OBJECTIVE: To explore the effect of chitosan (CS) hydrogel loaded with tendon-derived stem cells (TDSCs; hereinafter referred to as TDSCs/CS hydrogel) on tendon-to-bone healing after rotator cuff repair in rabbits. METHODS: TDSCs were isolated from the rotator cuff tissue of 3 adult New Zealand white rabbits by Henderson step-by-step enzymatic digestion method and identified by multidirectional differentiation and flow cytometry. The 3rd generation TDSCs were encapsulated in CS to construct TDSCs/CS hydrogel. The cell counting kit 8 (CCK-8) assay was used to detect the proliferation of TDSCs in the hydrogel after 1-5 days of culture in vitro, and cell compatibility of TDSCs/CS hydrogel was evaluated by using TDSCs alone as control. Another 36 adult New Zealand white rabbits were randomly divided into 3 groups ( n=12): rotator cuff repair group (control group), rotator cuff repair+CS hydrogel injection group (CS group), and rotator cuff repair+TDSCs/CS hydrogel injection group (TDSCs/CS group). After establishing the rotator cuff repair models, the corresponding hydrogel was injected into the tendon-to-bone interface in the CS group and TDSCs/CS group, and no other treatment was performed in the control group. The general condition of the animals was observed after operation. At 4 and 8 weeks, real-time quantitative PCR (qPCR) was used to detect the relative expressions of tendon forming related genes (tenomodulin, scleraxis), chondrogenesis related genes (aggrecan, sex determining region Y-related high mobility group-box gene 9), and osteogenesis related genes (alkaline phosphatase, Runt-related transcription factor 2) at the tendon-to-bone interface. At 8 weeks, HE and Masson staining were used to observe the histological changes, and the biomechanical test was used to evaluate the ultimate load and the failure site of the repaired rotator cuff to evaluate the tendon-to-bone healing and biomechanical properties. RESULTS: CCK-8 assay showed that the CS hydrogel could promote the proliferation of TDSCs ( P<0.05). qPCR results showed that the expressions of tendon-to-bone interface related genes were significantly higher in the TDSCs/CS group than in the CS group and control group at 4 and 8 weeks after operation ( P<0.05). Moreover, the expressions of tendon-to-bone interface related genes at 8 weeks after operation were significantly higher than those at 4 weeks after operation in the TDSCs/CS group ( P<0.05). Histological staining showed the clear cartilage tissue and dense and orderly collagen formation at the tendon-to-bone interface in the TDSCs/CS group. The results of semi-quantitative analysis showed that compared with the control group, the number of cells, the proportion of collagen fiber orientation, and the histological score in the TDSCs/CS group increased, the vascularity decreased, showing significant differences ( P<0.05); compared with the CS group, the proportion of collagen fiber orientation and the histological score in the TDSCs/CS group significantly increased ( P<0.05), while there was no significant difference in the number of cells and vascularity ( P>0.05). All samples in biomechanical testing failed at the repair site during the testing process. The ultimate load of the TDSCs/CS group was significantly higher than that of the control group ( P<0.05), but there was no significant difference compared to the CS group ( P>0.05). CONCLUSION TDSCs/CS hydrogel can induce cartilage regeneration to promote rotator cuff tendon-to-bone healing.
Rabbits ; Animals ; Rotator Cuff/surgery* ; Chitosan ; Hydrogels ; Rotator Cuff Injuries/surgery* ; Wound Healing ; Tendons/surgery* ; Collagen ; Stem Cells ; Biomechanical Phenomena

OBJECTIVE: To analyze the effect of local unstable atherosclerotic plaque on plaque formation in the carotid and aortic arteries of rabbits. METHODS: Thirty New Zealand white rabbits were randomly divided into atherosclerosis model group, highfat diet feeding group, and normal chow feeding group (blank control group). In the model group, carotid artery balloon injury was induced after 4 weeks of high-fat diet feeding. Eight weeks later, all the rabbits were euthanized for histopathological examination of the carotid artery and abdominal aorta, and the mean intimal thickness and plaque to lumen area ratio were measured using image analysis software. Venous blood samples were collected from the rabbits for blood lipid analysis. RESULT: At the ends of 4, 8 and 12 weeks, the rabbits in the model group and high-fat feeding group, but not those in the blank control group, all showed significant weight gain compared with their body weight at 0 week (P < 0.05). The mean intimal thickness was significantly greater in atherosclerosis model group than in the other two groups (P < 0.05). In atherosclerosis model group, the mean intimal thickness and plaque to lumen area ratio in the injured carotid artery were significantly greater than those in the contralateral carotid artery and abdominal aorta (P < 0.05). At the end of the 12 weeks, the levels of triglyceride (TG), total cholesterol (TC), low-density cholesterol (LDL-C), high-density cholesterol (HDL-C) and highsensitivity C-reactive protein (CRP) were all significantly higher in the model group and high-fat feeding group than in the blank control group (P < 0.05); the levels of TG, TC, LDL-C, or HDL-C did not differ significantly between the model group and high-fat feeding group (P>0.05), but the level of CRP was significantly higher in arteriosclerosis model group (P < 0.05). CONCLUSION Local unstable atherosclerotic plaque can increase the level of CRP and promote the formation of atherosclerotic plaques in the carotid artery and abdominal aorta in rabbits.
Rabbits ; Animals ; Plaque, Atherosclerotic ; Aorta, Abdominal ; Cholesterol, LDL ; Atherosclerosis ; Carotid Arteries

BACKGROUND: Hair follicles are easily accessible and contain stem cells with different developmental origins, including mesenchymal stem cells (MSCs), that consequently reveal the potential of human hair follicle (hHF)-derived MSCs in repair and regeneration. However, the role of hHF-MSCs in Achilles tendinopathy (AT) remains unclear. The present study investigated the effects of hHF-MSCs on Achilles tendon repair in rabbits. METHODS: First, we extracted and characterized hHF-MSCs. Then, a rabbit tendinopathy model was constructed to analyze the ability of hHF-MSCs to promote repair in vivo . Anatomical observation and pathological and biomechanical analyses were performed to determine the effect of hHF-MSCs on AT, and quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemical staining were performed to explore the molecular mechanisms through which hHF-MSCs affects AT. Furthermore, statistical analyses were performed using independent sample t test, one-way analysis of variance (ANOVA), and one-way repeated measures multivariate ANOVA as appropriate. RESULTS: Flow cytometry, a trilineage-induced differentiation test, confirmed that hHF-derived stem cells were derived from MSCs. The effect of hHF-MSCs on AT revealed that the Achilles tendon was anatomically healthy, as well as the maximum load carried by the Achilles tendon and hydroxyproline proteomic levels were increased. Moreover, collagen I and III were upregulated in rabbit AT treated with hHF-MSCs (compared with AT group; P  < 0.05). Analysis of the molecular mechanisms revealed that hHF-MSCs promoted collagen fiber regeneration, possibly through Tenascin-C (TNC) upregulation and matrix metalloproteinase (MMP)-9 downregulation. CONCLUSIONS hHF-MSCs can be a treatment modality to promote AT repair in rabbits by upregulating collagen I and III. Further analysis revealed that treatment of AT using hHF-MSCs promoted the regeneration of collagen fiber, possibly because of upregulation of TNC and downregulation of MMP-9, thus suggesting that hHF-MSCs are more promising for AT.
Animals ; Humans ; Rabbits ; Hair Follicle ; Achilles Tendon/pathology* ; Tendinopathy/pathology* ; Proteomics ; Collagen Type I ; Mesenchymal Stem Cells

Nearly a quarter of the world's population is infected with Mycobacterium tuberculosis and remains long-term asymptomatic infection. Rv2626c is a latent infection-related protein regulated by DosR of M. tuberculosis. In this study, the Rv2626c protein was prokaryotically expressed and purified, and its immunobiological characteristics were analyzed using RAW264.7 cells and mice as infection models. SDS-PAGE and Western blotting analysis showed that the Rv2626c-His fusion protein was mainly expressed in soluble form and specifically reacted with the rabbit anti-H37RV polyclonal serum. In addition, we found that the Rv2626c protein bound to the surface of RAW264.7 macrophages and up-regulated the production of NO. Moreover, the Rv2626c protein significantly induced the production of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6 and MCP-1, and induced strong Th1-tendency immune response. These results may help to reveal the pathogenic mechanism of M. tuberculosis and facilitate the development of new tuberculosis vaccine.
Animals ; Mice ; Rabbits ; Mycobacterium tuberculosis/genetics* ; Tuberculosis ; Antigens, Bacterial ; Cytokines ; Immunity, Cellular

c-Myc protein encoded by c-Myc (cellular-myelocytomatosis viral oncogene) gene regulates the related gene expression through the Wnt/β-catenin signaling pathway, and has received extensive attention in recent years. The purpose of this study was to express Helicoverpa armigera c-Myc gene (Ha-c-Myc) by using prokaryotic expression system, prepare the polyclonal antibody, examine the spatio-temporal expression profile of Ha-c-Myc, and investigate the possible function of Ha-c-Myc in regulating H. armigera sterol carrier protein-2 (SCP-2) gene expression. The Ha-c-Myc gene was amplified by PCR and cloned into a prokaryotic expression plasmid pET-32a(+). The recombinant plasmid pET-32a-Ha-c-Myc was transformed into Escherichia coli BL21. IPTG was used to induce the expression of the recombinant protein. Protein was purified by Ni²⁺-NTA column and used to immunize New Zealand rabbits for preparing the polyclonal antibody. The Ha-c-Myc expression levels in different developmental stages (egg, larva, prepupa, pupa, and adult) of H. armigera and different tissues (midgut, fat body, head, and epidermis) of the prepupa were determined by real-time quantitative reverse transcription PCR (qRT-PCR). Ha-c-Myc siRNA was synthesized and transfected into H. armigera Ha cells. The relative mRNA levels of Ha-c-Myc and HaSCP-2 in Ha cells were detected by qRT-PCR. Results showed that the pET-32a-Ha-c-Myc recombinant plasmid was constructed. The soluble Ha-c-Myc protein of about 65 kDa was expressed in E. coli. The polyclonal antibody was prepared. Western blotting analysis suggested that the antibody had high specificity. Enzyme linked immunosorbent assay (ELISA) showed that the titer of the antibody was high. Ha-c-Myc gene expressed at all developmental stages, with high levels in the early and late instars of larva, and the prepupal stage. Tissue expression profiles revealed that Ha-c-Myc expressed in various tissues of prepupa, with high expression level in the midgut, but low levels in the epidermis and fat body. RNAi results showed that the knockdown of Ha-c-Myc expression significantly affected transcription of HaSCP-2, leading to a 50% reduction in HaSCP-2 mRNA expression level. In conclusion, the Ha-c-Myc was expressed through a prokaryotic expression system, and the polyclonal anti-Ha-c-Myc antibody was obtained. Ha-c-Myc may promote the expression of HaSCP-2 and play an important role in the lipid metabolism of H. armigera. These results may facilitate further study on the potential role and function mechanism of Ha-c-Myc in H. armigera and provide experimental data for exploring new targets of green pesticides.
Animals ; Rabbits ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Moths/genetics* ; Blotting, Western ; Larva/genetics* ; Isoantibodies/metabolism* ; Antibody Specificity

A fluorescence endoscopic laser confocal microscope(FELCM) was used to direct the injection of sinomenine solid lipid nanoparticles(Sin-SLN) into the joint, and the in vitro effectiveness of Sin-SLN in the treatment of rheumatoid arthritis(RA) was evaluated. Sin-SLN was prepared with the emulsion evaporation-low temperature curing method. The Sin-SLN prepared under the optimal conditions showed the encapsulation efficiency of 64.79%±3.12%, the drug loading of 3.84%±0.28%, the average particle size of(215.27±4.21) nm, and the Zeta potential of(-32.67±0.84) mV. Moreover, the Sin-SLN demonstrated good stability after sto-rage for 30 days. The rabbit model of RA was established by the subcutaneous injection of ovalbumin and complete Freund's adjuvant. Five groups were designed, including a control group, a model group, a Sin(1.5 mg·kg~(-1)) group, a Sin-SLN(1.5 mg·kg~(-1)) group, and a dexamethasone(positive drug, 1.0 mg·kg~(-1), ig) group. The control group and the model group only received puncture treatment without drug injection. After drug administration, the local skin temperature and knee joint diameter were monitored every day. The knee joint diameter and the local skin temperature were lower in the drug administration groups than in the model group(P<0.05, P<0.01). FELCM recorded the morphological alterations of the cartilage of knee joint. The Sin-SLN group showed compact tissue structure and smooth surface of the cartilage. Enzyme-linked immunosorbent assay(ELISA) was employed to determine the serum le-vels of interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α). The findings revealed that the Sin-SLN group had lower IL-1 and TNF-α levels than the model group(P<0.05, P<0.01). Hematoxylin-eosin(HE) staining was employed to reveal the pathological changes of the synovial tissue, which were significantly mitigated in the Sin-SLN group. The prepared Sin-SLN had uniform particle size and high stability. Through joint injection administration, a drug reservoir was formed. Sin-SLN effectively alleviate joint swelling and cartilage damage of rabbit, down-regulated the expression of inflammatory cytokines, and inhibited the epithelial proliferation and inflammatory cell infiltration of the synovial tissue, demonstrating the efficacy in treating RA.
Animals ; Rabbits ; Tumor Necrosis Factor-alpha ; Fluorescence ; Arthritis, Rheumatoid/drug therapy* ; Interleukin-1 ; Arthritis, Experimental/drug therapy*

OBJECTIVE: To explore the effect of a modified three-point bending fracture device for establishing a rabbit model of closed tibial fracture. METHODS: The model of closed tibial fracture was established in 40 6-month-old male New Zealand white rabbits with a body weight of 2.5 to 3.0 kg, and the model was verified at 6 weeks after operation. Five rabbits underwent pre modeling without temporary external fixation before modeling, and then were fractured with a modified three-point bending fracture device;35 rabbits underwent formal modeling. Before modeling, needles were inserted, and splints were fixed externally, and then the fracture was performed with a modified three-point bending fracture device. The fracture model and healing process were evaluated by imaging and histopathology at 2 hours, 4 weeks, and 6 weeks after operation. RESULTS: Two hours after modeling, the prefabricated module showed oblique fracture in varying degrees and the broken end shifted significantly;Except for 1 comminuted fracture, 2 curved butterfly fractures and 2 without obvious fracture line, the rest were simple transverse and oblique fractures without obvious displacement in formal modeling group. According to the judgment criteria, the success rate of the model was 85.71%. Four weeks after modeling, the fixed needle and splint of the experimental rabbits were in good position, the fracture alignment was good, the fracture line was blurred, many continuous callus growths could be seen around the fracture end, and the callus density was high. Six weeks after modeling, many thick new bone trabeculae at the fracture, marginal osteoblasts attached, and a small number of macrophages were seen under the microscope. The intramembrane osteogenesis area was in the preparation bone stage, the medullary cavity at the fracture had been partially reopened, the callus was in the absorption plastic stage, and many osteoclasts were visible. The X-ray showed that the fracture line almost disappeared, part of the medullary cavity had been opened, the external callus was reduced around, the callus was in the plastic stage, and the bone cortex was continuous. It suggests that the fracture model showed secondary healing. CONCLUSION The improved three-point bending fracture device can establish a stable rabbit model of closed tibial fracture, and the operation is simple, which meets the requirements of closed fracture model in basic research related to fracture healing.
Rabbits ; Male ; Animals ; Bony Callus ; Fracture Healing ; Tibial Fractures/surgery* ; Osteogenesis ; Radiography

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.
Rabbits ; Male ; Animals ; Mice ; Antibody Specificity ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Recombinant Proteins ; Escherichia coli/genetics*

OBJECTIVE: To evaluate the antidiarrheal effect of ethanol extract of Glycyrrhiza uralensis Fisch root (GFR) in vivo and jejunal contraction in vitro. METHODS: In vivo, 50 mice were divided into negative control, positive control (verapamil), low-, medium- and high-dose GFR (250, 500, 1,000 mg/kg) groups by a random number table, 10 mice in each group. The antidiarrheal activity was evaluated in castor oil-induced diarrhea mice model by evacuation index (EI). In vitro, the effects of GFR (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/L) on the spontaneous contraction of isolated smooth muscle of rabbit jejunum and contraction of pretreated by Acetylcholine (ACh, 10 µmol/L) and KCl (60 mmol/L) were observed for 200 s. In addition, CaCl₂ was accumulated to further study its mechanism after pretreating jejunal smooth muscle with GFR (1 and 3 g/L) or verapamil (0.03 and 0.1 µmol/L) in a Ca²⁺-free-high-K⁺ solution containing ethylene diamine tetraacetic acid (EDTA). RESULTS: GFR (500 and 1,000 mg/kg) significantly reduced EI in castor oil-induced diarrhea model mice (P<0.01). Meanwhile, GFR (0.01, 0.03, 0.1, 0.3, 1, 3, and 10 g/L) inhibited the spontaneous contraction of rabbit jejunum (P<0.05 or P<0.01). Contraction of jejunums samples pretreated by ACh and KCl with 50% effective concentration (EC₅₀) values was 1.05 (0.71-1.24), 0.34 (0.29-0.41) and 0.15 (0.11-0.20) g/L, respectively. In addition, GFR moved the concentration-effect curve of CaCl₂ down to the right, showing a similar effect to verapamil. CONCLUSIONS GFR can effectively against diarrhea and inhibit intestinal contraction, and these antidiarrheal effects may be based on blocking L-type Ca²⁺ channels and muscarinic receptors.
Mice ; Rabbits ; Animals ; Antidiarrheals/adverse effects* ; Jejunum ; Glycyrrhiza uralensis ; Castor Oil/adverse effects* ; Calcium Chloride/adverse effects* ; Diarrhea/drug therapy* ; Plant Extracts/adverse effects* ; Verapamil/adverse effects* ; Muscle Contraction

Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia seen in clinical settings, which has been associated with substantial rates of mortality and morbidity. However, clinically available drugs have limited efficacy and adverse effects. We aimed to investigate the mechanisms of action of andrographolide (Andr) with respect to AF. We used network pharmacology approaches to investigate the possible therapeutic effect of Andr. To define the role of Andr in AF, HL-1 cells were pro-treated with Andr for 1 h before rapid electronic stimulation (RES) and rabbits were pro-treated for 1 d before rapid atrial pacing (RAP). Apoptosis, myofibril degradation, oxidative stress, and inflammation were determined. RNA sequencing (RNA-seq) was performed to investigate the relevant mechanism. Andr treatment attenuated RAP-induced atrial electrophysiological changes, inflammation, oxidative damage, and apoptosis both in vivo and in vitro. RNA-seq indicated that oxidative phosphorylation played an important role. Transmission electron microscopy and adenosine triphosphate (ATP) content assay respectively validated the morphological and functional changes in mitochondria. The translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the molecular docking suggested that Andr might exert a therapeutic effect by influencing the Keap1-Nrf2 complex. In conclusions, this study revealed that Andr is a potential preventive therapeutic drug toward AF via activating the translocation of Nrf2 to the nucleus and the upregulation of heme oxygenase-1 (HO-1) to promote mitochondrial bioenergetics.
Animals ; Rabbits ; Atrial Fibrillation/metabolism* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Signal Transduction ; NF-E2-Related Factor 2/pharmacology* ; Molecular Docking Simulation ; Oxidative Stress ; Energy Metabolism ; Mitochondria/metabolism* ; Inflammation/metabolism* ; Heme Oxygenase-1