Abstract: Objective　To explore the method of establishing the animal model of acute exacerbation of idiopathic pulmonary fibrosis in rat, and explore the usage and dosage of the inducer lipopolysaccharide. Methods　150 healthy male Wister rats were randomly divided into negative control group (Control group), remission control group (IPF group), acute exacerbation control group (BLM group), lipopolysaccharide low dose group (LPS-low group), lipopolysaccharide medium dose group (LPS-mid group), and lipopolysaccharide high dose group (LPS-high group). The Control group was induced by first intratracheal injection of 0. 9% sodium chloride injection combined with second intraperitoneal injection of 0. 9% sodium chloride injection(day 0, 0. 1 mL/100 g; day 28, 0. 5 mL/100 g). The IPF group was induced by the first intratracheal injection of bleomycin combined with the second intraperitoneal injection of 0. 9% sodium chloride injection (day 0, 5 mg/ mL, 0. 1 mL/100 g; day 28, 0. 5 mL/100 g). The BLM group was induced by two consecutive intratracheal injections of bleomycin (day 0, 5 mg/ mL, 0. 1 mL/100 g; day 28, 7 mg/ mL, 0. 1 mL/100 g). LPS-low group, LPS-mid group and LPS-high group were induced by the first intratracheal injection of bleomycin combined with the second intraperitoneal injection of lipopolysaccharide (day 0, bleomycin, 5 mg/ mL, 0. 1 mL/100 g; day 28, lipopolysaccharide, 2.5 mg/ mL, 5 mg/ mL, 7.5 mg/ mL, 0. 5 mL/100 g). Take samples on the 31st, 35th, and 42nd days after the first molding. Observe the survival status of rats, lung tissue wet to dry weight ratio (W/ D), lung coefficient, lung tissue HE staining, lung tissue Masson staining, lung CT, lung function, arterial blood gas analysis, bronchoalveolar lavage fluid (BALF) composition analysis (total cell count, neutrophil count, IL-6 mass concentration, TNF-α mass concentration), lung tissue hydroxyproline (HYP) content, and lung tissue α-SMA immunohistochemistry analysis. Results　(1) The LPS-low group, LPS-mid group, and LPS-high group rats showed significant wheezing, dyspnea, cyanosis of the paw lips, decreased body mass, and increased mortality rate. (2) The W/ D value and lung coefficient of rats in LPS-low group, LPS-mid group and LPS-high group increased significantly, and acute pulmonary edema appeared. The performance of LPSmid group and BLM group was more consistent (P> 0. 05). (3) The histopathological changes in the LPS-low, LPS-mid, and LPS-high groups were consistent with the classic pathological manifestations of the disease. (4) The LPS-low group, LPS-mid group, and LPS-high group had patchy cotton wool like shadows on CT scans of the lungs, accompanied by irregular density enhancement shadows. (5) The lung function and blood gas analysis structure of the LPS-low group, LPSmid group, and LPS-high group showed a downward trend, with the LPS-mid group and BLM group showing more consistent performance (P> 0. 05). (6) The total cell count and neutrophil count in the alveolar lavage fluid of rats in the LPS-low, LPS-mid, and LPS-high groups increased, while the mass concentration of TNF-α in the alveolar lavage fluid and IL-6 in the serum increased. Among them, the LPS-mid and BLM groups performed more consistently (P> 0. 05). (7) The LPS-low group, LPS-mid group, and LPS-high group showed an increase in HYP content and α-SMA expression in the lung tissue of rats. Among them, the LPS-mid group and BLM group showed more consistent performance (P> 0. 05). Conclusions　This method can successfully prepare a rat model of acute exacerbation of idiopathic pulmonary fibrosis. The optimal dosage of lipopolysaccharide is 5 mg/ mL, and the optimal observation time is the 31st day.

Abstract: Objective 　To establish an imiquimod compound chronic unpredictable mild stress ( CUMS) C57BL/6J mouse model of alopecia areata (AA). Methods 　Thirty female C57BL/6J mice aged 5 ~ 7 weeks were randomly divided into three groups of 10 mice each: control, IMQ, and CUMS (imiquimod combined with CUMS) groups. CUMS was administered to the CUMS group on days 1 ~ 21, whereas IMQ and CUMS groups were administered equal amounts of imiquimod cream topically at the same site on days 14 ~ 21. On day 21, overall and trichoscopic photographs of mice were taken, behavioral tests were performed, and skin lesions and peripheral blood were collected. CD4P>+P> and CD8P>+P> T cell infiltration into hair follicles was observed by immunohistochemical staining. Differentiation of Th1/ Th2 and Th17/ Treg cells from peripheral blood T lymphocyte subsets was detected by flow cytometry. Results 　Compared with blank and imiquimod groups, C57BL/6J mice in the CUMS group showed significant localized patchy hair loss, and trichoscopic photographs showed broken hair and exclamation mark-like hairs. The distance moved, number of times standing upright, and number of times entering the central area in the open field test were significantly reduced (P< 0. 05). Moreover, the rest time in the forced swim test was significantly increased (P< 0. 05). These data suggested that the mice were in an anxious and depressed state. Compared with blank and imiquimod groups, CUMS group mice had significant CD4P>+P> and CD8P>+P> T cells in skin lesions around hair follicles and in the hair follicle bulb. Flow cytometry showed that Th1 levels were significantly higher (P< 0. 01), Th2 levels were significantly lower (P< 0. 05), Th17 levels were significantly higher (P< 0. 05), the Th1/ Th2 ratio was significantly higher (P< 0. 001), and the Th17/ Treg ratio showed an upward trend in the CUMS group. Conclusions 　The C57BL/6J mouse model established by topical imiquimod combined with CUMS essentially simulates human AA manifestations. This method highlights the psychosomatic factors in AA pathogenesis, and to some extent, conforms to liver depression and spleen deficiency syndromes in TCM, providing a cultivation-friendly and more cost-effective modeling method for basic research of AA.

Abstract: Objective　To study the effect and possible mechanism of Zhenwu Decoction on renal inflammatory injury in mice with spleen-kidney yang deficiency-type diabetic nephropathy. Methods　Mice (db/ m mice) were treated with rhubarb solution and hydrocortisone to induce a db/ db mouse spleen and kidney yang deficiency syndrome model. Mice in the blank control group were untreated. After successful modeling, the db/ db mice were divided randomly into a model control group, irbesartan group (25 mg/ kg), and high-, medium-, and low-dose Zhenwu Decoction groups (33. 8, 16. 9, 8. 45 g/ kg, respectively) (n= 15 per group). Mice in all groups except the blank control and model control groups were given the corresponding drugs by gavage once a day for 8 weeks. Mice in the blank control and model control groups received the same volume of normal saline. Traditional Chinese medicine syndrome scores were measured in each group. Fasting blood glucose ( FBG), urinary creatinine ( Ucr), and 24 h urinary protein (24 h UTP) were measured. Testosterone (T), triiodothyronine (T3), tetraiodothyronine (T4), estradiol (E2), interleukin (IL)-1β, and IL-18 levels in renal tissues were measured by enzyme-linked immunosorbent assay. Histopathological changes in the kidneys were observed by hematoxylin/ eosin staining. NOD-like receptor thermal protein domain associated protein 3 ( NLRP3), Apoptosis-associated speckle-like protein(ASC), Cysteinyl aspartate specific proteinase-1(Caspase-1) mRNA and protein expression levels in renal tissues were detected by Real-time quantitative PCR ( RT-qPCR) and Western Blot. Results　Mice in the model control group showed symptoms of spleen-kidney yang deficiency, such as anorexia, fatigue, and decreased, loose stools compared with the blank control group, while FBG, 24 h UTP, IL-1β, IL-18, and E2 were significantly increased (P< 0. 05) and T, T3, T4, and Ucr were significantly decreased (P< 0. 05). Renal pathology showed glomerular hypertrophy, and glomerular mesangial and basal thickening. Protein and mRNA levels of NLRP3, ASC, and caspase-1 in renal tissue were significantly increased (P< 0. 05). In contrast, mice in the high-, medium-, and low-dose Zhenwu Decoction groups had increased body weight, activity, and stool molding compared with the model control group, as well as significantly increased levels of T, T3, T4, and Ucr (P< 0. 05) and significantly decreased levels of 24 h UTP, IL-1β, IL-18, and E2 (P< 0. 05). In addition, pathological damage to the renal tissue was significantly improved and the protein and mRNA expression levels of NLRP3, ASC, and Caspase-1 were significantly decreased in all treatment groups (P< 0. 05). Conclusions　Zhenwu Decoction can improve the general condition and renal function, and reduce the inflammatory reaction and renal pathological injury in db/ db mice with spleen-kidney yang deficiency, via a mechanism possibly related to inhibition of the NLRP3/ ASC/ Caspase-1 pathway.

Abstract: Objective　To determine if the Drosophila synaptic adhesion molecule neuroligin 4 (DNlg4) regulates the development of the larval neuromuscular synapse by interacting with the classic neuroligin (Nlg)-presynaptic ligand molecule Drosophila neurexin (DNrx). Methods 　Immunofluorescence staining and confocal microscopy were used to examine the morphology of the neuromuscular junction (NMJ) in Thrid-instar larvae in wild-type(WT), dnlg4 mutant, dnrx mutant, and dnlg4,dnrx double-mutant Drosophila. The length of the synaptic branches and number of synaptic boutons were used as parameters to assess the developmental status of the NMJ in the different genotypes. We determined the relationship between DNlg4 and DNrx in regulating the development of the NMJ by comparing the developmental defects in the different genotypes. Results　Deletion of DNrx result ed in more severe developmental defects in NMJs than DNlg4 deletion, characterized by shorter synaptic branches and fewer boutons. Loss of DNlg4 further aggravated the NMJ defects caused by DNrx deletion. The expression of exogenous DNlg4 in motoneurons partially rescued the developmental defects of NMJs caused by loss of DNrx. No in vivo physical interaction between DNlg4 and DNrx was detected. Conclusions　DNlg4 and DNrx have synergistic effects on the positive regulation of synaptic development in Drosophila larvae, and DNlg4 regulates synaptic development in a DNrx-independent manner.

Abstract: Objective　To observe the effects of different concentrations of Astragalus polysaccharides (APS) on hypoxia-inducible factor (HIF)-1 and S100A8/ A9 protein expression in the lung pre-metastatic niche(PMN), and to investigate the targets of APS in lung PMNs and its molecular mechanisms in relation to lung tumor metastasis. Methods Ninety specific-pathogen-free C57BL/6J mice were used, of which 15 were set as the blank control group. A PMN model was constructed in the remaining 75 mice by tail vein injection of 1 × 106 luciferase-labeled Lewis lung cancer cells. These 75 mice were divided randomly into five groups (n = 15 per group): model, low-dose (APS 50 mg/ kg), medium-dose (APS 100 mg/ kg), high-dose (APS 200 mg/ kg), and fingolimod (FTY720, injected intraperitoneally 1 mg/ kg) groups. APS was administered by gavage at the respective prescribed dose once a day on the day after inoculation of tumor cells and FTY720 was administered once every 2 days. The effects of different concentrations of APS on HIF-1 and S100A8/ A9 proteins in the PMN was observed and lung metastasis was assessed by hematoxylin/ eosin (HE) staining. On day 14, five mice from each group were randomly selected and lung tissues were removed and multifunctional proteoglycans, fibronectin, and lysyl oxidase were detected by Western Blot and Real-time qvantitative PCR (RT-qPCR), and histopathological changes in the lung were observed by HE staining. Protein and gene expression levels of HIF-1 and S100A8/ A9 in lung tissues taken at 28 days were also detected by Western Blot and RT-qPCR. Mice were fasted but had access to water for 12 h before sampling. Results　HE staining revealed alveolar wall thickening and granulocyte infiltration with a few tumor metastases in lung tissues in the model group. Tumor metastatic foci were significantly reduced and the lung tissues tended to be normal in the high-dose and FTY720 groups compared with the model group. HIF-1, S100A8, and S100A9 protein and gene expression levels were significantly higher in the lungs of mice in the model groups compared with the blank group (P< 0. 05), according to Western Blot and RT-qPCR. In contrast, the above protein and gene expression levels were decreased in the FTY720 group and all APS groups compared with the model group (P< 0. 05). Conclusions APS can reduce lung metastasis of tumor cells by regulating the expression levels of HIF-1, S100A8 and S100A9 in the PMN of lung tumors.

Abstract: Objective　To study the ability of human pluripotent stem cell-derived smooth muscle cells (hiPSCSMCs) to improve wound healing in diabetic mice. Methods　hiPSC-SMCs, positive control adipose-derived stem cells (ADSCs), and negative control cell-free phosphate-buffered saline (PBS) were added to type I rat tail collagen to prepare stem cell or cell-free collagen scaffolds. The scaffolds were applied to splinted back wounds in diabetic nude mice. The wound was photographed and harvested at various time points, and immunohistochemical staining and western blot analysis were performed. Results 　Compared with the cell-free control group, hiPSC-SMCs had the same ability as ADSCs to promote wound healing in diabetic mice. The wound size was significantly reduced the day after surgery. Wounds in hiPSCSMC and ADSC groups had significantly more cell proliferation, α-SMA-positive cells, CD31-positive cells, VEGF-Apositive cells, and VEGF-A and PDGF expression. Conclusions　hiPSC-SMCs promote the expression of angiogenic and regenerative cytokines, and promote angiogenesis and cell proliferation, thereby promoting wound healing in diabetic mice.

Abstract: Objective　To investigate the effects of three Tiao-Bu Fei-Shen therapies on interleukin (IL)-6, IL-33, and interferon (IFN)-γ in the lung and intestines of rats with stable chronic obstructive pulmonary disease (COPD). Methods　Sixty Sprague-Dawley rats were divided randomly into blank (Control), COPD model (Model), Bufei Jianpi (BJ), Bufei Yishen (BY), Yiqi Zishen ( ZS), and aminophylline ( Am) groups. A stable COPD rat model was established by cigarette-smoke exposure combined with lipopolysaccharide nasal drip in the first 8 weeks. The model rats were given normal saline, Bufei Jianpi Formula, Bufei Yishen Formula, Yiqi Zishen Formula, or aminophylline by gavage for 4 weeks. Morphological changes in the lung and colon were observed by light microscopy, and IL-6, IL-33, and IFN-γ protein levels in lung and colon tissues were localized and analyzed semi-quantitatively by immunohistochemistry. Results　Rats in the model group had more inflammatory cells and inflammatory exudates around the bronchi and alveolar spaces compared with control rats, and also had more inflammatory cell infiltration in the colon mucosa and significantly increased levels of IL-6, IL-33, and IFN-γ in the lung and colon tissues (P< 0. 05, P< 0. 01). Bronchial and alveolar inflammatory cell infiltration and colonic mucosal inflammatory cells were decreased in all treatment groups compared with the model group. IL-33 and IFN-γ in the lung and colon and IL-6 in the colon were significantly decreased (P< 0. 05, P<0. 01), and IL-6 in the lung was significantly reduced in the BY, ZS, and Am groups (P< 0. 01). There were positive correlations between lung IL-6 and colon IL-6, lung IL-33 and colon IL-33, and lung IFN-γ and colon IFN-γ levels. Conclusions　The three tested Tiao-Bu Fei-Shen therapies can reduce intestinal dysfunction in COPD rats. The mechanism may be related to inhibition of the release of IL-6, IL-33, and IFN-γ in lung and colon tissues, and reducing the inflammatory reaction between the lung and intestine.

Abstract: Objective　To summarize the modeling characteristics of animal models of ulcerative colitis (UC) and provide a reference to establish an ideal animal model of UC. Methods　We searched the China Knowledge Base for topics of “ulcerative colitis” and “animals”, screened the relevant literature on animal experiments of UC from January 2012 to July 2022, and constructed a database using Excel 2017. The research contents, animal strains, modeling method, and detection indexes were sorted to analyze the model characteristics and main application directions of UC animal models. Results　After comparative analysis, we found that SD rats (247 reports, 43. 03%) and Wistar rats (105 reports, 18. 29%) were the animal strain used for modeling, male (355 reports, 64. 43%) was the main sex, and TNBS-ethanol solution enema (231 reports, 39. 69%) and DSS-free drinking (225 reports, 38. 66%) were the most common modeling method for both drug efficacy verification and mechanistic studies. Among these tests, colon pathology (269 reports, 13. 63%), disease activity index (DAI) score (246 reports, 12. 46%), general condition (231 reports, 11. 70%) and serum biochemical indices (225 cases, 11. 40%) and colonic tissue protein expression (171 reports, 8. 66%) were predominant. Conclusions　UC animal experiments are often performed in male SD and Wistar rats, which are modeled by a TNBS-ethanol enema and DSS-free drinking method. These method ologies have the advantages of ease, a high model establishment rate, and low cost, combined with colon histopathology, DAI scores, general conditions, serum biochemical indexes, and Colonic tissue protein expression, which provide the basis for UC animal experiments.

Abstract: Objective 　Clinical evidence suggests that the risk of hyperuricemia is increased under high temperature-humidity conditions. The current study aimed to explore this effect and the possible molecular mechanism responsible for the environmental effect on serum uric acid (SUA) levels, using a recently established hyperuricemia rat model. Methods　Hyperuricemia was induced in rats by potassium oxonate (PO group) or a high-purine diet (HPD group) using traditional chemical-induction method. The rats were also irradiated in a high temperature-humidity incubator (37℃, 80% humidity) for 1 h/ d to produce the high temperature-humidity group (HTH group) and high purine diet +high temperature-humidity group (HPD + HTH group). A control group (CON group) was also included. The experiment lasted for 12 weeks. Anal temperature and body weight were measured during daily irradiation, and blood samples were collected every 4 weeks to examine SUA and creatine levels in each group. After the experiment, the kidneys were removed and expression levels of ATP-binding cassette super family G2 ( ABCG2) and urate transporter 1 ( URAT1) were determined by Western Blot and immunohistochemistry. Results　The anal temperature and dehydration rate of rats in the HTH and HPD + HTH groups were significantly increased after irradiation in a high temperature-humidity incubator (P<0. 05). SUA levels were significantly higher in the HTH group compared with the CON and PO groups, and in the HPD +HTH compared with the HPD group (P< 0. 05). There was no significant difference in serum creatinine among the five groups after 12 weeks (P> 0. 05). Expression levels of the uric acid secretion protein ABCG2 were significantly lower in the HTH and HPD + HTH groups compared with the CON group, in the HTH group compared with the PO group, and in the HPD + HTH group compared with the HPD group, as shown by Western Blot (P< 0. 05). Expression levels of the urate transporter URAT1 were significantly higher in the HTH and HPD + HTH groups compared with the CON group, in the HTH group compared with the PO group, and the HPD + HTH group compared with the HPD group (P< 0. 05). Immunohistochemistry indicated that both ABCG2 and URAT1 were expressed in proximal convoluted tubule epithelial cells. ABCG2 expression levels were lower while URAT1 expression levels were higher in the HPD + HTH and HTH groups compared with the other groups (P< 0. 05). Conclusions　A high temperature-humidity environment can influence the secretion and reabsorption of uric acid by disrupting the function of the uric acid transporters ABCG2 and URAT1, which are located in the proximal tubules of the kidney. This can lead to increased SUA levels, potentially aggravating the occurrence and development of hyperuricemia.

Abstract:The mouse hind limb ischemic model is a classic model used to study peripheral arterial disease. Six modeling method have been used to date: arterial ligation, interventional embolization, photochemical embolization, chemical injury, physical injury, and ameroid constrictor. This article reviews the anatomy of the murine hind limb artery and unifies the names of the major branches of the hind limb. We also introduce the modeling and evaluation method used in the mouse hind limb ischemia model, and compare the differences, advantages, and disadvantages of the existing method. Notably, mouse strain, sex, age, and method of anesthesia may affect the model. This report thus provides a comprehensive review of the modeling method of the mouse hind limb ischemia model and provides a reference for researchers to help them to select appropriate models according to their research direction.

Abstract:Mesenchymal stem cells ( MSCs) are adult stem cells with self-replication and multi-directional differentiation abilities. MSCs can differentiate into various human tissues such as bone, cartilage, and muscle, and thus have broad clinical application prospects in the endogenous repair of human tissues. However, increased cell replication and aging mean that MSCs inevitably face the problem of senescence, which affects and limits their clinical application. To allow MSCs to be applied more effectively in basic and clinical research, it is necessary to study the mechanisms responsible for their senescence and develop strategies to prevent its occurrence. This report highlights the role of epigenetics in MSC senescence.

Abstract:With the increasing age of the population, and a large amount of bone calcium loss, osteoporosis has become a major public health problem. Osteoporosis is a “silent disease”, with a high incidence, high disability, and high death rates, which seriously affect quality of life. There are many animal models of osteoporosis, but there is a lack of relevant TCM disease and syndrome factors. Therefore, it can be combined with models that are highly consistent with clinical practice to make it closer to clinical research. In this article, we systematically review the advantages and disadvantages of various method of osteoporosis modeling, the advantages and disadvantages of animal species selection, the time of modeling, the drugs selection, and the application of assessment indicators to provide a reference for the standardization and improvement of animal model research.

Abstract:Drug-induced liver injury is one of the most common adverse clinical drug reactions, potentially resulting in acute liver failure or even death. Clinically however, there is still a lack of specific diagnostic and treatment method for this kind of injury. The construction of corresponding animal models is an important process to support in-depth mechanistic research and screening of effective diagnostic and treatment method. Numerous animal models of drug-induced liver injury have been established and are widely used, including liver injury models induced by non-steroidal antiinflammatory, anti-bacterial, anti-epileptic, and anti-thyroid drugs, with mice and rats being the most commonly used animals. In this report, we review the preparation method, modeling standards, and characteristics of these types of animal models in combination with recent literature reports, to provide references for the future construction of related models.

Abstract:In recent years, with the introduction of the concept of metagenomics, human metagenomics has also become a new approach to reveal the development and health of humankind. As the largest microflora in the human body, the gut microbiome has been found to play an important role in the normal physiological functions of the human body and the occurrence and development of diseases. The variety and quantity of gut microbiome are abundant. In studies of the gut microbiome, the application of germ-free animals makes the experiments more accurate and convenient. It is an ideal basic animal model, which is of great significance to the research of microecology. This review summarizes the current understanding of germ-free animals, the application of germ-free animals in experimental research, and the prospects of germ-free animal models to review the application and development of germ-free animals in research of the gut microbiome.

Abstract:Alcoholic liver disease (ALD) is a broad-spectrum disease caused by long-term heavy drinking, including alcoholic fatty liver, hepatitis, liver fibrosis, cirrhosis, and liver cancer. With the development of research, we have a certain understanding of the pathogenesis of ALD, but clinical treatment of ALD remains very limited. An appropriate ALD animal model is of great significance to elucidate ALD pathogenesis by seeking effective therapeutic targets and evaluating the therapeutic effects of related drugs. This review discusses the current research and development status of ALD animal models, introduces the modeling ideas, characteristics, and limitations of related models, and reviews the relevant new modeling ideas to provide a reference for researchers to establish appropriate ALD animal models.

Abstract:With the introduction and implementation of China’ s three-child policy, the productivity of elderly mothers in China has increased yearly. Advanced elderly mothers is prone to a variety of difficult and high-risk conditions during pregnancy and delivery, with a subsequent increase in the birth rate of high-risk neonates. High-risk neonates are treated and cared for in the neonatal intensive care unit after birth and separated from their mothers. Maternal separation impairs cognitive functions, but no effective care has been found in clinical care. Numerous animal experiments have shown that enriched environment can improve cognitive dysfunction caused by diseases such as alzheimer's disease, depression, and ischemic stroke, but there are few reports and disagreements about the role and mechanism of the enriched environment in the model of maternal separation. Therefore, we reviewed the research progress on the effects of an enriched environment on cognitive functions in rodent models of maternal separation and the mechanisms of action with oxidative stress, epigenetics, synaptic plasticity, and the hypothalamic-pituitary-adrenal axis to provide new ideas for the improvement and care of cognitive functions in high-risk neonates.