Objective To establish an orthotopic transplantation tumor model of pancreatic cancer derived from transgenic LSL-KrasG12D/+ LSL-Trp53R172H/+ Pdx1-Cre(KPC)mice.To provide a stable and reliable drug preclinical research animal model to study the developmental mechanism and treatment strategies of pancreatic cancer.Methods Tumor tissue derived from KPC transgenic mice with spontaneous pancreatic cancer was transplanted into the C57BL/6J mouse pancreas.Ultrasound was used to monitor tumor growth.HE and immunofluorescence staining was used to evaluate the pathological characteristics of this model.Results The tumor derived from KPC mice grew steadily on the pancreas of C57BL/6J mice.Tumor cell proliferation index Ki67,matrix fibrosis marker αSMA,and immune cell markers CD45 and CD206 were all stably expressed in the tumor.The model stably retained the pathological features of primary pancreatic cancer.Widespread tumor metastases,which were similar to those observed in patients with pancreatic cancer,developed in this model.Conclusions An orthotopic transplantation model derived from a transgenic mouse with spontaneous pancreatic cancer was established successfully.The model simulates the stromal environment and immune cell infiltration of pancreatic cancer and retains strong stability and uniformity with the original tumor.It can be used as an effective drug preclinical research model to study pancreatic cancer progression and treatment strategies.

Objective A feasible and stable mouse model of thoracic aortic dissection(TAD)combined with acute lung injury(ALI)was established using β-aminopropionitrile monofumarate(BAPN)1 g/(kg·d)administered in drinking water.The mouse model of TAD combined with acute lung injury(ALI)was established to provide a rational animal model to study TAD combined with ALI.Methods Forty-five SPF-grade 3-week-old C57BL/6J male mice were selected and randomly allocated to a CON group(normal dietary water;15 mice)or BAPN group(administration in sterile water at 1 g/(kg·d);30 mice)for 4 weeks.During the experimental period,the general condition and modeling rate of mice were observed.TAD model mice were validated,and the BAPN group was divided into TAD and non-TAD groups by measuring the maximum diameter of the thoracic aorta and HE staining of aortic tissues.HE pathological staining,the wet/dry weight(W/D)ratio,total protein level in bronchioalveolar lavage fluid(BALF),and interleukin(IL)-1 β,IL-6,and tumor necrosis factor-α(TNF-α)in BALF)were used to validate the TAD combined ALI model in mice.Results BAPN treatment significantly delayed the increase in body mass and water intake of mice.Compared with CON and non-TAD groups,the maximum diameter of the thoracic aorta of mice in the TAD group was significantly thickened(P＜0.05).HE staining of the aorta showed significant thickening of the middle aortic layer,and the structure of the aortic wall was damaged and disordered.HE staining of lung tissues showed significant interstitial edema and inflammatory exudation accompanied by enlargement of alveolar lumen,alveolar wall epithelial exfoliation and hyaline membrane formation,and a significant increase in the pathological scores of lung injury(P＜0.05).Total protein levels and expression of IL-1β,IL-6,and TNF-α in lung tissue,W/D ratio,and BALF were also significantly increased(P＜0.05),whereas no significant difference was observed in the above indexes between the other two groups.Conclusions A mouse model of thoracic aortic dissection combined with acute lung injury can be established by BAPN administration in drinking water.

Objective To overcome the limitations of existing human respiratory syncytial virus(hRSV)animal models,such as semi-permissiveness and short duration of infection,this study established an IL2rg gene knockout(IL2rg-/-)rat model using TALEN gene editing technology.Methods The animal model was infected with hRSV intranasally.Clinical characteristics,body weight,and temperature changes were observed over the infection period(0～35 days).The total viral loads in respiratory organs,such as the nasal tissue,trachea,and lungs,were measured at various time points(4,11,20,and 35 days post-infection).Pathological analysis was conducted on target organs at the endpoint of observation(35 days post-infection).Changes in peripheral blood T,B,NK,and NKT cells and various cytokines were assessed at various time points(4,20,and 35 days post-infection).Results(1)IL2rg/-knockout rats sustained high viral loads in the nasal cavity upon intranasal inoculation with hRSV.The average peak titer rapidly reached 1 × 1010 copies/g in nasal tissue and 1 × 107 copies/g up to 5 weeks post-infection.(2)However,no significant pathological changes were noted in nasal,tracheal,or lung tissues.(3)An increase was observed in the content of peripheral blood B cells in hRSV-infected IL2rg--rats.(4)IL-6 and MCP-1 were increased in the early stage of infection and then decreased at the end of the observation period.Conclusions This study established a new IL2rg-/-rat model using TALEN technology and found that this model effectively supported high-level replication and long-term infection of hRSV,providing a good basis for antiviral drug screening and in vivo efficacy evaluation of anti-hRSV antibodies.

Objective To investigate the mechanism of icariin regulating the NLRP3 inflammasome in the treatment of cerebral ischemia-reperfusion injury in rats.Methods A rat model of focal cerebral ischemia-reperfusion was induced using the thread embolism method.At 24 hours post-operation,the rats were randomly allocated into a sham operation group,model group,butylphthalide group(70 mg/kg),ICA-low dose(20 mg/kg),ICA-middle dose(40 mg/kg),and ICA-high dose(80 mg/kg)groups.The corresponding drugs were administered by gavage at 10 mL/kg once a day for 13 consecutive days.One hour after the last administration,neurological function was scored.The cerebral cortex was observed by hematoxylin-eosin(HE)staining.Expression of interleukin(IL)-1β and IL-18 in the cerebral cortex was determined by immunohistochemistry.Expression of NLRP3,ASC,and Caspase-1 in the cerebral cortex was determined by Western Blot.Results In contrast to the sham operation group,there was a notable increase in neural function scores within the model group.The ischemic area around the visible cerebral cortex showed neuron necrosis at various level or glial cell proliferation,and the number of intact neurons was significantly reduced.IL-1β and IL-18 positive cells were significantly increased.Expression of NLRP3,ASC,and Caspase-1 was significantly increased(P＜0.01,P＜0.05).After treatment with icariin,the neural function score was decreased significantly.The degree of neuronal necrosis in the peri-ischemic area was significantly reduced,and the number of intact neurons was significantly increased.IL-1 β and IL-18-positive cells were decreased significantly.Expressions of NLRP3,ASC,and Caspase-1 were significantly decreased(P＜0.01,P＜0.05).Conclusions Treatment of cerebral ischemia-reperfusion injury by icariin may be related to regulation of the NLRP3 inflammasome.

Objective To explore the mechanism underlying the anti-hemorrhoid effects of Qingji Liangxue mixture(QLM).Methods Models of acute hemorrhoid in rats were established using a croton oil preparation(COP)and glacial acetic acid.A total of 80 SD female rats were randomly divided into two major groups:the COP group and the glacial acetic acid group,each consisting of 40 rats.In the COP group,rats were further randomly assigned to the following subgroups:control group 1(group CA),model group 1(group CB),low-dose QLM group 1-1(group CC),high-dose QLM group 1-2(group CD),and diosmin group 1(group CE),with eight rats in each subgroup.In the glacial acetic acid group,rats were randomly divided into the following subgroups:control group 2(group GA),model group 2(group GB),low-dose QLM group 2-1(group GC),high-dose QLM group 2-2(group GD),and diosmin group 2(group GE),with eight rats in each subgroup.Except for the control groups,all other subgroups were treated with COP or glacial acetic acid then treated with various drugs for 7 days.HE staining was used to observe anorectal histomorphology induced by COP and glacial acetic acid.ELIS As were used to measure serum levels of IL-6 and TNF-α induced by COP.Additionally,the area of perianal ulcers induced by glacial acetic acid was recorded.Twenty-four C57 mice were randomly divided into a control group(group A),low-dose QLM group(group B),high-dose QLM group(group C),and adrenal chromazone tablet group(group D).Results QLM significantly improved the pathological injury of animal models with similar hemorrhoids,reduced the score of pathological changes induced by COP(P＜0.05),reduced the serum levels of IL-6 and TNF-α(P＜0.05),reduced the area of perianal ulcers induced by glacial acetic acid(P＜0.05),and shortened the time of coagulation and bleeding(P＜0.05).Conclusions QLM has a good anti-hemorrhoid activity,which may be achieved by anti-inflammatory effects,hemostasis,and reducing tissue damage.

Objective Total thyroidectomy of C57BL/6 and KM mice was performed by two different surgical methods to verify the success of mouse hypothyroidism model modeling,and compared the success rate of different surgical methods.Methods C57BL/6 and KM mice underwent total thyroidectomy by ligation(operation method Ⅰ)or hemostasis(operation method Ⅱ),and the detailed operation processes were recorded.Serum TT3,TT4 and TSH levels detected by enzyme-linked immunosorbent assay,body weight,and hematoxylin-eosin(HE)-stained neck tissue were compared before and after surgery to verify the model.Results Serum TT3 and TT4 levels were decreased(P＜0.05)and TSH was increased(P＜0.001)in both model groups.The 28-day postoperative survival rates were 40％and 60％in groups Ⅰ and Ⅱ,respectively,and 50％and 40％in KM mice.Body weights were significantly higher in both model groups compared with the sham control group.HE staining and microscopic observation showed that the cervical tissue in both strains was thyroid tissue,and the back membrane of the thyroid remained intact after isolation.Conclusions Both surgical method can induce hypothyroidism in C57BL/6 and KM mice;however,it is necessary to consider the anatomical relationship of the thyroid gland to the surrounding tissue,improve the proficiency of the surgical operation,prevent the occurrence of postoperative hypocalcemia and infection,and thus improve the survival rate of the model mice.

Objective Exploring the antidiarrhea effect of new Ershen pills composed of nutmeg koji based on the spleen and kidney yang deficiency diarrhea mouse model.Methods KM mice were randomly divided into normal,model,Ershen pills Ⅰ(salt psoralen+bran-stewed nutmeg),Ershen pills Ⅱ(salt psoralen+nutmeg koji),Ershen pills Ⅲ(salt psoralen+nutmeg raw product),salt psoralen,and nutmeg koji groups.The combined modeling method of hydrocortisone+senna leaf was used to establish the diarrhea mouse model with spleen-kidney yang deficiency.General signs and pathological changes of each organ were observed.Various organ indexes,the small intestine propulsion rate,gastric residual rate,serum motilin(MTL),gastrin(GAS),adrenal ketone(CORT),thyroid stimulating hormone(TSH),testosterone(T),tumor necrosis factor-α(TNF-α),and interleukin 1β(IL-1β)were assessed.16S rDNA sequencing and data analysis were conducted for fecal microorganisms.Results After modeling,compared with the normal group,the weight and activity of mice in the model group were reduced,the small intestine propulsion rate was significantly increased,the gastric residual rate and organ indexes were significantly decreased,serum GAS,CORT,TSH,and T levels were significantly decreased,MTL,TNF-α,and IL-1β levels were significantly increased,and intestinal flora species diversity was decreased.After administration,the above indexes and symptoms were improved by various degrees in each administration group,and the Ershen pills Ⅱ group was better than Ershen pills Ⅰ,Ershen pills Ⅲ,salt-psoralea,and nutmeg koji groups.Conclusions Combined use of nutmeg koji and salt psoralen has a remarkable effect on diarrhea of spleen-kidney yang deficiency.Fermented nutmeg reduces its toxicity risk and enhances its effect of warming the spleen and preventing diarrhea,which facilitates the development of nutmeg koji.

Objective To explore the protective effect and possible mechanism of Periplaneta americana powder on rats with spinal cord injury.Methods Forty-eight male SD rats were randomly divided into sham operation,saline,Periplaneta americana powder,and Toll-like receptor 4(TLR4)inhibitor groups.Except for the sham operation group,a rat spinal cord hemi-transection injury model was established in the other three groups.The sham operation group received no treatment after the operation,saline and drug groups were subjected to intragastric administration of equal volumes of normal saline and Periplaneta americana powder(630 mg/kg),respectively,and the TLR4 inhibitor group was administered an intraperitoneal injection of TLR4 inhibitor(3 mg/kg).On days 1,3,7,and 14 after the operation,the motor function of rat hind limbs was evaluated by the Basso,Beattie,and Bresnahan(BBB)score.Histopathological changes of the spinal cord were observed by hematoxylin-eosin staining.Changes in the number of neurons were observed by immunohistochemistry.The levels of inflammatory factors IL-1,IL-6,IL-10,and TNF-α were measured by ELISA,and expression of TLR4,myeloid differentiation factor 88(MyD88),and NF-κB p65 was detected by Western Blot.Results Compared with the sham operation group,the BBB score and number of neurons in the saline group were significantly decreased,while the degree of pathological damage,and IL-1,IL-6,TNF-α,TLR4,MyD88,and NF-κB p65 levels were significantly increased(P＜0.05).Compared with the saline group,periplaneta americana powder and TLR4 inhibitor groups showed an increase in BBB scores and the number of neurons,and decreases in the degree of pathological damage and IL-1,IL-6,TNF-α,TLR4,MyD88,and NF-κB p65 levels(P＜0.05).Compared with the TLR4 inhibitor group,the periplaneta americana powder group had better increases in the BBB score,number of neurons and decreases in the degree of pathological damage and expression of IL-1 and TNF-α.Conclusions Periplaneta americana powder reduces the production of inflammatory factors after spinal cord injury by inhibiting the TLR4/MyD88/NF-κB pathway,protects nerves,and promotes motor recovery.

Objective To observe the distribution and characteristics of lymphatic vessels in normal and injured mouse spinal cord,and to determine if lymphatic vessels participate in the repair of spinal cord injury.Methods Thirty-nine adult male KM mice were divided randomly into a normal group(n=6)and an injured group(n=33).Mice in the injured group were further divided randomly into mice examined on postoperative days 1,3,5,7,and 14.Three mice in the injury group died after acupuncture injury,and the sample was subsequently supplemented randomly.Spinal cord damage was induced in the injured group by acupuncture,while mice in the normal group had no spinal cord damage.The distribution of lymphatic endothelial cells(LECs)in the spinal cord was detected by immunohistochemistry,and expression levels of the lymphatic endothelial cell markers prospero-related homobox-1(prox-1),lymphatic vessel endothelial cell hyaluronic acid receptor-1(lyve-1),and flat foot protein(podoplanin),and the vascular endothelial cell marker CD34 were observed in the spinal cords in normal and acupuncture-injured mice.Spinal cord samples were examined by immunofluorescence staining,and the source of new LECs was explored by observing the co-expression of lyve-1/prox-1,lyve-1/podoplanin,and CD34/prox-1.Results Lymphangioid structures were present in the spinal cord in normal mice and were distributed in segments,laterally between the white matter and gray matter.Nascent lymphangioid-like structures appeared in the spinal cord at the site of acupuncture injury,and prox-1,podoplanin,lyve-1,and CD34 were expressed simultaneously;however,these nascent lymphangioid-like structures disappeared after scarring during spinal cord injury.Conclusions Segmental,transversely distributed lymphangioid-like structures are present in the spinal cord in normal adult mice,and neonatal lymphangioid-like structures are involved in the reconstruction of spinal cord injury.These nascent LECs may originate from the surrounding existing lymphatic vessels or from vascular endothelial cells.

Objective To establish and evaluate a method for rapid and sensitive S.xylosus detection using qPCR(real-time quantitative PCR).Methods A gehM gene fragment was selected as the target for S.xylosus.A set of specific primers was synthesized and a qPCR method was established to detect S.xylosus.A S.xylosus standard strain and other non-target strains were chosen for analysis.DNA of S.xylosus was diluted 10-fold to determine its sensitivity.Clinical samples were tested,and positive products were sequenced.The result were compared with those of bacterial culture.Results S.xylosus had a specific amplification curve,whereas other non-S.xylosus species did not,indicating that the primers were specific for S.xylosus.Sensitivity was 100 fg/μL DNA.Repeatability within and between groups was less than 3％.A total of 60 clinical samples were analyzed,of which five samples had a typical S curve.qPCR products were sequenced and BLAST searched.The similarity of the gene sequences was 99.63％,indicating that the sample was positive for the S.xylosus gehM gene with a positivity rate of 8.3％.However,the positivity rate of bacterial culture was 6.7％.The positivity rate of qPCR was slightly higher than that of the culture.Conclusions The established qPCR method is rapid with high sensitivity and specificity,and can be used to detect S.xylosus.