups were extended (P<0.05), the OT% and OE% of the high dose paeoniflorin group and the OT% of the middle dose paeoniflorin group decreased significantly (P<0.05 or P<0.01), the expression levels of CaMKⅡ and pho-CaMK Ⅱ proteins in the high dose paeoniflorin groups were significantly reduced (P<0.05 or P<0.01), and the expression level of BDNF protein was significantly increased (P<0.05). Conclusion Bayk8644 can induce depression and anxiety behavior of rats. Paeoniflorin can antagonize the negative effect of Bayk8644 and exert its pharmacological effect by inhibiting the L-type calcium channel and influenceing the activation of CaMKⅡ protein.



Molecular Mechanism of Resveratrol Against Myocardial Ischemia-Reperfusion Injury in Rats Based on Network Pharmacology

ZHANG Xiaojie, JIANG Lei

2020, 40(6):  496-505.  DOI: 10.3969/j.issn.1674-5817.2020.06.006

Asbtract ( 300 )   PDF (2307KB) ( 415 )  

Figures and Tables | References | Related Articles | Metrics

Objective To investigate the protective effect of resveratrol (RSV) against myocardial ischemia-reperfusion (MI/R) injury and its mechanism in rats based on the network pharmacological information screening method. Methods TCMSP database was used to screen the target genes of RSV, and GeneCards database was used to screen MI/R injury-related target genes. R programming language was used to achieve the intersection of the two sets, indicating the possible target genes for RSV treating MI/R injury, then a gene network was drawn by Cytoscape software to screen out the core target genes. Finally, the GO function and KEGG pathway of the screened gene targets were analyzed by R language. Thirty SD rate were randomly divided into a sham operation group (Sham group), an ischemia-reperfusion group (MI/R group) and a RSV administration group (RSV group). The MI/R injury rat model was induced by ligating the anterior descending coronary artery. The serum contents of lactate dehydrogenase (LDH), creatine kinase (CK) and cardiac troponin Ⅰ (cTn Ⅰ) as well as the size of myocardial infarction area in the MI/R injuny model rats induced by ligature of left anterior descending of coronary artery were determined. Hematoxylin-eosin staining (HE) was used to observe the structure of myocardium, TUNEL staining was used to detect cardiomyocyte apoptosis, immunohistochemistry and Western blotting were used to detect the expressions of apoptosis-related proteins (caspase-3 and Bcl-2) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Results According to the screening of pharmacology information, there were 86 core genes in RSV treatment of MI/R injury, which mainly involved the biological processes such as cytokine receptors, phosphatase binding, and death receptors, as well as the AGE-RAGE signaling pathway, apoptosis and other signaling pathways regulating diabetic complications. Compared with the Sham group, the expression of TRAIL protein, the contents of LDH, CK and cTn Ⅰ, and the infarct size increased (P<0.01, or P<0.05), while the arrangement of myocardial muscle fibers was disordered, and the apoptosis rate and caspase-3 protein expression level increased (P<0.05), but the expression of Bcl-2 was down-regulated (P<0.05). However, in the RSV group, the arrangement of myocardial muscle fibers was relatively regular, while the expression of TRAIL protein, the contens of LDH, CK and cTn Ⅰ, as well as the infarct size and apoptosis rate were significantly reduced (all P<0.05). Conclusion RSV may inhibit apoptosis and reverse MI/R injury by regulating TRAIL protein experssion.



Isolation and Identification of Exosomes from Skin Fibroblasts of Naked Mole Rats

FENG Yan, WU Wenqing, ZHANG Jingyuan, LI Yu, LI Leichen, YUAN Zheng, CUI Shufang

2020, 40(6):  506-512.  DOI: 10.3969/j.issn.1674-5817.2020.06.007

Asbtract ( 400 )   PDF (1380KB) ( 399 )  

Figures and Tables | References | Related Articles | Metrics

Objective To identify skin fibroblasts isolated and cultured from naked mole rats, and explore whether the skin fibroblasts have the ability to secrete exosomes. Methods The back skin samples of germ-free newborn naked mole rats were collected and were cut into pieces to a size of 1-2 mm². The samples were spread evenly over the plate, cultured in low-sugar DMEM (containing 20% fetal bovine serum) after cell adhesion, and treated by trypsin digestion for purification. The vimentin expressions were detected by immunofluorescence and Western blotting. When the cells were grown to about 80% confluency, they were moved to low-sugar DMEM (containing 20% fetal bovine serum without exosomes) and cultured for 48 h. Next the exosomes in supernatant were seperated by ultra-high-speed centrifuge, and determined vesicular structures under transmission electron microscope, particle size by nano flow cytometry, and specific proteins related to exosomes by Western blotting, respectively. Results A small amount of cells were seen free from the tissues after culturing for 30 h, and a large number of cells after 5 d. The cells were elongated spindle or irregular polygonal, growing radially. Immunofluorescence and Western blotting analysis confirmed that the skin fibroblasts expressed vimentin. Saucer-like membranous exosomes were observed under transmission electron microscopy, with particles size between 46.75-206.75 nm. Western blotting showed that the proteins specific to exosomes included the lysosmal membrane associated glycoprotein 3 (CD63), tumor susceptibility gene 101 (TSG101) and heat shock protein 70 (Hsp70). Conclusion The cells isolated from naked mole rats have typical fibroblast morphology and express actually vimentin. The skin fibroblasts are successfully isolated from the naked mole rats, and can secrete the exosomes, which provides a basis for studying the bioactive molecules carried by naked mole rat exosomes and their biological functions.



Effects of ⁶⁰Co γ-ray Radiation on Kidney, Lung and Skeletal Muscle Tissues of Naked Mole Rats

ZHANG Jingyuan, ZHANG Qianqian, ZHAO Yining, YAGN Rong, ZHANG Chengcai, HE Chenjiong, YUAN Zheng, CUI Shufang

2020, 40(6):  513-518.  DOI: 10.3969/j.issn.1674-5817.2020.06.008

Asbtract ( 383 )   PDF (1366KB) ( 376 )  

Figures and Tables | References | Related Articles | Metrics

Objective To observe and analyze the changes of the kidney, lung, and skeletal muscle morphology and the related indicators of oxidative stress in naked mole rats after total body irradiation with ⁶⁰Co γ-rays. Methods After 7 d and 14 d of total body radiation with ⁶⁰Co γ-rays at a one-time dose of 10 Gy, the kidney, lung and gastrocnemius muscle samples of naked mole rats were collected. The microstructures of the samples were observed under an optical microscope after HE staining. Indicators such as malondialdehyde (MDA), total antioxidant capacity (T-AOC), glutathione (GSH) and oxidized glutathione (GSSG) were detected with the corresponding biochemical test kits. Results Microscopic examination showed that there were no obvious pathological changes in the kidney, lung and skeletal muscle tissues of the naked mole rats after the designed irradiation. Biochemical examination showed that the content of MDA in kidney increased significantly after radiation (P<0.01), the content of GSH was significantly increased at first and then declined (P<0.05), and the contents of GSSG and T-AOC did not changed dramatically (P>0.05). The content of MDA in lung was significantly increased (P<0.05), and the contents of GSH, GSSG and T-AOC decreased at first and then increased over time (P<0.05). The content of MDA in skeletal muscle was significantly increased (P<0.01), while the contents of GSH and GSSH decreased firstly and then increased over time (P<0.01). The content of T-AOC decreased significantly on the 7th day after irradiation (P<0.01), and was maintained till the 14th day. Conclusion The kidney, lung and skeletal muscle of naked mole rats showed a certain degree of tolerance to ⁶⁰Co γ-ray radiation, and the specific tolerance mechanism is worthy of further study.



Effect of ⁶⁰Co γ-ray Radiation on Spleen of Naked Mole Rats

YANG Rong, ZHAO Yining, YANG Wenjing, ZHAO Shanming, CHAI Yujing, ZHANG Chengcai, YUAN Zheng, CUI Shufang

2020, 40(6):  519-522.  DOI: 10.3969/j.issn.1674-5817.2020.06.009

Asbtract ( 385 )   PDF (646KB) ( 371 )  

Figures and Tables | References | Related Articles | Metrics

Objective To observe the changes of immune system and tissue biochemical indexes in the spleen of naked mole rats exposed to ⁶⁰Co γ-ray radiation, for probing the radiation tolerance characteristics of the spleen of naked mole rats. Methods The whole body of naked mole rats was irradiated with a radiation dose rate of 1.163 Gy/min and a total dose of 10 Gy ⁶⁰Co γ-rays. The spleens of the normal control and the exposed naked mole rats after 7 d, 14 d, and 21 d of irradiation were collected. The proportions of B lymphocytes and macrophages in the spleens were measured by flow cytometry. Malondialdehyde (MDA), total antioxidative capacity (T-AOC), glutathione (GSH) and oxidative glutathione (GSSG) in the spleen tissues were measured with corresponding biochemical assay kits. Results Flow cytometry results showed that the numbers of both B lymphocyte and macrophages increased significantly after irradiation (P<0.001). Biochemical examination results showed that the MDA level in spleen was significantly increased (P<0.05) after irradiation, the GSH and GSSH levels were significantly decreased (P<0.05), the T-AOC level was first significantly decreased (P<0.01) and then raised 21 days after irradiation, which was significantly higher than the indexes 7 days and 14 days after irradiation (P<0.01). Conclusion The spleens of naked mole rats can tolerate ⁶⁰Co γ-ray radiation to some extent, but the mechanism of tolerance is not clear.



A Preliminary Study on Effects of Lipopolysaccharide on Lung Tissues of Naked Mole Rats

ZHANG Chengcai, LIU Yinhang, CHEN Chao, JIANG Wenzheng

2020, 40(6):  523-527.  DOI: 10.3969/j.issn.1674-5817.2020.06.010

Asbtract ( 349 )   PDF (645KB) ( 375 )  

Figures and Tables | References | Related Articles | Metrics

Objective To detect the effects of lipopolysaccharide (LPS) on lung morphology and pathological parameters, and explore the LPS tolerance in naked mole rats. Methods Ten male naked mole rats were randomly divided into an experimental group and a control group. The naked mole rats of the experimental group were injected with LPS at 10 mg/kg according the body weight, while the naked mole rats of the control group were injected with the same volume of normal saline. Six hours after the administration, lung tissues were collected to detect the ratio of wet to dry weight; paraffin sections were prepared and stained with HE, and the lung microstructure was observed under optical microscope; the level of reactive oxygen species (ROS) and the apoptosis ratio of lung tissue cells were detected by flow cytometry. Results Microscopic examination showed that the lung tissue structure of naked mole rats in the experimental group was intact, and there were no obvious exudate and inflammatory cell infiltration in the alveolar foramen. Compared with the control group, the ratio of wet to dry weight of lung tissues in the experimental group and the proportion of early apoptosis had no significant differences, but the proportion of late apoptosis and the content of ROS increased significantly (P < 0.01, and P < 0.05, respectively). Conclusion Naked mole rats can tolerate the lung injury induced by LPS to some degree, and the specific mechanism needs to be further studied.



Establishment and Applications of Tumor Organoid Models

LUO Baohua, ZHANG Yongbin, LIU Xiaoqiu, SHI Changhong

2020, 40(6):  540-546.  DOI: 10.3969/j.issn.1674-5817.2020.06.015

Asbtract ( 451 )   PDF (435KB) ( 513 )  

Figures and Tables | References | Related Articles | Metrics

The organoid is derived from specific stem cells for in vitro three-dimensional culture to form a spatial structure, which keeps similar biological characteristics to the original organ. By this culture technology, the tumor organoids can be established. It well retains the feature in vivo and heterogeneity of the original tumor tissues, displays the advantages of short culture cycle and stable genomes after multiple passages, which compensates the defects of traditional tumor models. These tumor organoids can be used to study the mechanism of tumor development, drug screening and individualized treatment, immunotherapy, liquid biopsy, etc. In this paper, the development history, establishment methods, application prospects and limitations of tumor organoid models are reviewed.

Research Progress in Construction and Application of Diabetes Model in Zebrafish

JIANG Xia, QIAN Haojie, WEI Xun, ZHENG Yuxuan, ZHOU Zhengyu

2020, 40(6):  547-552.  DOI: 10.3969/j.issn.1674-5817.2020.06.016

Asbtract ( 573 )   PDF (410KB) ( 544 )  

References | Related Articles | Metrics

Diabetes mellitus is a group of metabolic disorders, and its pathogenesis has not been fully elucidated. Diabetes mainly includes type 1 diabetes, type 2 diabetes and gestational diabetes. Many animals such as mice, rats, rabbits, mini-pigs and macaques are commonly used as diabetes models. As a model organism, zebrafish (Danio rerio) has been widely used to simulate human disease in many fields, including type 1 and type 2 diabetes mellitus. By searching a large amount of literature, this article summarized the current construction methods of zebrafish models of diabetes and its application progress in order to provide references for its future research and application.