Abstract: Objective To optimize the method of combining azomethane oxide (AOM) and dextran sodium sulfate (DSS) to create a colitis-associated colon cancer (CAC) model, and to explore the pathogenesis of the intestinal flora in CAC. Methods Model groups A and B were established by one and two injections of AOM, respectively, combined with free drinking of DSS, and a normal control group was injected intraperitoneally with normal saline combined with purified water (n = 10 mice per group). The better modeling scheme was selected by comprehensive evaluation of the disease activity index score, colon length, tumor rate, and mortality. Serum levels of interleukin-6 (IL-6), tumor necrosis factorα (TNF-α), and tumor markers CA199, CEA, and CA724 were detected by enzyme-linked immunosorbent assay. Colon lesions were evaluated by hematoxylin and eosin (HE) staining. Changes in the intestinal microbiota in CAC mice were detected by 16S rDNA high-throughput gene sequencing analysis of mouse feces. Results Both single and enhanced AOM injections combined with DSS induced CAC mice; however, colon growths were larger, more closely arranged, and their morphological size was more consistent in group B compared with group A, with a tumor-formation rate of 100%. IL-6 levels were increased in the model group compared with the normal group (P< 0. 05). TNF-α levels were increased in the model group compared with the normal group (P> 0. 05). The CA199 and CEA levels were also significantly increased (P< 0. 05), but CA724 levels were not. Infiltration of inflammatory cells in the colon detected by HE pathology was accompanied by high-grade intraepithelial tumor-like changes on the surface of the lumen. The diversity and abundance of intestinal bacteria were decreased in CAC mice compared with normal mice: phyla Verrucomicrobiota and Actinobacteriota were significantly increased (P< 0. 05), Bacteroidota and Campilobacterota were significantly decreased (P< 0. 05).Akkermansia, Prevotellaceae, Ruminococcus, and Bifidobacterium were significantly increased (P< 0. 05), and Roseburia, Rikenellaceae_RC9_gut_group, Anaeroplasma, and Muribaculaceae were significantly decreased (P< 0. 05). Conclusions Two injections of AOM combined with 1. 5% ( 1. 5 g / 100 mL) DSS induced CAC model mice with a high colontumorigenesis rate, uniform tumor morphology, and low mortality, and may thus be the preferred modeling scheme for pharmacodynamic experiments. Disorders or dysfunction of the intestinal flora may lead to increased permeability, loss of intestinal mucosal barrier function, and the release of enterogenic endotoxins, Resultsing in a sustained inflammatory response, as an indirect or direct cause of CAC pathogenesis.

Abstract: Objective Cervical disc herniation (CDH) is one of the common orthopaedic diseases. With the indepth study of it and the development of cervical implants, the establishment of cervical fusion animal models has become an indispensable part. Notably however, studies of the establishment and evaluation of cervical fusion animal models in China are currently lacking. This study aimed to provide a suitable animal model and evaluation scheme for implants for cervical spine-related research. Methods Small-tailed Han sheep were chosen for anterior cervical discectomy fusion(ACDF) after modified surgery, and a polyetheretherketone (PEEK) interbody fusion cage (Cage) (control group), 3Dprinted Ti6Al4V Cage (group 1), and new method Ti6Al4V Cage (group 2) were implanted in different cervical segments(C2 / 3 ~ C4 / 5) in each sheep, respectively. Hematology and histopathological analyses were carried out after surgery to evaluate recovery of sheep and the biosafety of the materials. Bone in-growth and bone fusion were assessed by X-ray,computed tomography (CT), Micro-CT and quantitative analysis, hard tissue section staining, and biomechanical tests. Results The modified ACDF ovine model was established successfully. There were no significant differences in important hematology indexes (P> 0. 05) and histopathological analysis showed no pathological changes, such as inflammatory cell infiltration. The implants had good biosafety. Furthermore, X-ray and CT examinations showed that the position of internal fixation and the interbody fusion were good. Micro-CT and quantitative analysis at 3 and 6 months after operation showed that compared with PEEK Cage group, the bone volume / total volume and trabecular number were significantly increased (P< 0. 01) while the trabecular spacing was significantly decreased in the new method Ti6Al4V and 3D-printed Ti6Al4V groups compared with the PEEK Cage group (P< 0. 01). Moreover, the new method new method Ti6Al4V Cage group had more bone growth (P< 0. 01). Hard tissue section staining demonstrated that the pores of the new method Ti6Al4V Cage and 3D-printed Ti6Al4V Cage had obvious bone growth and relatively dense pores in the new method Ti6Al4V and 3Dprinted Ti6Al4V groups, and the combination was slightly better than that of PEEK Cage. Biomechanical evaluation indicated that the new method Ti6Al4V Cage and 3D-printed Ti6Al4V Cage reduced the range of cervical flexion-extension,lateral bending, and axial rotation (P< 0. 05) compared with the PEEK cage, as well as enhancing the stability of the cervical vertebra, and the new method Ti6Al4V Cage was more advantageous ( P< 0. 05). Conclusions After the establishment of the modified ACDF ovine model, reasonable and effective assessment method were used to demonstrate the suitability and effectiveness of the model and the good biosecurity of all three Cage materials. Compared with the PEEK Cage, the new method Ti6Al4V Cage and 3D-printed Ti6Al4V Cages showed better performances in terms of bone growth and bone fusion, which could enhance the stability of the cervical vertebrae. The new method Ti6Al4V Cage was particularly advantageous.

Abstract: Objective To compare animal models of chronic heart failure ( CHF) prepared by three different protocols, to establish a stable, reliable, and reproducible mouse model of CHF. Methods Twenty-five male C57BL/ 6J mice were divided randomly into four groups: a blank group, model A group (MA group), model B group (MB group), and model C group ( MC group). The model groups adopted different preparation protocols for continuous injection of isoprenaline. The MA group and MB group were dose-decreasing models: MA group: subcutaneous injection of 10 mg / kg on day 1, 5 mg / kg on day 2, 2. 5 mg / (kg·d) on days 3 ~ 30, total 30 days; and MB group: subcutaneous injection of 20 mg / kg on day 1, 10 mg / kg on day 2, 5 mg / (kg·d) on days 3 ~ 14, total 14 days. The MC group used a constant dose of intraperitoneal injection of 7. 5 mg / (kg·d) for 28 days. The day after the final injection, the survival and model-formation rates for each group of mice were calculated. Cardiac function was measured by cardiac ultrasound and serum levels of Nterminal pro B-type natriuretic peptide, interleukin-6, and tumor necrosis factor-α were measured. Results CHF was successfully induced in all the model groups after all injections at the end of the fourth week. However, comprehensive test result showed that the MC model was the most stable. Conclusions An isoprenaline-induced mouse model of CHF using constant intraperitoneal injection of 7. 5 mg / (kg·d) for 28 days may be the most suitable model for subsequent research on traditional Chinese medicine.

Abstract: Objective To induce an NLRP3P>- / -P> mouse model of ulcerative colitis ( UC ) using different concentrations of dextran sulfate sodium ( DSS) and different administration times, and to analyze and evaluate the advantages and disadvantages of the preparations to provide a more suitable animal model for the study of UC pathogenesis in humans and the development of therapeutic drugs. Methods Forty-eight male NLRP3P>- / -P>specific-pathogen-free mice were divided randomly into blank, 2. 5% 7 d, 3% 7 d, and 3% 5 d groups (n = 12 mice per group). UC mouse models were induced using combinations of different concentrations and administration times of DSS. Body weight, DAI( disease activity index)score, hematoxylin and eosin (HE) staining, colon length, and related indicators (interleukin IL-6, tumor necrosis factor (TNF)-α, and tight junction protein (ZO-1)) were observed and evaluated. Results (1)UC membrane type was induced in each group with different concentrations and administration times. (2)Mouse body weight decreased, the fecal occult blood became more positive, the DAI score increased, and more mice died with increasing DSS concentration and administration time. (3)Longer administration time and higher concentration of DSS were also associated with more severe damage to the intestinal mucosa, as shown by HE staining. (4) Immunohistochemistry showed that the inflammatory factors TNF-α and IL-6 were increased in the model group compared with the blank control group, while expression of ZO-1 was decreased compared with the blank group. Conclusions (1)Administration of 2. 5% or 3% DSS for 7 days or 3% DSS for 5 days can induce UC in NLRP3P>- / -P> mice. (2)The combination of DAI score, HE staining, the detection of related indicators, and mouse survival rate indicated that NLRP3P>- / -P> mice treated with 3% DSS for 5 days produced the most suitable UC model to study the clinical manifestations and drug treatment of UC.

Abstract: Objective To explore the possible mechanism of Xiebaisan in protecting against allergic asthma in rats from the perspective of host intestinal flora metabolism. Methods SPF SD rats were divided into normal group ( NC group), model group ( M group), and Xiebaisan group. The allergic asthma rat model was established by ovalbumin.Changes in lung histopathology were observed by HE staining. Colon contents were harvested for 16S rDNA high-throughput sequencing to assess changes in the intestinal flora structure and function. Serum and lung tissue samples were collected for non-targeted metabolomics by Ultra-high performance liquid-time-of-flight mass spectrometer. Results HE staining showed some improvement of lung histomorphology in asthmatic rats in the Xiebaisan group compared with that in the M group. 16S rDNA high-throughput sequencing showed that the diversity of intestinal flora was decreased in the M group and increased in the Xiebaisan group compared with the M group, the microecosystem of intestinal was improved. Non-targeted metabolomics of serum showed regulation of amino acid metabolism and the mTOR pathway in the Xiebaisan group, and partially reversed differential metabolite expression in the M group. Non-targeted metabonomics of lung tissue samples showed regulation of carbon metabolism, vascular smooth muscle and cAMP signaling pathways in the Xiebaisan group, and partially reversed differential metabolite expression in the M group. Conclusions The protective effects of Xiebaisan on allergic asthma in rats may be related to improvement of the morphological structure of lung tissue, the diversity of intestinal flora, and regulation of mTOR, vascular smooth muscle contraction, and cAMP pathways, which affect amino acid and carbon metabolism.

Abstract: Objective Given that psychosocial stress can contribute to a series of diseases, such as inflammatory bowel disease and irritable bowel syndrome, we aimed to establish an experimental chronic restraint mouse intestinal stress injury model as a basis for exploring the pathogenic mechanism of chronic restraint stress-induced gastrointestinal diseases,and for developing preventive and curative measures. Methods Eighteen male SPF-grade BALB/ c mice were acclimatized for 7 days and then divided into a control group and a chronic restraint stress group according to body weight, using a randomized numerical table method. The mice were subjected to restraint stress for 3 hours per day for 14 days to establish an intestinal injury model. The model was evaluated by observing body weight, pathological changes in intestinal histomorphology, expression of tight junction proteins, apoptosis of intestinal epithelial cells, and mRNA expression levels of inflammatory cytokines. Results After 14 days of chronic restraint stress, model mice showed weight loss, shortened duodenal villus height, abnormal crypt structure, a decreased villus/ crypt ratio, colonic mucosal inflammatory cell infiltration, and irregular crypt structure. Protein immunoblotting, immunohistochemistry, and immunofluorescence staining showed that the expression levels of the duodenal and colonic tight junction proteins Occludin and Claudin-1 were significantly decreased in mice after chronic restraint stress (P< 0. 05), while expression levels of the apoptotic protein cleaved-caspase-3 in intestinal epithelial cells were significantly increased (P< 0. 05). Regarding the mRNA expression levels of intestinal inflammatory factors and chemokines, chronic restraint stress for 14 days significantly increased the gene expression levels of interleukin ( IL)-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α,and IL-10 in the duodenum of mice (P< 0. 05), and significantly increased the gene expression levels of IL-1β, IL-6, and MCP-1 in the colon (P< 0. 001). Conclusions The use of a behavioral restriction device to restrain mice continuously for 14 days led to abnormal intestinal tissue structure, intestinal barrier dysfunction, and intestinal epithelial cell apoptosis,and triggered an intestinal inflammatory response in the stressed mice, indicating successful establishment of a mouse model of intestinal injury by chronic restraint stress.

Abstract: Objective To construct plasmids and knock out HIF-1α gene expression in an naked mole rat skin fibroblasts (NSF)cell line using CRISPR/ Cas9 genomic editing technology, to provide an in vitro cell model for studying the mechanism of hypoxia tolerance and the occurrence and development of hypoxia-related diseases in naked mole rats. Methods We designed four pairs of single guide RNA ( sgRNA) sequences targeting exons 1 ~ 4 of the NSF HIF-1α gene and successfully constructed an expression plasmid. The plasmid with the optimal sgRNA was identified and transfected into 293T cells, and the supernatant was used for detecting the virus titer. Lentivirus particles carrying sgRNAs of HIF-1α were transfected into NSF cells which express Cas9 protein, based on a previous protocol. After transfection,fluorescence signals were observed under a fluorescence microscope, and HIF-1α expression in NSF cells was detected by Western Blot and T7 endonuclease 1 (T7E1) analysis. Results Sanger sequencing showed that the designed sgRNA was successfully inserted into pX459 and pKLV2-U6-sgRNA2 vectors, demonstrating successful construction of a recombinant plasmid for transfection. T7E1 digestion successfully Methodsremoved three bands and the target efficiency of sgRNA was 54%.Western Blot showed that the HIF-1α gene was successfully knocked out and its protein level was significantly reduced in NSF cells from naked mole rats (p= 0. 0019). There were no obvious morphological changes in HIF-1α-knockout cells under the microscope, and gene knockout had no obvious effect on cell proliferation. Conclusions We successfully constructed an HIF-1α-knockout cell line using CRISPR/ Cas9 technology, to provide an experimental basis for further studies of the biological function of HIF-1α, as well as the mechanism of hypoxia tolerance in naked mole rats. The result also provide a theoretical foundation for the prevention and treatment of hypoxia-related diseases.

Abstract: Objective To investigate the effect of WNK2 on the ERK1 / 2 / ROS / SHP2 signaling pathway in hepatocellular carcinoma (HCC) and to explore its role in cell proliferation and migration in HCC. Methods HepG2 cells were transfected with WNK2-mimic, sh-RNA WNK2, and corresponding negative control. The effect of WNK2 on the proliferation of HCC was examined by subcutaneous tumorigenesis assay in BALB/ c nude mice. The expressions of WNK2,p40, gp90, p-SHP2, p-AKT, and p-ERK1 / 2 in tumor tissues were detected by Western Blot. After treatment with SHP2 inhibitor PHPS1, the expressions of WNK2, P40, gp90, p-SHP2, p-AKT, and p-ERK1 / 2 in HepG2 cells were detected by Western Blot. The migration ability and invasion ability of HepG2 cells were detected by cell scratch assay and Transwell. The proliferation ability of HepG2 cells was detected by monoclonal proliferation assay. Results Compared with the sh-NC group, the tumor volume of nude mice in the sh-RNA WNK2 group was significantly increased ( P<0. 01); Compared with the NC-mimic group, the tumor volume of nude mice in the WNK2-mimic group was significantly reduced (P< 0. 01). Western Blot result showed that compared with the sh-NC group, the expression of WNK2 in the shRNA WNK2 group was significantly decreased (P< 0. 01), while the expressions of p40, gp90, p-SHP2, p-AKT and pERK1 / 2 were significantly increased (P< 0. 01). Compared with the NC-mimic group, the expression of WNK2 was significantly increased in the WNK2-mimic group (P< 0. 01), and the expressions of p40, gp90, p-SHP2, p-AKT, and p-ERK1 / 2 were significantly decreased (P< 0. 01). In vitro experiment, compared with the sh-NC group, the expression of WNK2 was significantly decreased in the sh-RNA WNK2 group (P< 0. 01), while the expressions of p40, gp90, pSHP2, p-AKT and p-ERK1 / 2 were significantly increased in the sh-RNA WNK2 group (P< 0. 01). Compared with the sh-NC + PHPS1 group, the expression of WNK2 was significantly decreased in the sh-RNA WNK2 + PHPS1 group (P<0. 01), while the expressions of p40, gp90, p-SHP2, p-AKT, and p-ERK1 / 2 were reversed and had no significant differences compared with the sh-NC + PHPS1 group (P> 0. 05). The cell scratch assay and Transwell result showed that the migration and invasion ability of HepG2 cells in the sh-RNA WNK2 group was significantly increased compared with the sh-NC group (P< 0. 01). The migration and invasion ability of HepG2 cells in the sh-NC + PHPS1 group and sh-RNA WNK2 + PHPS1 group were significantly decreased with no significant difference ( P> 0. 05 ). The result of the monoclonal proliferation experiment showed that the proliferation capacity of HepG2 cells in the sh-RNA WNK2 group was significantly increased compared with the sh-NC group (P< 0. 01), while the proliferation ability of HepG2 cells in the shNC + PHPS1 group and sh-RNA WNK2 + PHPS1 group was significantly decreased with no significant difference (P>0. 05). Conclusions WNK2 can inhibit the ERK1 / 2 / ROS / SHP2 signaling pathway, thereby inhibiting ERK1 / 2 / Akt signaling and delaying the proliferation and migration of HCC.

Abstract: Objective To analyze and explore the analgesic effect of Angelica dahurica in neuropathic pain and its regulatory effect on the Mas-related G-protein coupled receptor member D (MrgprD)-transient receptor potential ankyrin 1(TRPA1) signaling pathway, using a mouse model of sciatic nerve chronic constriction injury (CCI). Methods A CCI mouse model was prepared by sterile surgical ligation and wrapping of the sciatic nerve in 30 mice. Pain-related behavioral changes induced by mechanical stimulation were detected by the VonFrey method, and the thermal hyperalgesic effects of Angelica dahurica were evaluated by thermal radiation experiments. The effects of Angelica dahurica on the protein expression levels MrgprD and TRPA1, the number of dorsal root ganglion (DRG) positive neurons, and mRNA levels of MrgprD and TRPA1 in mice were detected by Western Blot, immunofluorescence, and reverse transcription-polymerase chain reaction, respectively. Differences in fluorescence signal intensity in HEK293 cells after single transfection and cotransfection with MrgprD and TRPA1 plasmids, respectively, were analyzed by calcium imaging experiments. Results A total of 25 CCI mouse models were successfully prepared, with a modeling rate of 83. 33% ( 25 / 30). The mechanical threshold and foot retraction latency were significantly higher in CCI mice treated with Angelica dahurica compared with the control group (P< 0. 05). Expression levels of MrgprD and TRPA1 proteins were significantly lower in CCI mice treated with Angelica dahurica than in the control group (P< 0. 05). The number of MrgprD- and TRPA1-positive neurons in the DRG was significantly lower group (P< 0. 05) and the mRNA levels of MrgprD and TRPA1 were also significantly lower in CCI mice treated with Angelica dahurica than in the control group (P< 0. 05). The fluorescence intensity was significantly higher in HEK293 cells co-transfected with MrgprD and TRPA1 plasmids than in single-transfected and blank control cells (P< 0. 05 ). Conclusions This study demonstrated that the MrgprD-TRPA1 pathway is an important target for neuropathic pain, and indicated that Angelica dahurica can inhibit neuropathic pain by regulating this signal transduction pathway. These result provide a foundation for further research on the development of new clinical analgesic drugs and analgesic mechanisms.

Abstract: Objective To investigate changes in coagulation function and inflammation levels during sepsis. Methods A rat model of sepsis was established using the multiple infection sepsis model (MIM) based on cecal ligation and puncture. Forty-eight male Sprague-Dawley rats were assigned randomly to the following groups: control group, sham group, 4 h sepsis group, 8 h sepsis group, 12 h sepsis group, and 16 h sepsis group (n= 8 per group). Inflammatory markers and coagulation-related indicators were measured by enzyme-linked immunosorbent assay and coagulation analysis. Results (1)Lipopolysaccharide (LPS) and interleukin-6 (IL-6) levels were significantly higher in the model rats at all time points compared with the sham group (P< 0. 001). LPS and IL-6 levels increased gradually with disease progression,with no further changes in LPS after 12 hours. (2)Prothrombin time (PT) was significantly prolonged in the middle and late stages of the sepsis model, starting from 8, compared with the sham group ( P< 0. 01). ( 3) Partially activated prothrombin time (APTT) time was significantly prolonged in the 8, 12, and 16 h groups compared with the sham group (P< 0. 05, P< 0. 01). APTT gradually lengthened from 8 h, but approached control levels thereafter. (4) Fibrinogen (Fbg) content was significantly higher in all sepsis groups, except for the 8 h group, compared with the sham group (P<0. 01). (5)Fibrin degradation products (FDP) differed significantly between the control and sham groups (P< 0. 01),but not between the sham and sepsis groups. (6)Antithrombin-Ⅲ(AT-Ⅲ) levels decreased significantly throughout each stage of sepsis progression compared with the sham group ( P< 0. 01), and AT-Ⅲ showed a downward trend with the course of disease, with significant differences among the 4, 8, and 16 h groups. Conclusions The MIM rat model can reflect the development of inflammatory and blood coagulation disorders and their relationship during the course of sepsis,and may thus provide a good foundation for further research into the disease course of sepsis.

Abstract:Irritable bowel syndrome ( IBS) is one of the most common functional gastrointestinal disorders, of which diarrhea-predominant IBS (IBS-D) accounts for the largest proportion. The pathogenesis of IBS-D is complicated and diverse, and there is currently a lack of clinically effective drugs. The establishment of animal models is an essential tool for further studies of the disease mechanisms, evaluation of clinical efficacy, and drug development, and the preparation and evaluation standards of models are important factors affecting the quality of the research. Based on the currently accepted pathogenesis of IBS-D and the previous modeling experience of our research group, this review systematically summarizes the evaluation method used in animal models of IBS-D in terms of diarrhea observation, visceral sensitivity tests, and intestinal motility tests, to provide a reference for future studies.

Abstract:Pigeons show flocking and homing behaviors, which require characteristics including long-distance weight-bearing and continuous flight, with excellent navigation and spatial cognitive abilities. Pigeons have been widely used in animal robot research in recent years. Pigeon robots achieve motor behavior control by applying neural information intervention to specific neural targets in the pigeon’ s brain. This review summarizes research progress in pigeon robots based on the sensory system, motivation and emotional system or cortex and midbrain motor area respectively, according to the distribution of hierarchical multi-level neural regulatory targets in the pigeon’ s brain, with the aim of providing reference and guidance for further applied research into the use of pigeon robots in space perception, reconnaissance, and anti-terrorism search and rescue.

Abstract:Diminished ovarian reserve (DOR) is associated with a reduced quantity and / or quality of retrieved oocytes, usually leading to low numbers of retrieved oocytes and poor reproductive outcomes. DOR may potentially progress to premature ovarian insufficiency and premature ovarian failure, which have adverse impacts on women’s health. There is currently no effective clinical treatment to rescue ovarian function. The limited availability of human ovarian tissues and medical ethics issues mean that animal models are crucial for improving our understanding of the molecular pathogenesis of DOR and identifying preventive and therapeutic targets. This review thus aims to summarize the techniques and strategies used to establish rodent models of DOR, to provide a reference for future studies.

Abstract:Transgenic 5 × FAD mice are APP / PS1 transgenic mice carrying five familial Alzheimer’ s disease (AD) gene mutations. Beta-amyloid precursor protein ( amyloid precursor protein, APP) expression is related to the K670N/ M671L (Swedish), 1716V (Florida), and V7171 (London) mutations, and presenilin 1 (PS1) is affected by the M146L and L286V mutations. 5 × FAD mice express high levels of β-amyloid in the brain at 1. 5 months old, and neuritic plaques began to appear at 2 months old. The pathological phenotypes of 5 × FAD mice include amyloid plaque aggregation, neuronal loss, gliosis, and memory dysfunction, while their biological characteristics include changes in the formation of brain β-amyloid plaques, hyperphosphorylation of Tau protein, synaptic dysfunction, neuroinflammatory response, mitochondrial dysfunction, blood-brain barrier injury, neuronal injury, endoplasmic reticulum stress, and eye lesions. As a classic animal model of AD, 5 × FAD transgenic mice can simulate the neuropathological process and behavioral manifestations of late-stage AD in humans, and these mice are thus widely used in research into the pathogenesis of AD and the development of new drugs. In this review, we summarize the model construction, biological background, and biological characteristics of 5 × FAD transgenic mice, and the development and application of drugs for the prevention and treatment of AD, to provide references for the application of 5 × FAD transgenic transgenic mice in AD research.