Abstract: Objective To establish a fast, stable, and sensitive zebrafish model of osteoporosis ( OP) using different method. Methods OP models were induced by iron overload or prednisolone (Pred), and bone formation and mortality were observed. The groups were divided into: Control group, model group (include FAC group and Pred group),and positive control group (AC group). Ammonium ferric citrate was used as the model drug in the iron-overload induction method. For the Pred induction models, the modeling time for the Pred-3 days post-fertilization ( dpf) method was 3 ~9 dpf, the modeling time for the Pred-5 dpf method was 5 ~ 10 dpf, and Pred was administered from 3 dpf and removed from 7 ~ 9 dpf for the Pred withdrawal method. To compare the anti-osteoporosis (OP) effects of commonly used drugs such as Alfacalcidol (AC), Calcitriol ( CA), and Alendronate ( AL), it’ s important to select a stable and sensitive positive control drug and to further optimize different staining Methods and conditions. Results There was no significant effect of ammonium ferric citrate 500 μg / mL on bone formation. Bone formation and the length of the first vertebra were significantly decreased in the Pred group induced by Pred-3 dpf compared with those in the control group (P<0. 01, P<0. 05), but zebrafish mortality was higher. There was no significant difference between the Pred-5 dpf method, but bone formation was significantly reduced in the Pred withdrawal group (P<0. 01), with no mortality. Alfacalcidol, calcitriol, and alendronate all had anti-OP effects, with CA having the most sensitive and stable anti-OP effect. Alizarin red staining showed that the optimal dye parameters were 0. 02% concentration for dyeing 2 h, with washing in 0. 5% KOH and glycerol under the conditions of a 3 ∶1 ratio for 3 h followed by a 1 ∶1 ratio for 14 h. The result of staining showed that calcein was more sensitive for staining bone nodes and ARS staining was more sensitive for staining the first vertebra. Conclusions The Pred withdrawal method can be used to establish a rapid, stable, and sensitive OP model in zebrafish as a reliable model for studying OP.

Abstract: Objective To investigate the effect of Ba Bao Dan(BBD) on cardiac injury induced by doxorubicin in zebrafish. Methods We induced a zebrafish myocardial injury model using the chemotherapeutic drug, doxorubicin. We then examined the effects of different concentrations of BBD on pericardial edema and heart rate under an in vivo microscope. We also examined the inhibitory effects of BBD on neutrophil infiltration in the heart in Tg ( mpx:EGFP) transgenic zebrafish. The impacts of BBD on superoxide dismutase, catalase, and malondialdehyde were observed. mRNA expression levels of ferroptosis-related factors, including glutathione peroxidase 4a ( gpx4a), prostaglandin-endoperoxide synthase 2 (ptgs2), arachidonate 5-lipoxygenase (alox5a), and acyl-CoA synthetase long-chain family member 4 (acsl4) were determined by Real-time quantitative polymerase chain reaction. The accumulation of ferrous ions in zebrafish heart was assessed using a fluorescent probe for ferrous ions. Results BBD alleviated doxorubicin-induced pericardial edema and bradycardia in zebrafish, reduced neutrophil infiltration in the heart ( P<0. 05 ), decreased malondialdehyde concentration (P<0. 05), and enhanced the activities of superoxide dismutase and catalase ( P<0. 05). BBD also significantly inhibited ferroptosis, reduced the accumulation of ferrous ions in the zebrafish heart, suppressed the expression of ptgs2, alox5a, and acsl4 (P<0. 05), and promoted the expression of gpx4a (P<0. 05). Conclusions BBD can attenuate doxorubicin-induced zebrafish myocardial injury and improve cardiac function by inhibiting lipid peroxidation and regulating ferroptosis.

Abstract: Objective To explore whether voluntary wheel running affects liver oxidative stress by regulating the Nrf2 / HO-1 pathway, thereby alleviating HFFC diet-related lipid deposition in the liver. Methods Eight-week-old C57BL/ 6J mice were randomly divided into a normal diet group ( NC group, n= 10) and high-fat, fructose, and cholesterol diet group (HFFC group, n= 20) after 1 week of adaptive feeding. Ten weeks of feeding later, mice in the HFFC group were divided into a quiet group (HFFC group, n= 10) and HFFC combined with exercise group (HFFC+EX group, n= 10). HFFC + EX group mice were caged with voluntary running wheels for free movement, and the number of running wheels was recorded every day for 8 weeks. After the last treatment, the mice were sacrificed by fasting for 12 hours at an interval of 24 hours, and the blood and liver were collected for analysis. Results ( 1) Body weight, liver weight, and liver index of mice fed the HFFC diet were significantly higher than those of the NC group, which significantly decreased after exercise (P<0. 05). ( 2) Compared with the NC group, HDL-C and LDL-C in the HFFC group were significantly increased, and the LDL-C level was significantly decreased after 8 weeks of exercise (P<0. 05). (3) The liver fat droplet area and liver TG content in the HFFC group were significantly higher than those in the NC group, whereas those in HFFC + EX group were significantly decreased (P<0. 05). (4) Compared with the NC group, the content of oxidase MDA in the HFFC group were significantly increased, and nuclear translocation and gene expression of Nrf2 were significantly decreased. After exercise, the activities of SOD and T-AOC were significantly increased, and the nuclear translocation and gene expression of Nrf2 and expression levels of HO-1 and SOD-1 were significantly increased ( P<0. 05). (5)The number of apoptotic hepatocytes and CHOP expression in the HFFC diet group were significantly higher than those in the NC group, whereas the number of apoptotic hepatocytes, and CHOP and Bax / Bcl-2 expression in the exercise group were significantly lower than those in the NC group ( P<0. 05). Conclusions Voluntary wheel can alleviate HFFC diet induced liver lipid deposition by regulating the Nrf2 / HO-1 pathway, thereby alleviating oxidative stress and reducing apoptosis in liver cells.

Abstract: Objective To investigate the differences in blood pressure phenotypes, renal pathological changes, and related pathogenic pathways in genetically diverse hypertensive mice obtained from 13 strains. Methods The genotypes ofCckbr^{+ / +},Cckbr^{+ / -}and Cckbr^{- / -}were obtained by hybridization of 13 strains of genetically diverse mice with Cckbr^{- / -} mice. Blood pressure was measured with a noninvasive blood pressure analysis system (BP-2000). The expression of CCKBR protein in mouse kidney tissue was detected by Western Blot, and the pathological changes in mouse kidney tissue were detected by hematoxylin-eosin ( HE) staining and immunohistochemistry ( IHC). The pathogenic pathways related to essential hypertension were screened by RNA sequencing. Results In three specific mouse strains (A/ J, LOT,and FIM), the systolic blood pressure( SBP) was significantly different between the Cckbr^{- / -}andCckbr^{+ / +} groups. HE staining and IHC showed that hypertension caused a certain degree of renal injury in the mice. Gene Ontology(GO) and pathway enrichment analysis showed that differentially expressed genes were enriched in metabolic processes and circadian rhythm regulation. Conclusions Genetically diverse mice can effectively simulate the genetic background of the population and provide a new resource for studying the pathogenic genes related to essential hypertension.

Abstract: Objective To investigate the effects of the estrogen synthesis inhibitor letrozole on the composition, diversity, and abundance of the intestinal microbiota in rats with polycystic ovary syndrome (PCOS). Methods A rat model of PCOS was induced using the estrogen synthesis inhibitor letrozole. The gut microbiota structures of normal and PCOS rats were analyzed 16S rRNA sequencing. Results At the phylum level, rats in the PCOS group had a lower abundance of Firmicutes (73. 29% ± 5. 34% vs. 77. 17% ± 4. 01%) and a higher abundance of Bacteroidetes (23. 85% ± 6. 22% vs. 17. 21% ± 6. 90%) compared with the normal group. At the genus level, the abundances of Rochella (5. 61% ± 3. 20% vs. 16. 13% ± 6. 20%) and Zurichella (1. 75% ± 2. 23% vs. 9. 98% ± 7. 34%) were lower in the PCOS group than in the normal group, but the abundances of Lactobacillus (18. 83% ± 11. 50% vs. 15. 88% ± 7. 06%), Prevotella(4. 58% ± 1. 09% vs. 0. 46% ± 0. 15%), and Ruminococcus (2. 97% ± 1. 56% vs. 1. 16% ± 0. 60%) were increased.Analysis of Kyoto Encyclopedia of Genes and Genomes signaling pathways of the differentially expressed bacteria showed that signaling pathways related to bile acid biosynthesis in the intestine were changed in the PCOS rat model. Conclusions The structure of intestinal flora in PCOS rat model constructed by intragastric administration of letrozole is significantly disordered, which may be related to the signaling pathways related to bile acid biosynthesis.

Abstract: Objective The DNA methylation microhaplotype ( DMH) refers to the combination of multiple methylation sites within a very short range, and these haplotypes show wide diversity. We carried out screening and validation of age-related DMHs in mouse blood. Methods We initially constructed a theoretical dataset of DMHs based on the mouse reference genome. We then screened age-related DMHs by Spearman’ s rank correlation analysis, using highthroughput sequencing information for DNA methylation in mouse blood from a network database. Finally, cross-validation was performed using a validation dataset. Results A total of 6787 142 DMH sites were identified within 50 bp in the mouse genome, including 98. 64% of single-digit CpG sites. A total of 5835 age-associated DMHs were screened in 58 mouse blood samples ( |rho | > 0. 5, P<0. 01), accounting for 0. 086% of DMHs. Finally, we validated the top 100 ageassociated DMHs with high correlation in 95 independent samples, Results ing in 44 loci. Conclusions The age-associated DMHs screened in this study may be useful in future studies of apparent age prediction using mouse blood and in aging studies.

Abstract: Objective This study aimed to develop an experimental animal platform for evaluating the feasibility and safety of intelligent acupuncture robots and to lay the foundation for further research. Methods Six 2-month-old Guangxi Bama miniature pigs were used as experimental subjects for acupuncture verification after anesthesia. First,manual acupuncture verification was carried out. Six acupoints were selected for each experimental animal and the needles were left for 20 min after the lifting, inserting, and twisting manipulation. Before and after controls were included. The experiment was carried out for 28 days, and each experiment was conducted once every 2 days for a total of 10 times. After verification of manual acupuncture, a point 10 mm from each of the six selected acupoints was selected, with a total of 12 points, and acupuncture operations were carried out on the experimental animals using the intelligent acupuncture module of the acupuncture robot at different frequencies and angles, to further verify the stability and feasibility of the animal platform. Results Routine safety-related blood indicators and blood biochemistry indicators after the procedure were normal and stable compared with those before the procedure. The average heart rate of the animals was 124 beats/ min, the average blood pressure was 87 / 36 mmHg, and the average body temperature of was 36 ℃ at a room temperature of 25 ℃ ,with no significant change in body temperature during and after the experiment. On the basis of this experimental platform,acupuncture manipulation using the intelligent acupuncture module of the acupuncture robot was completed successfully,with no abnormalities related to acupuncture such as bending, breaking, or stagnation of needles during the experimental process, and the experimental animals showed no obvious abnormalities. Conclusions This study established a stable experimental animal platform for evaluating the feasibility and safety of acupuncture carried out by intelligent acupuncture robots, based on the existing experimental method of miniature pigs. These result lay a foundation for further research related to the use of intelligent acupuncture robots.

Abstract: Objective To establish an immortalized tree shrew corneal stromal cells (CSCs) line and to study its response to virus infection. Methods Primary tree shrew CSCs were isolated and cultured by the tissue block adhesion method. CSCs were then transfected with a lentivirus carrying the SV40T gene and monoclonal cells were selected for passage culture. The characteristics of the CSCs were investigated by morphological observation and compared with 40 generations until the 50 generations or more, immunofluorescence identification of vimentin and SV40T genes, karyotype examination, and cell proliferation curve. The CSCs were infected with herpes simplex virus-1 (HSV-1)(McKrae strain),Zika virus (ZIKV, GZ01 strain), Dengue virus type Ⅱ, and H1N1 (PR8). Results The immortalized tree shrew CSCs after > 50 passages appeared spindle-shaped with good cell morphology and structure compared with 40 generations.Positive immunofluorescence expression of vimentin and SV40T genes. The cell growth curve showed that the cells were in logarithmic-phase growth on days 4 ~ 5 and grew vigorously. The number of chromosomes in the primary cells was stable at 62, while immortalized CSCs had 64 chromosomes at P21 and P56. The virus titer Results showed that the immortalized tree shrew CSCs were sensitive to HSV-1( McKrae strain), ZIKV (GZ01 strain), Dengue virus type Ⅱ, and H1N1 (PR8),with virus titers of 1. 32 ×10⁵、5. 62 × 10⁶、2. 69 × 10⁷、7. 76 × 10⁴ CCID50 / mL, respectively. Conclusions Theimmortalized tree shrew CSCs were established successfully, suggesting that this cell line is suitable for studies of the mechanisms of HSV, ZIKV, Dengue virus, and influenza A virus infection in relation to corneal diseases and antiviral drugs.

Abstract: Objective To observe the effects of different ligation sites and fasting method on a C57BL/ 6J mouse model of partial bile duct ligation (pBDL)-induced cholestasis, to establish a pBDL modeling method with a high modeling rate, typical symptoms, and good stability. Methods C57BL/ 6J mice were subjected to selective ligation of the left hepatic bile duct (L-pBDL) and left-to-median bile duct junction ligation (ML-pBDL) for modeling, and the effects of different pBDL ligation method on serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase (ALP), total bilirubin, total bile acid, and liver histopathology were observed. The effects of different fasting method on symptoms and liver injury in the ML-pBDL model were also observed after fasting for 12 and 16 h before surgery, and for 4 h after surgery. Results (1)The incidence of jaundice in the ML-pBDL group was 52. 94% and the survival rate within 3 weeks after surgery was 64. 71%, while the incidence of jaundice in the L-pBDL group was 11. 76% and the survival rate within 3 weeks after surgery was 82. 35%. Compared with those in the sham surgery group, serum liver function indicators were significantly increased in the L-pBDL and ML-pBDL groups (P<0. 01), and ALP activity was significantly higher in the ML-pBDL group than in the L-pBDL group (P<0. 05). Compared with mice in the L-pBDL group, mice in the MLpBDL group had more severe liver fibrosis at 3 weeks post-surgery (P<0. 01). (2)In addition, the incidence of jaundice in the 16 h fasting group was 93. 33% and the survival rate within 3 weeks after surgery was 73. 77%, while the incidence of jaundice in the 12 h fasting group was 42. 86% and the survival rate within 3 weeks after surgery was 71. 42%.Compared with those in the normal group, ALP activity, alanine aminotransferase / aspartate aminotransferase ratio, total bile acid level, and proportion of collagen fiber area were all significantly increased in the 16 h and 12 h fasting groups (P<0. 05). Although the observed indicators were higher in the 16 h fasting group compared with those in the 12 h fasting group, the difference was not significant (P>0. 05). Mice in the 12 h and 16 h fasting groups both showed significant bile duct hyperplasia and liver fibrosis (P<0. 01), with more severe liver fibrosis in the 16 h fasting group (P<0. 01). Conclusions Both L-pBDL and ML-pBDL ligation method can be used to establish a mouse model of cholestasis;however, symptoms in the L-pBDL model only exhibit transient damage characteristics, while the liver lesions in the MLpBDL model are typical and stable. Prolonging the preoperative fasting time can improve the modeling rate and stability of the ML-pBDL model and produce more-typical pathological symptoms.

Abstract: Objective To investigate the effects of cultured mycelium Cordyceps sinensis (CMCS) on the AMPK/SirT1 signaling pathway in carbon tetrachloride (CCl₄ )-induced liver fibrosis in mice. Methods Forty male SPF-grade C57BL/ 6 mice were divided randomly into a normal control group, CMCS control group (3. 0 g / kg), model control group,CMCS 1. 5 g / kg group, and CMCS 3. 0 g / kg group. Mice were injected intraperitoneally with 10% CCl4 (2 mL/ kg) to induce liver fibrosis. Two weeks later, serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and total bilirubin(TBil) were measured. Inflammation and collagen deposition in liver tissue were observed by hematoxylin and eosin (HE) and Sirius red staining, respectively. The content of hydroxyproline in liver tissue was detected by Jamall’ s hydrochloric acid hydrolysis method. Levels of interleukin ( IL)-6, monocyte chemoattractant protein-1 ( MCP-1), interferon, tumor necrosis factor (TNF), IL-10, and IL-12p70 in liver tissue were detected using a cytometric bead array analysis system. Collagen Ⅰ and SirT1 expression in liver tissue were detected by immunohisotochemistry, and Prkaa1,Prkaa2, Lkb1, and p53 gene expression were detected by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction. Results Serum levels of ALT, AST, and TBil were significantly increased in the model control group compared with those in the normal control group (P<0. 05). HE and Sirius red staining showed extensive inflammatory cell infiltration and collagen deposition in the liver, respectively. Hydroxyproline content and expression levels of IL-6,MCP-1, and TNF in the liver were significantly increased (P<0. 05), while IL-10 and IL-12p70 levels were significantly decreased (P<0. 01). Immunohistochemical staining revealed an increase in Collagen Ⅰ expression and SirT1 staining was decreased in the hepatic sinusoidal space, while collagen deposition was increased. Prkaa1, Prkaa2, and Lkb1 gene expression levels were decreased and p53 was increased in liver tissue (P<0. 05). CMCS significantly reduced serum ALT and AST levels, decreased IL-6, MCP-1, and TNF expression in liver tissue (P<0. 05), up-regulated IL-10 and IL12p70 ( P<0. 05), alleviated liver inflammation, collagen deposition, and hydroxyproline content, up-regulated the expression of SirT1 in the hepatic sinusoidal space, enhanced Prkaa1, Prkaa2, and Lkb1 expression (P<0. 05), and down-regulated Collagen Ⅰ and p53 ( P<0. 05) in the liver. Compared with CMCS 1. 5 g / kg, CMCS 3. 0 g / kg significantly inhibited liver inflammation and collagen deposition and up-regulated AMPK/ SirT1 expression (P<0. 05). Conclusions CMCS could improve CCl₄-induced liver fibrosis via up-regulation of the AMPK/ SirT1 signaling pathway.

Abstract: Objective To evaluate the protective effect of epigallocatechin-3-gallate (EGCG) on cisplatin (CIS)-induced acute kidney injury (AKI) in rats based on network pharmacology and in vivo animal model experiments. Methods Targets of EGCG and AKI were collected from the TCMSP, Gene Cards, and OMIM websites, and a protein-protein interaction network was constructed based on the intersection. The intersection targets were analyzed visually using Cytoscape 3. 9. 1 and the key targets were screened out. Kyoto Encyclopedia and of Genes and Genomes and Gene Ontology enrichment analyses were carried out using the DAVID database. Thirty-two male Wistar rats were divided randomly into four groups: CON group, EGCG group, CIS group, and CIS + EGCG group. Rats in the control and CIS groups were given normal saline every day, rats in the EGCG and CIS + EGCG groups were given EGCG (40 mg / kg) every day for 28 d, and rats in the CIS and CIS + EGCG groups received an intraperitoneal injection of CIS (7 mg / kg) on the 26th day. Blood and tissue samples were obtained on the 29th day. Serum urea nitrogen and creatinine levels were detected, renal pathology was observed by HE staining, and apoptosis in renal tissue was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling. Western Blot, qRT-PCR, and immunohistochemistry were used to verify the result. Results Eighty-seven genes intersecting EGCG and AKI and 25 core targets were screened from network pharmacology, which influenced the development of AKI via signal pathways such as PI3K/ Akt and various biological processes. EGCG pretreatment significantly reduced serum levels of serum urea nitrogen and creatinine, improved the renal pathology, and reduced renal tissue apoptosis in AKI rats. Western Blot, qRT-PCR and immunohistochemistry showed that pretreatment with EGCG activated the PI3K/ Akt pathway. Conclusions EGCG alleviates CIS-induced AKI in rats via the PI3K/ Akt signaling pathway.

Abstract: Objective To optimize the simulation of the pathological characteristics and seizure behavior of medial temporal lobe epilepsy ( MTLE), we aimed to establish a chronic epilepsy model of MTLE by unilateral, single hippocampal injection of kainic acid ( KA) via stereotactic surgery, and to validate this epilepsy model in terms of behavior, electrophysiology, and pathology. Methods A total of 22 healthy C57BL/ 6 wild-type male mice were divided randomly into a control group (n= 6) and an experimental group, which received KA injections (n= 16). Both groups underwent microinjection of equal doses of saline or KA in the hippocampal CA3 area via stereotactic surgery. One week later, all mice were implanted with electrodes in the hippocampal CA3 area to facilitate electroencephalogram ( EEG) recording. Seizure frequency and duration were analyzed statistically. The chronic epilepsy model was assessed in terms of behavior, electrophysiology, and pathology. Results No mice in the control group experienced seizures, while all surviving mice in the experimental group developed seizures. Adult model mice exhibited chronic spontaneous seizure behaviors, such as staring, chewing, head and facial muscle twitching, and limb spasms. Two mice died as a result of the surgery, four mice died during the acute seizure phase, and ten model mice were successfully established. EEG recordings showed epileptiform changes consistent with MTLE. Immunofluorescence staining revealed neuronal loss in the CA3 area and astrocytic changes, consistent with characteristic pathological changes of hippocampal sclerosis. Conclusions The model constructed by single unilateral intrahippocampal KA injection demonstrated several advantages such as being timeefficient, easy to operate, and reproducible. This model exhibited EEG, behavioral, and neuropathological changes similar to human MTLE, making it valuable for studying effective treatments for temporal lobe epilepsy and serving as an ideal animal model for predicting outcomes of epilepsy surgery.

Abstract:Alzheimer’s disease (AD) is a multifactorial degenerative disorder of the central nervous system that mainly manifests as cognitive dysfunction and loss of speech. In recent years, the zebrafish has attracted extensive attention because of its high homology with humans in terms of brain structure and function, nerve conduction, and pathogenic genes of AD. This article reviews the advantages of the zebrafish as an animal model of AD, covering topics in the pathogenesis of AD and the evaluation and screening of drugs for treatment of AD. The overall goal is to provide new insights into the pathogenesis of AD and development of novel drugs.

Abstract:Alzheimer’s disease (AD) is a multifactorial degenerative disorder of the central nervous system that mainly manifests as cognitive dysfunction and loss of speech. In recent years, the zebrafish has attracted extensive attention because of its high homology with humans in terms of brain structure and function, nerve conduction, and pathogenic genes of AD. This article reviews the advantages of the zebrafish as an animal model of AD, covering topics in the pathogenesis of AD and the evaluation and screening of drugs for treatment of AD. The overall goal is to provide new insights into the pathogenesis of AD and development of novel drugs.

Abstract:Micro-computed tomography (Micro-CT) is a non-invasive technology that is widely used in animal experiments to assist in the detection of bone, lung, oral, metabolic, middle and inner ear diseases, as well as tumors, and in other animal disease models. The technique can provide diverse scientific and reliable imaging data for animal experiments and has accordingly become an indispensable experimental method in animal experiments. In this review, we introduce the imaging principles of Micro-CT, review its application in the study of animal disease models, summarize the limitations of Micro-CT technology, and consider its future prospects.