Abstract: Objective　To observe changes in blood glucose, bone strength, and intestinal microflora in db/ db mice. Methods　Six male db/ db mice and six wild-type(WT)mice at 20 weeks of age were used to assess fasting blood glucose. A three-point bending test was performed to assess mechanical parameters of the femur. 16S rRNA high-throughput sequencing technology was used to sequence the V3-V4 gene region of intestinal contents and analyze the bioinformation. Results　The body weight(P< 0. 01) and blood glucose(P< 0. 05) of mice in the db/ db group were increased significantly. Tibial stiffness, maximum displacement, maximum energy absorption, and maximum load of db/ db mice were significantly lower than those of WT mice(P<0. 01). The Chao1 index, ACE index, and observed species in the α-diversity index of db/ db mice were decreased significantly(P<0. 05). There were significant differences in the intestinal bacterial community composition between the two groups(ANOSIM: P=0. 02). Lachnospira was significantly decreased in db/ db mice(P<0. 01). Desulfovibrionales, Proteobacteria, and Deltaproteobacteria were significantly increased(P<0. 05), and Alloprevotella was increased significantly(P<0. 01). Conclusions　db/ db mice with hyperglycemia and decreased bone strength are suitable animal models of T2DOP. The diversity of intestinal flora and the abundance of specific flora were different between db/ db and WT mice, which provides a theoretical basis to study intestinal flora and diabetic osteoporosis.

Abstract: Objective　To explore the mechanism of Shenfu injection improving cognitive dysfunction in vascular dementia model mice. Methods　Male Kunming mice were randomly divided into a Sham operation group, VD model group, SFI group(10 mL/ kg), L-Arg group(25 mg/ kg), and L-NAME group(10 mg/ kg)with nine in each group. The VD mouse model was established by repeated clipping and reperfusion of bilateral common carotid arteries and intraperitoneal injection of sodium nitroprusside. After modeling, each group was administered drugs for 21 days, and Sham operation and VD model groups were administered the same amount of physiological saline. The Morris water maze test assessed learning and memory abilities of mice, HE and Nissl staining was used to observe pathological changes. The Griess method was used to assess the NO concentration. ROS, MDA, and GSH contents were assessed by kits. iNOS, nNOS, and eNOS protein expression was assessed by Western blot analysis. Results　Compared with the Sham operation group, the latency time of the VD group was prolonged in the Morris maze test, pathological damage in the hippocampal Ca1 region was obvious, NO, ROS, and MDA contents were increased, GSH activity was decreased(P<0. 01, P<0. 05), nNOS and iNOS expression was significantly increased, and eNOS protein expression was decreased(P<0. 01, P<0. 05). Compared with the VD group, L-Arg treatment group showed no significant improvements in learning or memory abilities, pathological damage in the hippocampal CA1 region, or expression of NO, ROS, MDA, and GSH. Compared with the VD group, learning and memory abilities in SFI and L-NAME groups were improved, pathological damage of the hippocampal Ca1 region was significantly improved, NO, ROS, and MDA contents were decreased, GSH activity was increased, eNOS protein expression was increased, and iNOS and nNOS protein expression was significantly decreased(P<0. 01, P<0. 05). Conclusions　SFI improves cognitive dysfunction in VD mice, which may be related to the NOS/ NO pathway.

Abstract: Objective　To investigate the effect of electroacupuncture(EA) at Zusanli and Neiguan points on neuroplasticity factors GAP-43, SYN and MAP-2 in cerebral ischemic rats. Methods　Eighty rats were divided into Sham, EA 3 d, middle cerebral artery occlusion(MCAO)3 d, EA 14 d, and MCAO 14 d groups by a random number table with 16 rats per group. Except for the Sham group, a permanent MCAO rat model was established using the Longa thread embolization method . The EA group was treated at Neiguan and Zusanli points for 20 min daily after surgery. The motor function of rats was assessed by the balance beam test, and protein expression of neuroplasticity factors GAP-43 and SYN in the cerebral cortex contralateral to the infarct was assessed by Western blot. mRNA expression of GAP-43 and SYN was assessed by Real-time quantitative PCR, and colocalized expression of GAP-43/ MAP-2 and SYN/ MAP-2 was determined by immunofluorescence. Results　Compared with the Sham group, the MCAO group had significantly higher balance beam test scores at each time point(P<0. 05), GAP-43 and SYN mRNA expression was significantly lower in MCAO 3 d(P<0. 05)and 14 d(P<0. 05) groups, a significantly reduced SYN/ MAP-2 colocalized mean optical density was found in the MCAO 14 d group(P<0. 05), and GAP-43 and SYN protein expression was not significantly different in MCAO 3 d and 14 d groups. Compared with the MCAO group, no significant differences were seen in balance beam test scores at 1, 3, and 7 days in the EA group, and the balance beam test scores were significantly lower at 14 days(P<0. 05). Compared with the MCAO 3 d group, GAP-43 and SYN mRNA expression was significantly increased in the EA 3 d group(P< 0. 05). Compared with the MCAO 14 d group, GAP-43 and SYN mRNA expression was significantly increased(P<0. 05)GAP-43 and SYN protein expression was significantly increased in the EA 14 d group(P<0. 05), and the colocalized mean optical density of GAP-43/ MAP-2 and SYN/ MAP-2 were significantly increased(P<0. 05). Conclusions 　EA stimulation of Zusanli and Neiguan points improves motor functions after ischemic injury, and the mechanism may be related to promoting expression of neuroplasticity factors to induce axon sprouting and activating neuroplasticity after injury.

Abstract: Objective　To observe the effect of liuwei dihuangwan on the mitochondrial quality control system in the hippocampus of aged mice and its neuroprotective mechanism. Methods　Fifty 20-month-old C57BL/6J mice were randomly divided into five groups with 10 mice in each group, including a model group liuwei dihnangwan low, medium, of treatment, behavioral tests were carried out. Immunofluorescence was used to assess TNF-α and GFAP expression. Transmission electron microscopy was used to observe mitochondria and the number of autophagosomes. Proteins of the mitochondrial quality control system were assessed by Western blot. Results　Compared with the model group, behavioral tests showed that liuwei dihuangwan improved learning and memory abilities, enhanced the exercise ability, and reduced anxiety. Immunofluorescence showed decreases in TNF-α and GFAP expression. Transmission electron microscopy showed that the number of normal mitochondria was increased. PINK1, PARKIN, ATG3, ATG7, LC3B, MFN2, OPA1, TFAM, and PGC-1α protein expression was upregulated in the high-dose group, while P62, DRP1, and TNF-α protein expression was downregulated. Conclusions 　Liuwei dihuangwan improves the cognitive learning ability of mice, reduces neuroinflammation in the hippocampus, and improves the quality control system of mitochondria.

Abstract: Objective　To establish hematopoietic system-specific Marcksl1 knockout mice and explore the effect of Marcksl1 gene deletion on hematopoiesis. Methods　Using fetal liver competitive transplantation of E15. 5 Marcskl1 gene knockout mice, we analyzed the proportions of various hematopoietic cell types in peripheral blood at 1, 2, 3, and 4 months after fetal liver transplantation. KSL cells were sorted by flow cytometry and analyzed by RNA-seq. We used CRISPR/ Cas9 technology to generate Marcksl1 conditional knockout mice and obtained hematopoietic system-specific Marcksl1 knockout mice by crossing with Vav1-Cre mice. The genotype of the produced mice was confirmed by PCR and Sanger sequencing. Real-time PCR was used to assess Marcskl1 mRNA expression in bone marrow and hematopoietic stem cells. Flow cytometry was used to analyze the proportions of various hematopoietic cell types in bone marrow, peripheral blood, spleen, and thymus. The effect of Marcksl1 gene knockout on hematopoietic reconstitution was analyzed by competitive bone marrow transplantation. Results　Marcksl1 gene knockout decreased the proportion of B cells, increased the proportion of myeloid cells, decreased CLP and CMP, and increased GMP after fetal liver transplantation. RNA-seq showed that 252 genes were upregulated and 400 genes were downregulated. GO analysis showed that the differentially expressed genes were significantly enriched in the immune response, plasma membrane, and low-pressure gate calcium channel activity. KEGG analysis showed that the differentially expressed genes were significantly enriched in hematopoietic lineage differentiation and cytokine-cytokine receptor interaction-related signaling pathways. We generated hematopoietic system-specific Marcksl1 knockout mice successfully. Flow cytometry showed that Marcksl1 deletion did not affect the steady-state hematopoietic functions of adult mice, but it did affect the hematopoietic reconstitution ability after competitive bone marrow transplantation. Conclusions　We successfully established hematopoietic system-specific Marcksl1 knockout mice and found that Marcksl1 gene knockout does not affect stable hematopoiesis, but affects hematopoietic reconstitution. The underlying mechanism requires further investigation. This study provides an animal model to examine the gene function of Marcksl1 in adult hematopoiesis and other aspects.

Abstract: Objective　To explore the mechanism of LINC00662 in regulation of temozolomide resistance of glioma cells through miR-144/ COX-2 signaling. Methods 　qRT-PCR were used to measure the mRNA expression levels of LINC00662, miR-144, and COX-2 in glioma tissues, normal tissue adjacent to cancer, U251 cells, and U251/temozolomide ( TMZ) cells. The dual-luciferase reporter system were used to assess the regulatory relationship of LINC00662, miR-144, and COX-2. The five groups were a blank control, knockdown LINC00662 negative control(si-NC), knockdown LINC00662, and simultaneous knockdown of LINC00662 and inhibition of miR-144 expression. Cell proliferation were analyzed by CCK-8 and Edu assays. Apoptosis was evaluated by flow cytometry. Expression levels of target proteins were analyzed by Western blot. Results　Compared with adjacent normal tissue, the mRNA expression levels of LINC00662 and COX-2 were significantly upregulated and the expression level of miR-144 was downregulated in glioma tissues(P< 0. 05). Compared with U251 cells, the mRNA expression levels of LINC00662 and COX-2 were significantly upregulated and the expression level of miR-144 was downregulated in U251/ TMZ cells. Dual-luciferase reporter assays showed that LINC00662 targeted miR-144, and miR-144 targeted COX-2. Compared with the si-NC group, cell proliferation of the knockdown LINC00662 group was significantly decreased, the apoptosis rate of the knockdown LINC00662 group was increased(P<0. 05), COX-2, PCNA, MRP1 and Bcl-2 protein expression levels in the knockdown LINC00662 group were significantly downregulated, and the Bax protein expression level in the knockdown LINC00662 group was upregulated(P<0. 05). Inhibiting miR-144 expression reversed the effects of LINC00662 knockdown on U251/TMZ cell proliferation and apoptosis(P<0. 05). Conclusions　LINC00662 regulates the proliferation and apoptosis of glioma cells through miR-144/ COX-2 signaling and is closely related to temozolomide resistance of glioblastoma cells.

Abstract: Objective　To observe changes in retinal functions, structure, and glutamate content after retinal ischemia-reperfusion(RIR)injury in Long Evans rats to provide a reference for the study of retinal injury and possible protective mechanisms. Methods　Thirty adult SPF level Long Evans rats were randomly selected, and the left anterior chamber of their left eye was perfused with high-pressure normal saline for 60 minutes to establish an RIR injury model, while the right eye was untreated as a control. At 1, 3, 7, and 14 days after modeling, changes in retinal electrophysiological functions were assessed by flash electroretinogram. The retinal thickness was measured by optical coherence tomography(OCT)before and at 3, 7 and 14 days after modeling. Changes in fundus vessels were observed by fundus angiography. Rats were sacrificed at 14 days after modeling, and the retinal morphology, apoptosis, and distribution were observed by hematoxylin-eosin(HE) and TdT-mediated dUTP nick end labeling(TUNEL) staining. The content of glutamate in the retina was detected by ELISA. Results 　Compared with control eyes, the B wave amplitude of the electroretinogram in the model eyes were decreased significantly(P<0. 01)and latency was delayed significantly(P<0. 01) from the first day. OCT showed that the thickness of the retinal ganglion cell complex(GCC)was significantly thinner from day 3(P<0. 01), the thickness of the whole retina was significantly thinner from day 7(P<0. 01), and both of them became thinner over time(P<0. 05). Fundus images showed that the retina had obvious ischemia from day 3 and did not recover to the normal level until day 14. On day 14, the HE staining showed retinal atrophy, obvious thinning of the inner layer and a reduction of retinal ganglion cells. TUNEL staining showed obvious apoptosis in all retinal layers. ELISAs showed that the glutamic acid content in the retina was increased after modeling(P<0. 05). Conclusions　RIR injury in Long Evans rat causes serious damage to visual electrophysiological functions, retinal atrophy, an obvious reduction in the GCC thickness over time that becomes irreversible, RGC apoptosis, fundus vascular ischemia, and an increased retinal glutamate content, thereby providing a good animal model to study retinal injury diseases.

Abstract: Objective　To investigate the effect and mechanism of exogenous cholecystokinin octapeptide(CCK-8)on neuronal apoptosis induced by glutamate in the rat cerebral cortex in vitro. Methods　Rat cortical neurons cultured in vitro were treated with various concentrations of CCK-8. An MTT assay was used to assess cell survival and screen the appropriate concentration. Rat cortical neurons were treated with glutamic acid(Glu)to establish a neuron injury model and The Ca²⁺ level was assessed by Flou-4 staining. Bcl-2 and Bax mRNA expression was assessed by Real-time quantitative PCR. Bcl-2, Bax, and cleaved caspase-3 protein expression was assessed by Western blot. Results　Compared with the control group, the survival rate of neuronal cells, the distribution of G1 phase cells, Bcl-2 mRNA and protein expression levels were decreased, and the distributions of S and G2 / M phase cells, the apoptosis rate, calcium ion level, Bax and cleaved caspase-3 protein expression levels were increased in the model group(P<0. 05). Compared with the model group, the neuronal cell survival rate, G1 phase cell distribution, snf Bcl-2 mRNA and protein expression levels were increased, whereas S and G2 / M phase cell distributions, apoptosis rate, calcium ion level, Bax and cleaved caspase-3 protein expression levels were decreased in the CCK-8 group(P<0. 05). Conclusions　CCK-8 has an inhibitory effect on neuronal apoptosis induced by glutamate by inhibiting calcium influx and regulating Bax/ Bcl-2 expression in cells.

Abstract: Objective　To study the effect of cordycepin on invasion and metastasis of Hela cervical cancer cells by regulating miR-135b-5p expression and its regulatory mechanism. Methods　Hela cells were treated with 50 and 100 μmol/ L cordycepin, and Hela cell growth was measured by MTT assays. Transwell assays were used to assess Hela cell invasion, a scratch assay was used to assess the metastasis ability of Hela cells, and Real-time PCR was used to measure the miR-135-5p expression level in human immortalized epidermal cell line Hacat and Hela cells, and the effect of various cordycepin concentrations on miR-135-5p expression level. miR-135b-5p mimic was transfected into Hela cells, and the invasion and metastasis of Hela cells were assessed by Transwell and scratch assays. Results　After cordycepin treatment, Hela cell growth was significantly inhibited, and the inhibitory effect of the high concentration cordycepin was more obvious(P<0. 05). The miR-135b-5p expression level in Hela cells was (1. 97±0. 07), which was significantly higher than that in Hacat cells(1. 01±0. 03), and the difference between groups was statistically significant(t=28. 187, P=0. 000). After cordycepin treatment, the invasion and metastasis rates of Hela cells and miR-135b-5p expression were decreased significantly. After treatment with a high cordycepin concentration, the invasion and metastasis rates of Hela cells were lower, and the differences between groups were statistically significant(t= 138. 614~317. 100, P<0. 05). Overexpression of miR-135b-5p significantly increased the invasion and metastasis rates of Hela cells(t= 7. 145, 7. 465, P<0. 05), and vimentin expression was increased and E-cad expression was decreased(t= 8. 223, 7. 473, P< 0. 05). Conclusions Cordycepin inhibits the invasion and metastasis of Hela cells by inhibiting miR-135b-5p expression.

Abstract: Objective 　To investigate the effect of the JAK2/ STAT3 signaling pathway on the Th17/ Treg imbalance in acute lung injury(ALI)and its mechanism. Methods　Twenty-four C57BL/6 mice were randomly divided into control, model, JAK2 inhibitor, and STAT3 inhibitor groups with six mice in each group. The control group was instilled with normal saline in their airway, and the same amount of normal saline was injected intraperitoneally 8 h later. The model, JAK2 inhibitor, and STAT3 inhibitor groups were instilled with lipopolysaccharide(LPS) in their airway to establish the ALI model, and the same amount of normal saline, Fedratinib, and NSC74859 were injected intraperitoneally 8 hours later, respectively. The wet/ dry(W/ D) lung weight of mice was measured at 24 h after LPS infusion, the pathological changes of lung tissue were observed by HE staining, the proportion of Treg and Th17 cells in lung tissue was measured by flow cytometry, and IL-6, LL-10, IL-17A, and TGF-β1 levels in lung tissue homogenates were measured by ELISA. Protein expression of JAK2, STAT3, p-JAK2, and p-STAT3 in lung tissues was detected by Western blot. Results Compared with the control group, the W/ D ratio was increased(P<0. 01), lung tissue injury was severe, there was a degree number of inflammatory cell infiltration, the Th17/ Treg ratio, the levels of inflammatory factors IL-6, IL-17A, TGF-β1, p-JAK2, and p-STAT3 were significantly increased(P<0. 01), and the IL-10 level was significantly decreased in the model group(P<0. 01). Compared with the model group, the W/ D lung weight of JAK2 inhibitor and STAT3 inhibitor groups was decreased (P<0. 05), lung tissue injury was alleviated, inflammatory cell infiltration was reduced, the Th17/Treg ratio, IL-6, IL-17A, and TGF-β1 levels, p-JAK2 and p-STAT3 protein expression levels were significantly decreased in lung tissue(P<0. 01), and the IL-10 level was significantly increased(P<0. 01). Conclusions　Inhibiting activation of the JAK2/ STAT3 signaling pathway regulates the imbalance of Th17/ Treg cells in lung tissue of ALI mice induced by LPS, inhibits the inflammatory response, and reduces lung injury.

Abstract: Objective 　To analyze the influence of arbidol(ARB) on HCoV-OC43-induced activation of host signaling pathways and to explore the correlation between the anti-inflammatory activity of ARB and its effect on the neurotrophin signaling pathway. Methods　HRT-18 cells were infected with OC43 and treated with ARB. After 96 hours, total RNA was extracted, transcriptomic analysis was performed to identify differentially expressed genes(DEGs), and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed to identify potential biological processes and signaling pathways related to ARB treatment. RT-qPCR was used to verify the inhibitory effect of ARB on the expression of important molecules in the neurotrophin pathway. Results　ARB treatment at high, medium, and low doses(6, 2 and 0. 67 μg/ mL)resulted in 9459, 4186, and 1744 DEGs, respectively, compared with the virus-infected control group. GO analysis showed that ARB mainly affected biological processes of cotranslational protein targeting to membrane, cellular components such as focal adhesion and cell-substrate junction, and molecular functions such as cadherin binding. KEGG analysis showed that apoptosis and neurotrophin signaling pathways related to coronavirus infection were significantly enriched, and ARB had a significant inhibitory effect on MAPK, PI3K, and NF-κB in the neurotrophin signaling pathway. RT-qPCR analysis revealed that ARB significantly inhibited mRNA expression of PIK3CA, AKT, TRAF-6, Bax, p38, and c-Jun. Conclusions　This study suggests that ARB can be used for treatment of neuroinflammation caused by coronavirus infection.

Abstract:The student-centered “new three centers” education concept is the direction of teaching reform in colleges and universities. Medical laboratory animal science is an important foundation subject of medical education. To fully motivate student subjective initiative, develop their experimental skills and scientific research thinking, the teaching model of medical laboratory animal science gradually changed from traditional concept of teaching knowledge to centered on students, such as a flipped classroom, online and offline mixed teaching, MOOC, and other new modes. This article examines the knowledge framework and teaching important difficult points on the basis of the curriculum features, explores the advent of the era of the new three centers education concept on the basis of curriculum features, achieves improving teaching quality in course teaching and the scientific literacy in medical students by teaching method innovations and an intelligent teaching environment. “New three center” education promotes a combination of college ideological and political education with professional theory courses for a good synergistic effect with the teaching content of medical laboratory animal science.

Abstract:Autism spectrum disorder(ASD) is a kind of neurodevelopmental disorder that has attracted global attention because of its continued rise in incidence. There is a correlation between gut microbiota and ASD. Homeostasis of gut microbiota ensures normal life activities and may affect the occurrence and development of ASD through changing neurotransmitters, metabolites, the immune system, HPA axis, and intestinal permeability by losing its homeostasis. Gut microbiota treatment method, such as probiotics, prebiotics, and fecal microbiota transplantation, have a treatment effects on ASD. Additionally, traditional Chinese medicine may exert a certain treatment effect on ASD by through gut microbiota. The purpose of this review was to summarize the mechanism of gut microbiota disturbance affecting ASD as well as related research method and models to provide reference for the research and treatment of ASD.

Abstract:Cardiovascular disease(CVD) is hazardous to human health. Recent studies have found that gut microbiota participates in the occurrence and development of CVD through various mechanisms, among which the metabolite disorder is the main mechanism. To better understand the relationship between gut microbiota and CVD, this article focuses on the role and mechanism of metabolites from gut microbiota in the occurrence of CVD, which will play a positive role in the discovery of microbial markers and therapies of CVD.

Abstract:In recent years, exosomes have been used as a medium for cell-cell communication, providing a new perspective for information exchange between cells. Exosomes from various cell types, such as mesenchymal stem cells, osteoblasts, osteoclasts, and their precursors, are play an important role in bone remodeling. Many studies have shown that common bone metabolic diseases, such as osteoporosis, fracture, and osteoarthritis, have obvious correlations with exocrine bodies. Although bisphosphonate drug treatment, autologous and allograft bone grafting, and other treatment method achieve good result , they may lead to various complications and adverse reactions. Therefore, it is very meaningful to develop new targeted therapies with a strong bone regeneration ability, lower complication rate, and more accuracy. Therefore, this article reviews the mechanism of exosomes from various sources acting on bone tissue cells and the application of related bone metabolism disease treatments to provide ideas for developing new bone regeneration treatment method.

Abstract:Immune checkpoint inhibitors is currently the most important anti-tumor therapy, but immunotherapy resistance affects the therapeutic effect. This article reviews the related mechanisms of immune checkpoint inhibitor resistance and the mechanisms of the relationship between gut microbiota and immune checkpoint inhibitor resistance. By improving gut microbiota to increase treatment sensitivity and alleviate immunotherapy resistance, an effective method has been explored to address immune checkpoint inhibitor resistance.

Abstract:The modeling method of non-alcoholic fatty liver has always been a research focus. The selection of appropriate animal and cell models in accordance with experimental purposes is of great significance to study the pathogenesis of non-alcoholic fatty liver disease. The commonly used modeling method are high fat and sugar feeding, choline-methionine deficiency, drug induction, and saturated or unsaturated fatty acid induction. Rats, mice, genemodified mice, and cells are most frequently used to model non-alcoholic fatty liver. This review focuses on the research progress of rats, mice, genetically defective mice, and cell models of non-alcoholic fatty liver.

Abstract:Brain diseases are common in clinical practice. The blood-brain barrier prevents harmful substances from entering the brain and maintains homeostasis, but it also prevents drugs from entering the brain to exert effects. Nanotechnology has the advantage of tissue targeting. Active substances in Chinese traditional medicine have a good treatment effect on brain diseases. The combination of nanotechnology and active substances in Chinese traditional medicine for the treatment of brain diseases enhances targeting.