Abstract: Objective To study the role of intestinal epithelial Tlr4 in systemic immune effects by establishing a mouse model of conditional Tlr4 knockout in intestinal epithelia. Methods Intestinal epithelial cell-specific Tlr4 gene knockout mice ( Tlr4^{f/ f} Cre T) were established by CRISPR/ Cas9 technology. The genotype of Tlr4^{f/ f} Cre T mice was identified by PCR and immunofluorescence. The general biological characteristics, reproductive capacity, and offspring survival rate were also investigated. Differences in immune organ structures, the intestinal mucosal immune cell ratio, and cytokine secretion levels between wildtype and Tlr4^{f/ f} Cre T mice were compared by HE staining, flow cytometry, and ELISAs. Results Establishment of Tlr4^{f/ f} Cre T mice was verified at gene and protein levels. Compared with wildtype mice, Tlr4^{f/ f} Cre T mice had no significant difference in general biological characteristics and offspring survival rate of > 90%. No significant differences in the physiological structure of the thymus, spleen, or liver, proliferation of splenic lymphocytes, and serum cytokine level, and intestinal mucosa were found between the two groups. However, the number of CD4+ T and γδT cells appeared to be significantly low in Tlr4^{f/ f} Cre T mice. Conclusions The intestinal epithelial cell- specific Tlr4 gene knockout mouse model was successfully established, which provides the experimental means to study the regulatory roles of Tlr4 expression of intestinal epithelial cells in diseases.

Abstract: Objective The anti-hemorrhoid efficacy and adhesion of mussel adhesive protein (MAP) was studied and its mechanism evaluated. Methods Croton oil was used to stimulate the anorectal mucosa to establish the model. Forty specific-pathogen-free grade Sprague-Dawley rats were divided into four experimental groups. One hour after model creation, different treatments were applied to each group by rectal administration under anesthesia. General observation was performed on the fourth day. A 1% solution of EBD was injected into the tail vein 30 min before rats were sacrificed. Approximately 8 mm of rectum was cut off and weighed to calculate the anorectal coefficient. Vascular permeability was evaluated by measuring EBD concentrations. Western Blot was used to detect relative protein expression levels of Muc2 and Muc4, and ELISA was used to detect expression of serum tumor necrosis factor ( TNF-α), interleukin ( IL-6), and malonaldehyde (MDA) in rats. Fluorescence imaging of FITC-labeled MAP at different times was compared and observed by fluorescence microscopy. Results General observations revealed that MAP could reduce the redness, edema, mucosal secretion, and exudation in the rectal area of rats. Anorectal coefficient result suggested that MAP could reduce the degree of rectal swelling (P < 0. 01), and EBD contents suggested that MAP could reduce local vascular permeability (P< 0. 01). Relative expression levels of mucins Muc2 and Muc4 were increased (P< 0. 05), whereas expression levels of serum TNF-α, IL-6, and MDA were decreased ( P< 0. 01). Fluorescence imaging indicated that FITC-MAP could maintain good adhesion and stability in the rectum. Conclusions MAP could increase the expression of mucosal mucin, inhibit inflammation, repair the mucosal barrier, promote mucosal healing, and improve the symptoms of hemorrhoids in a rat model, which can provide a reliable basis for preclinical research of hemorrhoids drugs.

Abstract: Objective To investigate the effect of a neoadjuvant treatment strategy of an anti-PD-1 monoclonal antibody combined with cisplatin on postoperative recurrence and metastasis of non-small cell lung cancer (NSCLC) in a humanized mouse model. Methods Human peripheral blood mononuclear cells and human NSCLC cells were sequentially transplanted into the severe combined immunodeficiency mouse (NOD/ ShiLtJGpt-Prkdcem26Cd52 Il2rgem26Cd22 / Gpt, NCG) to establish an immune system-tumor double humanized NSCLC model. We elevated the treatment effect of various neoadjuvant strategies in the double humanized NSCLC model, including cisplatin, anti-PD-1 ( pembrolizumab), and combined treatments. Tumor recurrence and metastasis were observed by optical imaging. Ki67 expression in tumor tissue was analyzed by immunohistochemistry to assess tumor cell proliferation. Changes in the tumor immune microenvironment were detected by immunofluorescence. Results The double humanized NSCLC model was successfully established. Immune cells had infiltrated into peripheral blood, organs, and tumors. Optical imaging showed that neoadjuvant anti-PD-1 treatment inhibited tumor recurrence and metastasis, and the effect of combined treatment was more obvious. Moreover, combination treatment significantly decreased Ki67 expression in tumor tissues. Large numbers of CD8⁺ and CD45⁺ cells in tumors were observed after anti-PD-1 treatment, and many more CD8⁺ and CD45⁺ T cells were found in the combined therapy group. Granzyme B plays an important role in tumor killing by CD8⁺ T cells. Immunohistochemical staining showed that anti-PD-1 treatment increased granzyme B in tumor tissues, which was more obvious after combined treatment. Conclusions Neoadjuvant pembrolizumab combined with cisplatin effectively improves the anti-tumor effect in humanized mice and significantly inhibits tumor recurrence and metastasis.

Abstract: Objective To observe the changes in the hypoxia-inducible factor 1α (Hif-1α) / vascular endothelial growth factor (VEGF) signaling axis and specific type H vessels in the femoral head of steroid-induced osteonecrosis of the femoral head ( SONFH) model rats and explore the role of these changes in the development of osteonecrosis. Methods Thirty specific pathogen-free grade male Sprague-Dawley rats were randomly divided into a control group ( CG), model group ( MG) and deferoxamine group ( DFOG), with 10 rats in each group. The MG and DFOG were treated with methylprednisolone combined with lipopolysaccharide for SONFH modeling, the CG and MG received intraperitoneal injections of normal saline, and the DFOG received intraperitoneal injections of 250 mg / kg deferoxamine mesylate. Six weeks after modeling, micro-computed tomography was performed to observe the microstructural changes of the femoral head, hematoxylin / eosin staining was used to observe the histopathological changes of the femoral head, immunofluorescence staining was used to analyze the changes of type H vessels in the femoral head, and reverse- transcription polymerase chain reaction (RT-PCR) was used to analyze expression of HIF-1α/ VEGF signaling axis-related factors in the femoral head. Results Micro-computed tomography analysis showed that the femoral head in the MG and DFOG had sparse bone trabeculae, but the bone trabeculae in the MG exhibited fractures and the femoral head showed subchondral cystic degeneration. Hematoxylin / eosin staining showed that compared with the CG, both the MG and DFOG had obvious femoral head necrosis (P< 0. 01). Immunofluorescence staining showed that compared with the CG, the amount of type H vessels in the femoral head was significantly lower in the MG (P< 0. 05) and significantly higher in the DFOG (P< 0. 01). Compared with the CG, the amount of Osterix⁺ osteoblast (progenitor) cells was significantly lower in the MG (P< 0. 01) and significantly higher in the DFOG (P< 0. 01). RT-PCR showed that the expression of HIF-1α, VEGF, osterix, and Runx2 (mRNA) in the femoral head was significantly lower in the MG than CG (P< 0. 01) and significantly higher in the DFOG than MG ( P< 0. 01). Conclusions Steroid-induced HIF-1α/ VEGF signaling axis dysregulation and specific type H vessel damage were observed in this SONFH rat model, suggesting that bone-specific type H vessel damage may be a key pathogenetic factor of SONFH.

Abstract: Objective To analyze the sequence of mouse spermatogenesis associated protein 3 ( Spata3) and explore its structure and function. Methods The NCBI database was used to compare the homology of mouse Spata3. ExPASy ProtParam software was used to analyze its physical and chemical characteristics. SOPMA and GOR4 predicted the spatial conformation. NetPhos3. 1 and the STRING database were used to analyze protein modification sites and protein- protein interactions. Immunohistochemistry and immunofluorescence were used to observe the localization of Spata3 protein. Results Mouse Spata3 had 64% homology with humans in coding sequence. It was a basic unstable hydrophilic protein without a signal peptide. It was a non-transmembrane intracellular protein. It was mainly located in the nucleus and cytoplasm of sperm at all levels of testicular tissue, and its expression in round sperm cells was the highest. Spata3 contained an internal disordered domain, 30 potential phosphorylation sites, and 11 potential O-glycosylation sites, which may interact with proteins such as Spata46 and Spert. Conclusions Spata3 is a conserved protein in spermatogenesis and may regulate spermatogenesis.

Abstract: Objective To establish a rat model of polycystic ovary syndrome (PCOS) with endometrial insulin resistance(IR) by combining dehydroepiandrosterone ( DHEA) with a high fat diet ( HFDs). And by detecting the expression levels of insulin resistance related proteins and PI3K/ AKT pathway-related proteins in the endometrium, we can observe whether there is insulin resistance in the endometrium and explore its possible mechanism. Methods Thirty-two 21-day-old SD female rats were randomly divided into a Model group (Model group, n= 24) and Control group (Control group, n= 8). The Model group was subcutaneously injected with 6 mg / 100 (g·d) DHEA on the back of the neck and fed the high fat diet. The Control group was subcutaneously injected with the same volume of corn oil for injection on the back of the neck and fed a normal diet. The experimental period was 30 days. In accordance with the result of vaginal smear and ovarian tissue staining, PCOS rats were screened. And PCOS with IR rats ( PCOS-IR) were screened by detecting fasting blood glucose (FBG), serum insulin level (FINS), and insulin resistance index (HOMA-IR). Then by detecting the differences in the expression of IR-related proteins (IRS1, GLUT-4) and PI3K/ AKT pathway-related proteins (PI3K, PI3K p110α, p-AKT, AKT) in the endometrium between the two groups to explore the possible mechanism. Results Smears from rats in the Control group showed regular estrus and a normal ovarian tissue structure, whereas those of rats in the Model group had disordered estrous cycles and the ovaries had polycystic ovary manifestations. Compared with the Control group, FBG (P> 0. 05), FINS (P> 0. 05), and HOMA-IR (P< 0. 01) of rats in the Model group were all increased, among which 11 PCOS rats and eight PCOS with IR rats were screened from the Model group. Additionally, western blotting of IR-related proteins in endometrial tissue showed that IRS1 and GLUT-4 expression in the PCOS-IR were decreased (P< 0. 01), suggesting the existence of IR in the endometrial tissue. Compared with the Control group, the PI3K expression level was increased in the PCOS-IR group (P< 0. 05), and the PI3K p110α(P< 0. 01), p-AKT (P< 0. 01), AKT (P> 0. 05) expression levels were decreased in the endometrial tissues, suggesting that it may be related to the down-regulation of PI3K/ AKT pathway-related protein expression in the endometrium. Conclusions Rats with PCOS and IR have characteristic polycystic ovary tissue and abnormal glucose metabolism, and their endometrium is mostly insulin resistant, and may be related to the downregulation of PI3K/ AKT pathway-related protein expression.

Abstract: Objective To study the changes in platelets and coagulation function in a rabbit model of breast cancer lung metastasis. Methods Twelve female New Zealand white rabbits were randomly divided into two groups: normal group and model group. A VX2 breast cell suspension was injected into the third breast pad and injected intravenously through the ear marginal vein. The mental state, diet, body weight, and tumor size of the rabbits were observed over 42 days. Platelet- related parameters (PLT, MPV, PDW, and PCT) and coagulation-related indexes (PT, APTT, TT, FIB, and D-dimer) were detected at corresponding time points. The levels of CD63 and CD62P TXB₂ in serum, and GMP-140 in plasma, were detected by ELISA. The tumor tissue and lung tissue were fixed with 10% formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Morphological changes in the tumor and lung tissue were observed under an optical microscope. Results The rabbit model of breast cancer lung metastasis was successfully established by injecting a VX2 tissue suspension into the breast pad and a VX2 cell suspension intravenously. The lung metastasis rate was 100%. The blood of the model rabbits was in a hypercoagulable state. The levels of PLT, MPV, and PCT increased significantly from 14 to 42 days, the PT, APTT, and TT indexes were significantly shortened, and the contents of FIB and D-dimer were significantly increased from 10 to 30 days. The levels of CD63, CD62P and TXB₂ in serum, and GMP-140 in plasma, increased significantly. Tumor cells increased in number, appeared loosely arranged, and were infiltrated by inflammatory cells in the mammary gland of model rabbits. A tumor cell mass with hemorrhage and necrosis was observed in the lung tissue, which contained hemosiderin deposition. Conclusions The blood circulation in the rabbit model of breast cancer lung metastasis was abnormal, and this manifested as platelet activation and a hypercoagulable state in the blood.

Abstract: Objective The aim of this study was to establish a rat model of spontaneous short stature associated with female infertility. The main biological characteristics of the rat model were then identified. Methods The pedigree of mutant individuals with a short stature in the production group of SD rats was established by expanding the population. The proportion of offspring of mutant individuals with a short stature and that of the normal individuals was calculated and the genetic model was identified. Weight, body size, weight and index of main organs, blood physiology ( 18 items), biochemistry ( 20 items ), and reproductive performance of the mutant individuals were determined to clarify their phenotypic characteristics and biological characteristics. Results A family of SD rats with idiopathic autosomal dominant mutations was established, which was named SSR. Adult (12 weeks old) SSR rat weights were 57% of wildtype (?) and 65% (♀),Very significant difference (P< 0. 01) . The body length, tail length, tibia length, femur length and other body size data of adult SSR and the weight of main organs ( heart, liver, spleen, lungs, kidneys, testes/ ovaries) were significantly lower than those of wild type rats (P< 0. 01); There were no significant differences in the adult SSR rat shinbone length, femoral bone length index, or major organ indexes, compared with the wildtype ( P> 0. 05 ). Reproductive organ development of adult female rats was normal, but ovulation was not observed. Conclusions Spontaneous autosomal dominant mutations in dwarfism lines that can be stably inherited were established, which can be used as an applied study for human growth and infertility models. Female individuals were found to be infertile during the study, but further studies are needed to determine whether infertile individuals can have children.

Abstract: Objective To study the protective effect of various proportions of Scutellariae Radix-Forsythiae fructus in an acute pneumonia mouse model induced by lipopolysaccharide (LPS). Methods The Scutellariae Radix-Forsythiae fructus composition with various compatibility ratios was prepared by the heating reflux method . In total, 144 healthy KM mice were randomly divided into 12 groups ( normal control, model, dexamethasone positive drug, and nine groups of Scutellariae Radix-Forsythiae fructus at various proportions) with 12 mice in each group. Normal control and model groups were administered the same amount of normal saline and the other groups were administered corresponding drugs for 7 continuous days. On day 8, except for the normal control group, mice in other groups were treated with LPS (10 mg / mL) by dripping a solution into the nasal cavity to establish the acute pneumonia model. Samples were collected quickly at 6 hours after modeling. The protective effects of various proportions of Scutellariae Radix-Forsythiae fructus in the acute pneumonia mouse model were evaluated by clinical signs, average daily weight gain, feed intake, the viscera index, serum TNF-α, IL-1β and IL-6 contents, blood routine index, lung histopathological observation, and lung injury histopathological score. Results After modeling, the survival rate of mice in each group was 100%. The normal control group had normal clinical signs, breath sounds, and a sensitive reaction. Mice in the model group were unresponsive and huddled together with rales in the lungs detected by auscultation. Compared with the model group, the overall situation of the various administration groups was better. In particular, the Scutellariae Radix-Forsythiae fructus combination groups (1 ∶ 1, 2 ∶ 3, 1 ∶ 2) showed more activity and sensitive reactions. No significant differences were observed in daily body weight increase, daily feed intake, or kidney, liver, spleen, and thymus indexes (P> 0. 05). Compared with the normal control group, the lung index, serum contents of TNF-α, IL-1β and IL-6, blood biochemical indexes, and histopathological score of lung tissue injury were significantly increased in the model group ( P< 0. 01 or P< 0. 05) . The alveolar wall had significantly thickened, SFTPC protein expression in alveolar and interalveolar regions of lung tissue was significantly increased, and a large number of cells expressed SFTPC protein. Compared with the model group, the heart index of the Scutellariae Radix-Forsythiae fructus combination groups (3 ∶ 1, 3 ∶ 2, 2 ∶ 1, 1 ∶ 3, 3 ∶ 0, 0 ∶ 3) , the lung index of the Scutellariae Radix-Forsythiae fructus combination group ( 1 ∶ 2) , and the serum TNF-α, IL-1β And IL-6 content, blood routine index content of the Scutellariae Radix-Forsythiae fructus combination groups (1 ∶ 1, 2 ∶ 3, 1 ∶ 2) , lung tissue injury scores of the Scutellariae Radix-Forsythiae fructus combination groups ( 3 ∶ 2, 1 ∶ 1, 2 ∶ 3, 1 ∶ 2) were significantly decreased (P < 0. 01 or P < 0. 05) . The lung pathology of mice in the four Scutellariae Radix-Forsythiae fructus groups (3 ∶ 2, 1 ∶ 1, 2 ∶ 3, 1 ∶ 2) was reduced significantly, the expression level of SFTPC protein in the alveolar and interalveolar regions of lung tissue was reduced significantly, and the number of positive cells was low. Conclusions Combination of Scutellariae Radix-Forsythiae fructus(1 ∶ 1, 2 ∶ 3, 1 ∶ 2) has a significant protective effect on LPS-induced acute pneumonia.

Abstract: Objective To investigate the effects of passive smoking in adult female rats on behavior and expression of collapsin response regulatory protein 1 (CRMP1) and death-related protein kinase 1 (DAPK1) in the cerebral cortex of offspring. Methods Eighteen 8-week-old healthy female SD rats were randomly divided into the passive smoking group (n= 9) and control group (n= 9). The passive smoking group was treated with smoke to establish the passive smoking model, whereas the control group was treated normally. After 1 month of smoking, female rats were mated with males and continued smoking until delivery. Offspring (F1 generation) of passive smoking and control groups were evaluated by an open field test, rotary rod fatigue test, and Morris water maze test. These animal behavior tests were conducted to analyze the effects of passive smoking on anxiety, exercise tolerance, and spatial learning and memory abilities of the offspring. Moreover, CRMP1 and DAPK1 protein expression in cerebral cortex tissues of offspring was detected by Western Blot at various developmental stages (0, 7, and 21 days and 4 weeks ( adult)) after birth. Results The open field test showed that the total movement distance and average movement speed of the F1 generation in the passive smoking group were significantly increased in the total area ( P< 0. 05), and the number of entering the central area, and the movement distance and residence time in the central area were significantly increased compared with those of the control (P< 0. 05). The rotary rod fatigue test showed that the sustained movement time and movement distance of the F1 generation in the passive smoking group were significantly reduced compared with those in the control group (P< 0. 001). The Morris water maze showed that escape latency of the F1 generation had no significant difference between passive smoking group and control groups, but swimming speed was significantly decreased in the passive smoking group (P< 0. 001). Compared with the control group, the proportion of time and time of the F1 generation entering the target quadrant in the passive smoking group were significantly decreased (P< 0. 05), and the swimming speed and total swimming distance were also decreased significantly (P< 0. 01). Additionally, Western Blot showed that CRMP1 and DAPK1 protein expression in offspring of the passive smoking group was significantly lower than that of the control group at 0, 7, and 21 days. Conclusions Passive smoking in parental female rats promoted anxiety, reduced exercise tolerance, and impaired spatial memory of offspring, which may be related to regulation of CRMP1 and DAPK1 expression in the brain tissue of offspring.

Abstract: Objective To analyze the population genetic quality of the same closed KM mouse colony in 2013 and 2020 by microsatellites. Methods DNA was extracted from 30 KM mice in 2013 and 2020. Thirty pairs of microsatellites primers were used for PCR amplification and DNA sequencing to evaluate genetic quality by the number of alleles, heterozygosity and polymorphism content. Results Ninety-five alleles were found in 2013, average heterozygosity was 0. 4864 and average polymorphism content was 0. 4418. There were 122 alleles, average heterozygosity was 0. 5150, and average polymorphism content was 0. 4818. Conclusions The closed KM mice colony had genetic stability and genetic diversity, which satisfied the genetic characteristics of a closed colony of laboratory animals.

Abstract:Specific-pathogen-free ( SPF) pigs have been developed to prevent specific pig diseases and are important not only in the pig production industry, but also in life science research, and the production of biological products. Here, we review research progress on pathogen control and the quality control procedures applied to the production of SPF pigs throughout the world. We also provide an overview of the method for the purification of pathogens from SPF pigs, and briefly discuss the current applications of SPF pigs and potential future developments.

Abstract:The large number of smokers has had a broad effect on society. Nicotine is one of the most widely concerning components of cigarette smoke. Long-term intake of nicotine can lead to nicotine dependence that affects higher brain functions, which may involve anxiety, learning and memory impairments, and abnormal decision-making. To facilitate analyzing the molecular mechanisms of nicotine dependence, it is common to establish rodent nicotinoid dependence models to better understand the physiological changes. This review summarizes and evaluates studies of the behavior in nicotine-dependent animal models to provide a reliable reference for researchers to establish rodent nicotine- dependent models for evaluation.

Abstract:The mechanisms involved in learning and memory are complex and the forms of expression are diverse. The process of learning and memory involves signal recognition, information acquisition, consolidation, reproduction, and reconsolidation. Memory information includes image memory, sound memory, olfactory memory, and spatial location memory. Information can be divided into short-term memory and long-term memory according to how long it is stored. Behavioral experiments are the main method to evaluate learning and memory. To date, scientists have established a variety of behavioral experimental method in rats and mice to evaluate learning and memory. In this paper, the experimental method of behavioral learning and memory are classified and summarized for the first time, and different evaluation method are listed for the different experimental types. Our review provides a reference for behavioral experiment-based learning and memory research, and related product development.

Abstract:As a common neurodegenerative disease, Alzheimer’s disease (AD) seriously affects the physical and mental health, and the quality of life, of patients. The preparation of AD animal models is therefore of significance, especially models that can effectively replicate abnormal behavioral, biochemical, and pathological changes. In this review, the method of preparing AD animal models based on D-galactose alone, and D-galactose combined with an amyloid beta protein (Aβ) oligomer, AlCl₃ , and NaNO₂ were summarized. The mechanism of drug-induced AD, the presentation of impaired learning memory, and pathological changes such as oxidative stress, inflammatory reactions, neuron damage, and Aβ and Tau aggregation are evaluated and discussed to provide a reference for future studies involving AD animal models.

Abstract:As an emerging vertebrate animal model, zebrafish have been widely used in human disease research. Although simple in form, the larval pronephros is similar to the mammal kidney in terms of histological structure, gene expression, and functional complexity. Furthermore, the response of zebrafish larvae to kidney damage is similar to that of mammals. Because of their unique features and other advantages, zebrafish larvae present a useful model for research into kidney development and disease. This review discusses the applicability of zebrafish larvae in research into acute kidney injury based on the biological characteristics of the pronephros, the pathogenic mechanisms involved, and the advantages for kidney injury research.

Abstract:Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation, which can lead to joint deformity and dysfunction. Animal models of RA simulate the pathological and physiological processes of its occurrence and development, and are widely used in RA studies. Multimodal ultrasound technology allows qualitative and quantitative diagnosis of RA, monitors changes in conditions, evaluates drug efficacy, and guides local administration and targeted therapy, providing an accurate evaluation method for research of RA animals. This article reviews the application of multimodal ultrasound in the diagnosis and treatment of RA animal models.

Abstract:Traumatic brain injury (TBI) is a severe craniocerebral injury and a main cause of death and disability in young adults. Patients with severe TBI exhibit severe neurological deficits and behavioral disorders, but there is no effective treatment strategy. Neural stem cells (NSCs) are multipotent cells in the central nervous system, which secrete neurotrophic factors, differentiate into central and peripheral nervous system cell types, and play an important role in the treatment of TBI. Recent studies in animal models have demonstrated that the induction of endogenous NSCs and transplantation of regenerative neurons from exogenous NSCs can improve the symptoms in TBI animal models. Among the various TBI models, the cortical impact injury model has the most promising application in research of TBI treatments because of its accuracy and easy manipulation. Thus, in this review, we focus on cortical impact injury models to summarize their progress in the induction of endogenous NSCs to regenerate neurons and transplantation of exogenous NSCs to provide a new therapeutic strategy for TBI.