Table of Content

25 February 2020, Volume 40 Issue 1

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The Role of Villin-1 in Model of Habu Nephritis Mice with Unilateral Nephrectomy

JIANG Hongli, MA Hongye, XUE Jinhong, SUN Lingshuang, CHEN Lei

2020, 40(1):  1-8.  DOI: 10.3969/j.issn.1674-5817.2020.01.001

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Objective To investigate the differences of renal function, renal pathological manifestations and proteins expression between the Habu nephritis mice model with or without unilateral nephrectomy (UNX), as well as the molecular mechanism leading to these differences. Methods Twenty- four male SPF C57BL/6 mice (18-20 g) at the age of 6 weeks were randomly divided into two groups. One group (n=12) received UNX and then received tail vein injection of Habu snake venom (HSV-UNX group) after 1 week, and the other group (n=12) received sham surgery and then received tail vein injection of Habu snake venom (HSV group). The renal function of mice in the two groups was detected by blood biochemistry, and the renal histopathological changes of two groups were measured through periodinate-schiff staining. Furthermore, the expression levels of protein in glomeruli of the two groups were analyzed by comparative proteomics, and the differentially expressed proteins between the two groups were screened out. The levels of proteins, such as villin-1(IL1), caspase 3, Bax, p21 and p27, were validated by Western blotting. Results The renal function injury of the HSV-UNX group was worse than that of the HSV group (P<0.01), and the renal mesangiolysis level was higher than that of the HSV group (P<0.0001), while the mesangial proliferation was milder than that of the HSV group (P<0.001). It is confirmed by proteomic analysis and western blotting that VIL1 expression level in the HSV-UNX group was lower than that of the HSV group. In mouse mesangial cells (MMCs), cell apoptosis (P<0.01) and inhibiting cell proliferation (P<0.05) were protomed by VIL1 knockdown through up-regulating the expressions of caspase 3, Bax, p21 and p27 proteins. Conclusions VIL1 is down-regulated in the HSV-UNX mouse model, which impairs the self-repair ability of glomerular and aggravates the renal function damage by inhibiting cell proliferation and promoting cell apoptosis.



A Rabbit Model of Myocardial Infarction by Using Minimally Invasive Cardiac Catheter Technique

ZHOU Zhiwen, WANG Yucheng, ZHAO Lifang, CAO Jiaqi, DING Yueyou, LIU Huidong, HOU Lei, CHANG Wei

2020, 40(1):  9-14.  DOI: 10.3969/j.issn.1674-5817.2020.01.002

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Objective To establish a rabbit model of myocardial infarction (MI) by occluding coronary artery with cardiac catheter technique. Methods After general anesthesia, rabbit's carotid artery was isolated, and 5 F arterial sheath was inserted in carotid artery using Seldinger's method. Guiding catheter was send to the sinus at the root of the aorta through the arterial sheath. The microcatheter was inserted into the middle or distal left of anterior descending (LAD) by using a micro-guide wire. The middle or distal of LAD was occluded by injection of gelatin sponge embolic agent or spring coil through the microcatheter. After 1 month, the rabbit's heart was removed and the MI area was examined by pathology. Results Ninteen of 25 rabbits were successfully transported microcatheter into LAD. 10 rabbits were completed by using the spring coil for MI model, the other 9 rabbits were completed by using the embolic agent. In the spring coil group, 7 of them were successfully produced of MI model, 2 of them died in feeding. In the embolic agent group, 7 of them were successfully produced for MI models, one of them died in feeding. There was no significant difference in MI area and success rate between the two groups. Conclusions The rabbit MI model can be made by minimally invasive cardiac catheter technique with high success rate. It can avoid thoracotomy injury and be more similar with the pathological characteristics of clinical MI.



Comparative Pathological Analysis of Herpes Simplex Virus Type 2 Infection in BALB/c Mice by Different Routes

CHENG Jishuai, MOU Tangwei, LIU Lei, WU Huaye, LI Qihan

2020, 40(1):  15-21.  DOI: 10.3969/j.issn.1674-5817.2020.01.003

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Objective To compare the pathological changes of BALB/c mice infected with herpes simplex virus type 2 (HSV-2) by different routes. Method BALB/c mice were challenged with HSV-2 by three different ways , namely, through vagina, nasal cavity and cornea. The clinical manifestations of mice and the pathological changes of challenged site and nerve tissue were observed, and the viral load in nerve tissue was detected. Result There was no overt symptom in the nasal cavity of which the survival rate was 27.27% at the end of observation period. The vaginal group showed different degrees of vulvitis and weight loss and other symptoms, and the survival rate of the mice on day 8 was 0. Corneal group mice showed different degrees of keratitis, the survival rate at the end of observation period was still 63.64%. The results of pathological changes showed that there were obvious pathological changes in the brain tissue of nasal group mice. Both the vagina group and the cornea group mice showed significant pathological changes in the offending sites, and the brain and spinal cord of the vagina group mice showed obvious pathological changes. The results of viral load detection in the nerve tissue showed that, compared with control group, the viral load of HSV-2 in the brain and spinal cord of the three experimental groups was significantly increased with the increase of infection time. Conclusions The infection of HSV-2 by three different ways can cause different pathological changes in mice. This study provides a theoretical basis for the study of the pathogenesis of nervous system disease induced by HSV-2 infection.



Dynamic Behavioral Assessment of Vascular Dementia Rat Models Made by Bilateral Common Carotid Artery Occlusion

CONG Lin, ZHANG Zhilong

2020, 40(1):  22-27.  DOI: 10.3969/j.issn.1674-5817.2020.01.004

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Objective To observe the mortality of vascular dementia (VaD) rat models induced by bilateral common carotid artery occlusion (BCCAO), and to dynamically observe the cognitive function of rats after BCCAO via Morris water maze (MWM) test. Methods Sixty-eight male SD rats with no significant difference in intelligence were randomly divided into sham operation group (9 rats) and BCCAO group (59 rats). The mortality of the two groups was recorded within 30 days after operation. MWM behavioral tests were performed in 30-34, 60-64 and 90-94 days after operation to evaluate the cognitive function of the rats in BCCAO group and to detect the rate of VaD model forming in BCCAO group. Results Within 30 days after BCCAO, the mortality rate of rats was 20.3%. In MWM behavioral test, the average speed of rats in BCCAO group was basically same as that in sham operation group, with no significant difference (P>0.05); the escaping latency of rats in BCCAO group was significantly longer than that in sham operation group at 60-64 and 90-94 days after operation (P<0.05), and the VaD model rate was 97.9% and 95.7% respectively. Conclusions BCCAO is safe and reliable method to make VaD rat models. There is no obvious motor dysfunction in the rats after operation. VaD in the model rats can be basically formed between 35 and 60 days after operation and lasted till 90 days after operation.



Establishment of Atherosclerosis Model Induced by Feeding High-fat Diet Plus Carotid Artery Balloon Injury in Rabbits with Insulin Resistance

XU Zhenkun, REN Liwei, YUAN Tao

2020, 40(1):  28-32.  DOI: 10.3969/j.issn.1674-5817.2020.01.005

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Objectives To identify a quick, stable and reliable method for establishing atherosclerosis (AS) model in rabbit. Methods The atherosclerosis rabbit models were established by feeding high-fat diet plus carotid artery balloon injury. twenty healthy New Zealand white male rabbits weighting 2.0-2.5 kg were injured by carotid artery balloon and fed with high fat diet. Before and after 12 weeks feeding, blood from jugular vein were collected to detect total cholesterol (TC), triglyeride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), fasting blood glucose (FBG), fasting serum insulin (FSI), insulin sensitivity index (ISI) and homeostasis model assessment insulin resistance (HOMA-IR). Total carotid artery endometrium morphology was observed with naked eye and microscope. Results After feeding high fat diet for 12 weeks, the body weight, TC, TG, LDL-C of the rabbits were significantly higher than pre-treatment (P<0.01). The difference of FBG was not statistically significant compared with pre-treatment (P=0.550). FSI and HOMA-IR were significantly higher than pre-treatment (P<0.01). HDL-C and ISI were significantly lower than pre-treatment (P<0.01). The typical atherosclerosis mottling in common carotid artery lumen under macrography was visible and intima thickness increased significantly. Plaque and structure of fibrous cap had formed. Conclusions The typical carotid atherosclerosis lesion can be successfully induced by high fat diet combining with balloon injury in carotid artery. On the other hand, high fat diet can induce insulin resistance model.



Protective Effects of Different Sunscreen Standards on Skin Photodamage Induced by Ultraviolet Radiation in Rats

XU Yingyu, WANG Xiaohui, JIANG Yi, LI Min, PANG Zengxiong, LI Guangxian, CHEN Ziling, SUN Xia

2020, 40(1):  33-38.  DOI: 10.3969/j.issn.1674-5817.2020.01.006

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Objective To study the protective effects of different sunscreen standards on skin photodamage induced by ultraviolet radiation in rats, and to provide background data for the evaluation of sunscreen efficacy of sunscreen cosmetics. Methods The skin phototoxicity damage rat models were established by ultraviolet radiation of Ultraviolet A (UVA ,4.5 mJ·cm^-2·s^-1) + Ultraviolet B (UVB,0.036 mJ·cm^-2·s^-1). The rats were irradiated twice a week for 4 weeks, and the cumulative radiation time was 440 minutes. The sunscreen standard products (S1、S2、P2、P3 and P7)were applied 30 minutes before each exposure. After the experiment, the skin was observed histopathologically and the expression of Melanoma marker (human), mAb (HMB45) was detected by immunohistochemistry. Results Compared with the model group, the four indexes of epidermal thickening, sebaceous gland hyperplasia, skin injury score and HMB45 expression in group S2 were significantly alleviated; the three indexes of epidermal thickening, skin injury score and HMB45 expression in group P3 and group P2 were significantly alleviated compared with the model group; the two indexes of skin thickening and skin injury score in group S1 were significantly alleviated compared with the model group. In the group P7, only one index of epidermal thickening was significantly reduced compared with the model group. Generally speaking, the protective effect of the standard conforms to the values of PFA and SPF. Conclusions The protective effect of sunscreen cosmetics on skin photodamage caused by ultraviolet radiation is related to PFA and SPF values. This method can be used to evaluate the sunscreen efficacy of sunscreen cosmetics.



O-GlcNAc Glycosylation Level Positively Regulates Ang-2 Expression and Promotes Tumor Angiogenesis in Mice with Liver Cancer

WANG Aihong, WANG Mingquan, DU Juan

2020, 40(1):  39-46.  DOI: 10.3969/j.issn.1674-5817.2020.01.007

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Objective To explore the relationship between O-linked N-acetylglucosamine (O-GlcNAc) glycosylation and Angiopoietin-2(Ang-2) expression in the development of hepatocellular carcinoma. Methods C57BL/6 mice model of hepatocellular carcinoma was induced by Diethylnitroamine (DEN) to detect the changes of O-GlcNAc glycosylation, Ang-2 and cell adhesion molecule-1 (CD31) expression in hepatocellular carcinoma. HepG2 cells were intervened with O-linked Glc NAc-transferase (OGT) inhibitors and O-Glc NAcase (OGA) inhibitors to detect whether the expression of Ang-2 changed with the changes of O-GlcNAc glycosylation level. MTT was used to detect the relationship between O-GlcNAc glycosylation level and proliferation of hepatocellular carcinoma cells. Transwell method was used to detect relationship between the level of O-GlcNAc glycosylation and cell migration and invasion in HUVEC cells. Results In the DEN-induced hepatocarcinoma model, the liver volume of mice increased significantly, and tumors of different sizes could be seen in the liver. The results of immunohistochemistry showed that the glycosylation level of O-GlcNAc and the expression of Ang-2 increased significantly in the liver of the model group compared with the control group (P<0.05). The results of qRT-PCR showed that the level of O-GlcNAc glycosylation affected the expression of Ang-2 by detecting the levels of OGT, OGA and Ang-2. Western blotting showed that the expression of Ang-2 decreased with the decrease of the glycosylation level of O-GlcNAc after the intervention of OGT inhibitors on HepG2 cells. After the intervention of HepG2 cells with OGA inhibitors, the expression of Ang-2 increased with the decrease of the glycosylation level of O-GlcNAc. MTT results showed that the glycosylation level of O-GlcNAc was closely related to the proliferation of hepatocellular carcinoma cells. The proliferation ability of hepatocellular carcinoma cells was stronger when the glycosylation level of O-GlcNAc was higher,. The proliferation ability of hepatocellular carcinoma cells was significantly inhibited, when the glycosylation level of O-GlcNAc was low,. Transwell results showed that HUVEC cell migration and invasion ability was positively correlated with O-GlcNAc glycosylation level. Conclusions During the development of hepatocellular carcinoma, the glycosylation level of O-GlcNAc increases gradually, and the expression level of Ang-2 positively regulated by the glycosylation of O-GlcNAc, thus promoting neovascularization.



Evaluation of Monoiodoacetic Acid-induced Knee Osteoarthritis SD Rats with Diseasse Progression at Different Time Points

XIAO Kunlin, ZHANG Rui, Sun Hong, XIAO Kuntai, MA Jianbing

2020, 40(1):  47-52.  DOI: 10.3969/j.issn.1674-5817.2020.01.008

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Objective To evaluate disease progression of monoiodoacetic acid-induced knee osteoarthritis (OA) rat model at different time points. Methods Twenty-four male SD rats at the age of two month were randomly divided into model group (18 rats) and control group (6 rats). In the model group, 2 mg monoiodoacetic acid (MIA) dissolve in 50 μL saline was injected into the right knee joint of the mice, and the control group mice were injected with the same amount of saline. After 1 week, 2 weeks and 4 weeks respectively, 6 rats in each group were sacrificed, and the joint specimens were collected for morphological observation and histological analysis (HE, toluidine blue, and Safranin O staining). OA cartilage histopathology assessment system (OOCHAS) scoring and histological scoring were performed to evaluate the disease progression. Results The model group showed defects in articular cartilage, reduced number of chondrocytes, blurred tide line and lightened toluidine blue and safranin O staining. The severity was aggravated after 1 weeks, 2 weeks and 4 weeks. The OOCHAS gross score and histological score was significantly increased with time extended. Conclusions The rat model of knee OA was successfully induced by intra-articular injection of monoiodoacetic acid, and the results at different time points were correlated with the clinical disease process, which laid a fundament for later functional and mechanism experiments.



Effect of Runzao Mingmu Decoction on Expression of Caspase-3,Bcl-2 and Bax in Retina and Its Tissue of Rats with Acute High Intraocular Pressure

WAN Yu, LI Zhanmei, HUANG Hai, HUANG Xuewen, HE Yilan

2020, 40(1):  53-59.  DOI: 10.3969/j.issn.1674-5817.2020.01.009

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Objective To explore the possible mechanism of Runzao Mingmu Decoction in protecting optic nerve membrane cells in rats with acute high intraocular pressure. Methods Sixty male Sprague-Dawley rats were randomly divided into 6 groups. According to different treatment methods, they were divided into blank group, model group, positive drug group, Runzao Mingmu Decoction low dose group, medium dose group and high dose group. The changes of intraocular pressure and the expression of apoptosis-related factors in retinal tissue were detected and recorded in each group. Results (1) Immediately after modeling, there was no significant change in the blank group, and the intraocular pressure of the other groups was significantly increased (P<0.05). There was no significant difference in intraocular pressure between the model group, the low-dose group and the middle-dose group at 8 weeks after treatment (P>0.05), but the intraocular pressure of the high-dose group was significantly lower than that of the positive drug group (P<0.05). (2) The superoxide dismutase (SOD) level was the highest in the blank group, and the levels of malondialdehyde (MDA), nitric oxide (NO), glutamic acid (Glu) and Ca²⁺ were the lowest (P<0.05). Compared with the simple model group, there were significant differences between the drug-administered dose group and the positive drug group (P<0.05). Compared with the positive drug group, the levels of NO and Glu in the retinal tissue of the high-dose intervention group were Gradually decreasing trend, the difference was statistically significant (P<0.05). (3) The average integral optical density of Bcl-2 in rat retinal ganglion cells increased gradually with the increase of treatment dose, but there was no significant difference (P>0.05), while the average integral optical density of Bax and Caspase-3 decreased gradually, showing a dose-dependent (P<0.05). (4) Compared with the simple model group, the relative expression of Bcl-2 mRNA in the retinal ganglion cells of each group increased gradually, and the relative expression of Bax mRNA and Caspase-3 mRNA decreased gradually, all of which showed a dose-dependent difference. Statistically significant (P<0.05); the expression of Bcl-2 mRNA and Bax mRNA in the optic nerve tissue of the high dose intervention group was better than that of the positive drug group (P<0.05). Conclusions Runzao Mingmu Decoction has obvious protective effect on optic nerve in rats with acute high intraocular pressure. The mechanism may be related to the inhibition of Caspase-3/Bcl-2/Bax signaling pathway activity.



Effect of Astragalus Injection on mRNA Expression of IL-1β、MCP-1 and TNF-α in Kidney of db/db Mice

ZHU Likun, CAO Shuang, JIANG Shilin, YIN Xiaolin

2020, 40(1):  60-64.  DOI: 10.3969/j.issn.1674-5817.2020.01.010

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Objective To investigate the effects and the mechanism of Astragalus injection regulating immune cells and Inflammatory factors in db/db mice. Methods C57BL/6 mice were set as the control group, db/db mice were randomLy divided into diabetic group and astragalus-treated group. The mice of control group and diabetic group were intraperitoneally injected with PBS 100 μL, and the mice of astragalus-treated group were intraperitoneally injected with astragalus mongholicus 100 μL, every day for 12 weeks. Twelve weeks later, blood glucose and the counting and classification of leukocytes of the peripheral blood were detected. The expression of interleukin-10(IL-10) and tumor necrosis factor-α(TNF-α)was detected by ELISA in cell culture supernatant. The mRNA expression of interleukin-1β(IL-1β), TNF-α and monocyte chemoattractant protein-1(MCP-1) in mouse kidney was detected by real-time PCR. The percent of regulatory T cells(Tregs) was detected by flow cetometry. Results Blood glucose in diabetic group is higher than that in control group. There is no significant difference between astragalus-treated group and diabetic group. The counting leukocytes and the percentage of neutrophils and mmonocyte in diabetic group obviously were improved, and that in astragalus-treated group were decreased obviously. IL-10 in the serum in diabetic group was obviously lower than that in control group. However, IL-10 in astragalus-treated group was significantly higher than that in diabetic group. TNF-α in the serum in diabetic group was obviously higher than that in control group, and that in astragalus-treated group was significantly lower. mRNA expression of renal inflammatory factors in mice showed that the mRNA expression of IL-1β, MCP-1 and TNF-α in the diabetes group was significantly higher than that in the control group, and that in the astragalus-treated group was significantly lower than that in the diabetes group. The quantity of Tregs in diabetic group was improved, and that in astragalus-treated group was significantly higher. Conclusions Astragalus injection can reduce the number of inflammatory cells in db/db mice, increase the number of Tregs, reduce the expression of pro-inflammatory factors IL-1β, MCP-1 and TNF-α delay the occurrence of diabetic nephropathy.



Probe into Standardization of Acute Toxicity Test Method for Sea Urchin (Anthocidaris crassispina)

LAI Suqun, YU Lujun, WU Meihui, ZENG Jin, LI Jianjun

2020, 40(1):  65-69.  DOI: 10.3969/j.issn.1674-5817.2020.01.011

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Objective To optimize the sea urchin (Anthocidaris crassispina) acute toxicity test conditions and provide the scientific basis for standard test methods. Methods Cultivate sea urchins at a water temperature of 20 ℃, 22 ℃, 24 ℃, 26 ℃, 28 ℃ and 30 ℃ respectively, and record the larval development time and mortality. Calculate the 96 h LC₅₀ and the coefficient of variation of Cr⁶⁺, Zn²⁺, 3,5- Ddichlorophenol, Sodium dodecyl sulfate on the larvae at different developmental stages. Results In 24-26 ℃ water temperature, the survival rate was higher and the larval were growing more rapidly. The 96 h LC₅₀ of 12 day-old sea urchin for Cr⁶⁺, Zn²⁺, 3,5-Ddichlorophenol, Sodium dodecyl sulfate were 1.40, 1.69, 3.60 and 3.95 mg/L respectively. The sensitivity of 12 day-old sea urchin was moderate and the coefficient of variation of toxicity test was low. Conclusions The suitable temperature of acute toxicity test for sea urchin larvae is 24-26 ℃, and the best starting day age for the test is 12 days age. Anthocidaris crassispina is a species suitable for marine pollutant toxicity test.

Application of Sentinel Animals in Barrier Environment

ZHAO Yong, FAN Chun, ZHU Minjuan, JIN Yi, CHEN Guoqiang

2020, 40(1):  70-73.  DOI: 10.3969/j.issn.1674-5817.2020.01.012

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Sentinel animals in barrier environment should not only take into account the species, strain, sex, age, quantity, contact mode, placement position and detection frequency, but also the elimination of interference of animal stress on the monitoring results. Aaccording to the 3R theory of laboratory animals, make full use of sentinel animals as feed nutrition monitoring sentinel animals, put forward to set up the number of sentinel animals, that is the actual detection number of sentinel animals.

The Research Progress of Pathological Characteristics of Animal Models with Heart Failure with Ejection Fraction Preservation

DONG Guoju, LIU Jiangang, Guan Jie

2020, 40(1):  74-79.  DOI: 10.3969/j.issn.1674-5817.2020.01.013

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Heart failure with ejection fraction preservation (HFpEF) is the main type of heart failure at present, but there is no effective prevention and treatment method. To develop new drugs on preventingHFpEF and elucidate its pharmacodynamic and pharmacological characteristics, it is urgent to establish animal models that can reflect the pathogenesis and pathological characteristics of HFpEF. We reviewed the animal models that can reflect the pathological characteristics of HFpEF. And we expect more representative HFpEF animal models can be created further.

Microglia and Anxiety: The Key Role and Mechanism

LU Dengcheng, SHI Anhua, CHEN Shuai, WEI ShanShan

2020, 40(1):  80-86.  DOI: 10.3969/j.issn.1674-5817.2020.01.014

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Recently, the incidence of anxiety has increased, but its pathogenesis is still unclear and treatment results are unsatisfied. Microglia (MG) are innate immune cells which are widely distributed in the central nervous system (CNS) and participate in the immune process of CNS. Increasing studies have shown that over-activated MG play an important role in the development of anxiety. Stress-activated MG are divided into 2 polarization phenotypes: Classical activated MG (M1) and alternatively activated microglia (M2) according to their functions. M1 can promote the development of anxiety by inducing immune disorders, affecting neurotransmitter release, altering neuronal signaling, and activating endocrine systems; while M2 have the effects of promoting nerve regeneration, angiogenesis, remyelination to relieve anxiety.The study of MG function is expected to find a new perspective in mechanism of anxiety, and inducing the transformation of MG to M2 may be potentially benefited for the treatment of anxiety.