Abstract: Objective To observe changes in the intestinal flora in a Wuzhishan minipig model of atherosclerosis (AS) induced by a high-fat / high-sugar diet. Methods Twelve male Wuzhishan minipigs were divided into normal control (NC) and AS model groups according to body weight (n= 6 per group). The NC group was fed a normal diet and the AS model group was fed a high-fat / high-sugar diet. After 24 weeks of continuous diet, blood samples were taken from the anterior vena cava to determine biochemical indexes, including glucolipid metabolism, and a glucose to tolerance test was also performed. At the end of the experiment, the minipigs were euthanized and body fat deposition was observed. The aortic vessels were stained with Sudan IV and the abdominal aortic vessels were stained with hematoxylin and eosin to observe lipid deposition and histopathological changes, respectively. The cecal contents were used for 16S rDNA sequencing to analyze changes in the intestinal flora. Results Serum total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, blood glucose, and fructosamine levels were all significantly higher in the AS model group compared with the NC group (P< 0.05, P< 0.01). Body weight and body fat deposition were also significantly higher in the AS model group ( P< 0.01). The aortic vessels showed increased lipid deposition and AS lesions. DNA sequencing analysis showed significantly lower intestinal flora α-diversity in the AS model group compared with the NC group ( P< 0.05, P< 0.01). At the phylum level, the phyla Bacteroidetes and Actinomycetes were significantly reduced in the AS model group (P< 0.05, P< 0.01), while the phylum Proteobacteria and the Firmicutes/ Bacteroidetes ratio were significantly increased (P< 0.05). At the genus level, the relative abundances of 17 genera, including Hespellia, Ruminococcus, Lachnospiracea_incertae_sedis, Pseudobutyrivibrio, and Barnesiella, were significantly decreased (P< 0.05, P< 0.01), and the relative abundances of 11 genera, including Ramlibacter, Sphingorhabdus, Cloacibacillus, Oxalobacter, Sphingomonas, and Novosphingobium, were significantly increased (P< 0.05). Functional prediction analysis showed that multiple pathways such as lipopolysaccharide biosynthesis, lipopolysaccharide biosynthesis proteins, primary bile acid biosynthesis, and arachidonic acid metabolism may participate in the formation of AS. Correlation analysis showed that total cholesterol, intima media thickness, lipid deposition, and fructosamine were associated with 14 genera, including Oxalobacter, Sphingomonas, Cloacibacillus, Clostridialse _ unclassified, and Ruminococcus. Conclusions High-fat / high sugar diet-induced AS in Wuzhishan minipigs is characterized by obesity, disturbed glucolipid metabolism, and obvious AS lesions, possibly related to dysbiosis of the intestinal flora.

Abstract: Objective To investigate regulation of serum cyclic nucleotides (cAMP and cGMP) and the repair of dopamine neurons in mice with Yin deficiency and moving wind syndrome of Parkinson’ s disease ( PD) by Compound Dihuang Granule. Methods Overall, 24 male C57BL/ 6 mice were randomly divided into normal ( Normal), model (MPTP + NS), and treatment (MPTP + CDG) groups with eight mice in each group. The normal group was injected with normal saline and the model group was intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish the PD animal model. Both groups were injected once a day for 5 consecutive days. The treatment group was administered Compound Dihuang Granule (CDG) by gavage once a day for 7 days. The TCM syndrome attribute of the model was verified by etiology, symptoms, the objective index, and formula test in PD mice and the effect of CDG on expression of cyclic nucleotides and tyrosine hydroxylase in the serum of PD mice with Yin deficiency and moving wind syndrome was assessed. Results Significant differences in behavior, the tongue chroma value ratio, serum cyclic nucleotide content, and tyrosine hydroxylase content were found between model and normal control groups (P< 0.05). CDG treatment significantly improved the behavior and related objective indexes of mice (P< 0.05). Conclusions The mouse model of Yin deficiency and moving wind syndrome of Parkinson’ s disease prepared by intraperitoneal injection of MPTP is stable and reliable. CDG regulates the content of cyclic nucleotides in serum and effectively repairs damaged dopaminergic neurons.

Abstract: Objective To explore the effect of Naja atra venom on blood coagulation in rats. Methods Whole blood was incubated with Naja atra venom or various fractions in vitro and a thromboelastogram was obtained. In an in vivo experiment, Naja atra venom was injected via the tail vein or intramuscularly and whole blood samples were collected at various time points to obtain the thromboelastogram. LDH, hemoglobin, vWF, and P-selectin were measured in plasma. The activity of coagulation-related enzymes in plasma was measured by the chromogenic substrate method. Results The in vitro and in vivo result showed that Naja atra venom and some of its fractions obviously induced an abnormality in the coagulation function in rats, which was characterized by a lack of coagulation factors, low level of fibrinogen, and lowering of the platelet count and function, while the plasma hemoglobin content and LDH activity were increased significantly. In vitro, Naja atra venom and its fractions significantly decreased the enzymatic activities of cleaving S2238 and S2251. In vivo, after intramuscular injection, the content of P-selectin was significantly increased at 30 min and the enzymatic activity of cleaving S2765 was significantly increased within 6 h. After tail vein injection, the enzymatic activity of cleaving S2238 was increased significantly within 2 h and vWF content was increased obviously within 6 h. Conclusions Naja atra venom induces an obvious abnormality in the blood coagulation function of rats. Its effect is mainly related to the consumption of coagulation factors and fibrinogen caused by activation of the coagulation system and the consumption of platelets.

Abstract: 目的 探究 Clcn3 敲除对幼龄小鼠实质器官发育的影响,为 Clcn3 基因敲除动物应用提供理论参考。 方法 PCR 鉴定 FVB 小鼠基因型:野生型(Clcn3^{+ / +} )、杂合子(Clcn3^{+ / -})及纯合子(Clcn3^{- / -} );小鼠 3 周龄时,采集心、肝、脾、肺、肾和脑等主要实质器官并拍照,利用 Image J 软件测量其投影面积;中性福尔马林溶液固定各实质器官,石蜡切片进行 HE 染色,全切片利用全自动数字病理扫描分析系统扫描后观察器官细微结构。 结果 与 Clcn3^{+ / +}和 Clcn3^{+ / -}小鼠相比,Clcn3^{- / -}小鼠体重显著降低(P< 0.05);Clcn3 缺失会显著降低小鼠心、肝、脾、肺及肾的体积(P< 0.05),但不会影响小鼠脑的大小(P> 0.05);HE 染色显示,Clcn3^{- / -}小鼠各实质器官组织结构未见异常,实质细胞体积相对较小。 结论 Clcn3 敲除导致幼龄小鼠的实质器官减小,未对幼龄小鼠实质器官产生明显病理损伤。

Abstract: Objective To construct a muscle-specific Cas9-tracking expression vector to examine myoblast differentiation and establish a muscle-specific Cas9-expressing mouse model. Methods The muscle-specific promoter SP was synthesized to replace the CMV promoter in the PX459 vector. The editing efficiency of the recombinant vector in C2C12 cells was assessed by XbaI and T7E1 digestion. A DsRed fluorescent reporter protein was linked through homologous recombination to construct a muscle-specific Cas9-tracking expression vector (PX459-Rosa26-SP-DsRed). The SP-Cas9-DsRed fragment was connected between the left and right homologous arms of the Rosa26 site to construct a muscle-specific Cas9-tracking expression recombinant vector. The vector was cotransfected with PX459-Rosa26 into C2C12 cells that were selected by puromycin and fluorescence was observed. DNA was extracted for PCR and sequencing to detect integration of the vector in C2C12 cells. Results Enzyme digestion and sequencing showed that PX459-Rosa26-SP and Donor-Cas9-SP-DsRed were constructed successfully. Enzyme digestion showed that the editing efficiency of PX459- Rosa26-SP was 18. 38%. The transfected C2C12 cells emitted red fluorescence, indicating that Donor-Cas9-SP-DsRed was specifically expressed in muscle cells. PCR and sequencing showed that Donor-Cas9-SP-DsRed was successfully integrated at the Rosa26 site in C2C12 cells. Conclusions A muscle-specific Cas9-tracking expression vector was constructed successfully, which provides the basis to prepare a muscle-specific Cas9-expressing mouse model and new research ideas for gene therapy of muscle-related genetic diseases.

Abstract: Objective To observe the effects of a visceral hypersensitivity model in rats with irritable bowel syndrome established by neonatal maternal separation combined with colorectal distension and drugs with chronic stress, and to discuss their features. Methods Two-day-old and two-month-old Sprague-Dawley rats were randomly divided into CRD, control, and CUMS groups. After establishing the model, the general conditions, stools, visceral sensitivity, and anxiety-depression-like behavioral changes were evaluated. Colonic tissue was obtained for pathological examination. Results Both model groups had a higher AWR score, lower visceral pain threshold, and changed fecal characteristics and water content. The CUMS group had a shorter open arm residence time of the elevated plus maze test and shorter total distance and central area residence time of the open field test. Conclusions Both models showed visceral hypersensitivity and fecal changes, which agreed with the clinical symptoms of IBS, and are more inclined to the diarrhea type. Additionally, the CUMS group model had emotional changes.

Abstract: Objective To design and construct a rodent awake activity device for brain microdialysis recording and achieve the application and verification of brain microdialysis sampling in mice. Methods The rodent awake activity device for brain microdialysis recording was designed and constructed using relevant materials. Dopamine-related neurotransmitters in the substantia nigra-striatum neural circuit in mice were measured and verified by this device. Microdialysis probes were implanted into the substantia nigra and striatum. Recording and microdialysis sampling were performed for 7 days after operation. Dopamine and its metabolic intermediate DOPAC were analyzed in the substantia nigra and striatum. The microdialysis flow rate was 2 μL/ minute. Samples were collected every 20 minutes for 200 minutes to analyze DA and DOPAC concentration changes over time. Results ( 1) Mouse brain samples showed stable levels of neurotransmitters. (2)Dopamine concentrations were (0. 11 ± 0. 01) nmol / L in the substantia nigra and (0. 22 ± 0. 05) nmol / L in the striatum. DOPAC concentrations were (3. 38 ± 0. 59) nmol / L in the substantia nigra and (20. 35 ± 6. 87) nmol / L in the striatum. No significant differences were found between each sampling period. Conclusions The rodent awake activity device for brain microdialysis recording is practical and stable with good applications in time and space dimensions. Two national patents have been applied for and authorized: Patent for invention ( 202110525342. 5) and Patent for utility models (202121027247. 4).

Abstract: Objective To establish a simple and reproducible model and its evaluation system for bradycardiac arrhythmia in Zebrafish. Methods Wild-type and heart-labeled GFP transgenic zebrafish were employed in this study after 24 h of fertilization. Twenty-four hpf (hour post-fertilization) zebrafish embryos were treated with various concentrations of verapamil, propafenone, metoprolol, amiodarone, and mexiletine for 48 hours, then the cardiac function and cardiotoxicity, including survival rate, heart rate, stroke volume, cardiac output, ejection fraction, area difference between ventricular diastole and systole and the distance between sinus venosus and bulbus arteriosus (SV-BA), were observed in each group. Results To evaluate drug toxicity, the median lethal concentration ( IC₅₀ ) was ranked as propafenone < mexiletine < amiodarone < metoprolol and verapamil. After treatment with verapamil, the heart rate, cardiac output, ejection fraction and difference between ventricular diastolic and systolic areas significantly decreased in Zebrafish, while the distance of SV-BA increased. Treatment of propafenone reduced the heart rate and cardiac output in Zebrafish. Treatment of metoprolol decreased the heart rate in Zebrafish. Treatment of amiodarone decreased the heart rate and increased the distance of SV-BA in Zebrafish. Treatment of metoprolol increased the distance of SV-BA in Zebrafish. Conclusions Verapamil, propafenone, metoprolol and amiodarone all could be used to construct zebrafish models of bradycardiac arrhythmia, among which verapamil had the best efficacy. This study proposes the use of anti-arrhythmic drugs, particularly verapamil, to establish the bradycardiac arrhythmia model using zebrafish as well as the corresponding evaluation indicators. This study is of great significance to the research of bradycardiac arrhythmia and its drug development.

Abstract: Objective To use a modified protocol to obtain in vitro-cultured mouse ependymal cells (MEPCs) and to assess the morphological change of MEPCs during differentiation, the process of cilia formation, and the ciliary motility of mature MEPCs. Methods MEPCs with physiological functions were obtained by specialized culture and induced differentiation from isolated neonatal mouse telencephalon cells. Cilia formation and maturation of MEPCs were confirmed by immunocytochemistry. The ciliary beating pattern was recorded using a microscope equipped with a high-speed camera. Results The isolated telencephalon cells proliferated rapidly under culture conditions, and MEPCs with functional multicilia were obtained through induced differentiation. Conclusions Using a modified in vitro culture protocol, MEPCs with proper immunohistochemical characteristics and normal multicilia motility were successfully obtained. The process of multicilia formation at various stages of MEPC differentiation was revealed, providing a benchmark for further study using MEPCs as a model.

Abstract: Objective To study the role of deubiquitinating enzyme 16 ( Usp16 ) in spermatogenesis by establishing a mouse model of conditional Usp16 knockout in germ cells. Methods Vasa-cre transgenic mice were crossed with Usp16 transgenic mice (Usp16^{flox / flox}) to obtain germ cell-specific Usp16 knockout mice. To confirm that Usp16 had been conditionally inactivated in germ cells, genotyping by PCR and western blotting was performed to evaluate Usp16 expression. Initial characterization of the phenotype of conditional knockout (CKO) mice was then conducted by co-cage mating, computer-assisted sperm analysis, and HE staining. Results We confirmed that Usp16 expression was appropriate in the conditional knockout mouse. Compared with the wild type, the conception rate of female mice mated with CKO mice had decreased to only 7%, while no significant difference was observed in heterozygous knockout mice male mice. The sperm count of CKO mice was decreased by approximately 79%, motility was decreased by approximately 87%, and the rate of sperm deformity was as high as 83%. Multinucleated cells appeared in the seminiferous tubules of the testes and the number of precocious sperm in the epididymis had increased by approximately 76%. Conclusions Homozygous mutation of Usp16 in germ cells severely reduces sperm quality, thereby reducing male fertility.

Abstract: Objective To explore the effects and mechanism of different styles of music on the depression-like behavior of rats induced by chronic unpredictable and mild stress (CUMS). Methods We selected 39 depressed rats by sucrose preference tests (SPT) and forced swimming tests (FST). They were randomly divided into Control (no music), Light music, and Classical music groups. The Light music and Classical music groups were introduced to music by Hisaishi and Mozart, respectively, for 4 hours per day for 3 weeks, after which SPT, FST, and O-maze tests were conducted. A pathological change in the hippocampus was observed with HE staining. The expression level of hippocampal BDNF protein was detected by Western blot and immunohistochemical staining. Liquid chromatography-tandem mass spectrometry (LC- MS / MS) was used to measure the content of hippocampal CORT and 5-HT. BDNF, CORT, 5-HT, and IL-1 (interleukin- 1β, IL-1β) levels in serum were determined by ELISA. Results Compared with the Control group, the percentage of sucrose preference was significantly greater (P< 0. 05), the immobile time in FST was significantly less (P< 0. 01), and the time and staying time in the open arms of the O-maze were significantly greater (P< 0. 05) in the Light and Classical music groups. The hippocampal tissue of rats in the Light and Classical music group was relatively intact. The BDNF expression level in the hippocampus (P< 0. 01) and serum was significantly greater (P< 0.01) in the music groups. The CORT content in the hippocampus was significantly less in the music groups (P< 0. 01) and in the serum of only the Classical music group. The 5-HT content in the serum of the Light music group was greater (P< 0. 01) than the other groups. The content of IL-1β in the serum in both music groups was less than the Control group (P< 0. 01). Conclusions Light and classical music improve the behavior of depressed rats, indicating the potential value of music therapy in mental disease treatments.

Abstract: Objective To establish rat models of chronic skin ulcers by various methods and compare the differences and characteristics of the different models through various indexes. Methods Male SD rats were randomly divided into four groups and treated with various factors after creating a wound surface by removing back skin: skin defect, hydrocortisone (skin excision + hydrocortisone intervention), hydrocortisone plus bacterial interference (skin excision + hydrocortisone + bacterial intervention), and implantation of a foreign body (skin excision + embedded foreign body) groups. The body weight and wound healing of rats were measured each day and the wound state was observed. Materials were drawn from all groups at 14 days after the operation to assess histological changes, the immune organ index, blood leukocyte level, hydroxyproline (Hyp), total protein, and tumor necrosis factor-α (TNF-α) content in the skin ulcer. Results No significant difference in the body weight of rats in each group was found during the experimental period. The skin wounds of rats in the skin defect group healed faster, the symptom duration of chronic skin ulcers in the hydrocortisone group was shorter, and the color of granulation tissues in the implantation of the foreign body group was gray in the early stage, but the granulation grew faster in the later stage, while the hydrocortisone plus bacterial interference group had yellow pus in wounds and slow growth of granulation tissue. Pathomorphological observation showed severe inflammatory cell infiltration in hydrocortisone plus bacterial interference and implantation of foreign body groups. Moreover, compared with the skin defect group, the model groups showed increases in the spleen index, leukocyte level, and hydroxyproline content in wound tissue (P< 0.05 or P< 0.01), and in the skin lesion+ hormone +bacteria group, total protein content was decreased obviously (P< 0.05), while TNF-α content was increased significantly (P< 0.05). Conclusions After applying various intervention factors based on full- thickness skin resection, the healing speed of rat skin ulcers was slower than that of simple skin resection. Among them, the pathological characteristics of the skin lesion + hormone + bacteria model were more stable and long lasting. It can be used as the preferred model of chronic refractory skin ulcers, whereas the skin excision + hydrocortisone intervention and skin excision + foreign body embedded models may be more suitable to study short-term refractory wounds, and the embedded foreign body can be used to observe the growth of simple granulation in the early stage.

Abstract: Objective To establish a stable animal model of intrauterine adhesion ( IUA) using a minimally invasive method that recapitulates the clinicopathologic characteristics of IUA. Methods Overall, 20 female ICR mice were randomly divided into two groups (n= 10). After the cervix was relaxed with phloroglucinol, the uterine horn of mice in the model group was subjected to mechanical injury and lipopolysaccharide perfusion (0. 5 mg / kg) through the cervix to induce endometrial injury, while mice in the sham operation group only received the same volume of normal saline perfusion. Uteri of the mice were collected at 7, 14 and 21 days after the procedure. HE and Masson staining were used to assess uterine morphology and fibrosis. Immunofluorescence was performed to evaluate macrophage infiltration into the endometrium. qPCR and Western Blot were used to detect the expression of inflammatory factors ( IL-1β, TNF-α, IL-4, IL-10 and IL-6) and endometrial receptivity-related markers ( integrin β3 and LIF) in mice at 14 days after modeling. Furthermore, mouse fertility was evaluated in model and sham operation groups. Results Compared with the sham operation group, endometria in the model group were significantly thinner and exhibited severe necrosis, glandular loss, and incomplete luminal epithelial and glandular epithelial cell structures. Subsequently, severe tissue fibrosis was observed in endometria in the model group. Additionally, increased macrophage infiltration and upregulated expression of proinflammatory factors demonstrated an activated inflammatory response in endometria of the model group. Moreover, endometrial receptivity was significantly decreased after mechanical injury combined with lipopolysaccharide perfusion. Finally, significant decreases in pregnancy and the number of fetuses were observed in the model group. Conclusions This study demonstrated successful establishment of an IUA mouse model through a minimally invasive transcervical mechanical injury combined with lipopolysaccharide perfusion, which was consistent with the clinical characteristics of severe IUA. This model might support in-depth study of IUA pathogenesis and promote the development of IUA therapies.

Abstract: Objective To establish a transgenic humanized ACE2 nude mouse model against a BALB/ c-nude mouse background. Methods Female human ACE2 (hACE2) transgenic mice were hybridized with male BALB/ c-nude mice to produce an F1 generation. An F2 generation was then obtained by backcrossing the F1 generation hACE2 mice with BALB/ c-nude mice, and F3 generation hACE2 transgenic nude mice were obtained by intercrossing the F2 generation hACE2 mice. Finally, F3 hACE2 transgenic nude mice were compared with C57BL/ 6J wild-type mice, hACE2 mice, and BALB/ c-nude mice in terms of their phenotypes, and physiological and immunological characteristics. Results (1)There were no significant differences between hACE2 transgenic nude mice and C57BL/ 6J wild-type mice, hACE2 mice, or BALB/ c-nude mice in terms of their phenotypes. (2) hACE2 transgenic nude mice had similar physiological indexes to BALB/ c-nude mice and lacked thymuses. However, the spleen and liver indexes differed significantly between hACE2 transgenic nude mice and BALB/ c-nude mice ( P< 0.05). Routine blood indexes also showed that the neutrophil, lymphocyte, and monocyte percentages differed significantly between hACE2 transgenic nude mice and C57BL/ 6J wild-type mice and hACE2 mice (P < 0. 01). (3)There were also significant differences in immunological indexes between hACE2 transgenic nude mice and C57BL/ 6J wild-type mice and hACE2 mice in terms of the percentages of CD3, CD4, CD11B, and natural killer cells (P< 0.01), but no significant differences between hACE2 transgenic nude mice and BALB/ c-nude mice. Conclusions We successfully established a line of hACE2 transgenic nude mice with a BALB/ c-nude mouse background.

Abstract:Zebrafish are commonly used as model animals, and recent developments in techniques such as xenotransplantation for observation of tumor metastasis, and high-throughput screening and safety evaluation of drugs have furthered the study and understanding of diseases. Malignant melanoma is a common malignant skin tumor with high mortality and strong metastasis, and a low 5-year survival rate. In addition, the incidence rate of melanoma has been gradually increasing and the onset age has been getting younger. Zebrafish and human melanocyte development have certain similarities, and the signaling pathways involved in the occurrence and metastasis of malignant melanoma are highly conserved in zebrafish, making zebrafish a common model for studying the pathogenesis and metastasis of melanoma. In this paper, we summarize the application of new zebrafish models and analyze their value for melanoma research.

Abstract:Alcoholic liver diseases are closely related to gut microbiota. Alcoholic liver diseases can cause dysbiosis of gut microbiota that can affect the severity of liver disease progression. This review summarizes the research on the relationship and mechanism of gut microbiota and alcoholic liver diseases. Research of gut microbes, such as probiotics, prebiotics, synbiotics, postbiotics, and bacteriophages, and related products may improve or cure liver diseases.

Abstract:Data released by the Chinese Sleep Medicine Congress in 2021 showed that more than 300 million people in China have sleep disorders, of which insomnia is the most common. Research into the mechanisms of insomnia and candidate therapeutic drugs needs to involve insomnia animal models and appropriate evaluation method. This article summarizes aspects of the insomnia animal models commonly used in the past 10 years and classifies them from the perspective of physical, chemical, pathological, and multiple factors. The method used to evaluate the insomnia models are also outlined. This paper provides a resource for studies of insomnia.

Abstract:Intestinal flora form a specialized immune microenvironment that affects the occurrence and development of colorectal cancer through immunoregulation. Here, we review the research progress of intestinal flora in immune regulation therapy for colorectal cancer and summarize the relationship between intestinal flora and host or tumor immunity. Moreover, we analyzed the result of related animal experiments and clinical trials and introduce the mechanism and strategy of combination therapy based on intestinal flora immunomodulation for the treatment of colorectal cancer. Thus, this review may provide a reference for the treatment of colorectal cancer.

Abstract:Chronic kidney disease (CKD) is defined as a chronic structural and functional disorder of the kidney ( history of kidney damage >3 months) with various causes. CKD-associated pruritus, previously known as uremic pruritus, is a common symptom in patients with advanced CKD and end-stage renal disease worldwide, with an incidence of 28% to 70% in dialysis patients. Pruritus can cause skin damage and sleep disturbances, which can in turn seriously affect patient quality of life and sleep quality, and may even lead to depression and suicidal tendencies. There is thus an urgent need to investigate the pathogenesis of and develop new treatments for CKD-associated pruritus. However, animal models of CKD- associated pruritus are rare, and a reliable and effective animal model is urgently required. In this paper, we review the etiopathology of CKD-associated pruritus, consider chronic pruritus models, and assess pruritus behavior in animals.