Abstract:Objective The serum metabolic characteristics of a tree shrew model of H1N1 influenza virus infection were studied by UPLC-Q/ TOF-MS. Methods The H1N1 virus nasal drip method was used to prepare the tree shrew model. Then, the viral load and antibody hemostatic titer were measured. Pathological examination of the lung tissue was performed, and tree shrew serum samples were collected for untargeted metabolomics research. Results From day 3 to 7 of model preparation, the body temperature and viral load in the infected tree shrews peaked. Multivariate statistical analysisfound 24 differential ions in the serum of tree shrews infected with the H1N1 virus, which led to metabolic disorders such as phosphatidylcholine metabolism, sphingolipid metabolism, and arachidonic acid metabolism. Conclusions This study defined for the first time the metabolic characteristics of H1N1 virus-infected tree shrews, and provided evidence that the disordered metabolism in this animal model was related to inflammation.

Abstract:Objective To investigate the effect of different concentrations of Matrigel on ectopic subcutaneoustransplanted tumor of Lewis lung cancer in mice. Methods Twenty-four C57BL/6J Nifdc mice were randomly divided intoa Lung cancer group, 50% Matrigel group, 75% Matrigel group, and 100% Matrigel group, with six mice in each group.Lewis lung cancer cells (LL/2) were mixed with 0%, 50%, 75% and 100% Matrigel at a volume ratio of 1 ∶ 1. The micewere injected subcutaneously under the armpit of the right forelimb. The weight, diet and volume of drinking water of micein each group were measured every day. The tumor formation time, tumor formation rate and tumor size were observed. Thelong diameter and short diameter of the tumor were measured every day after the formation of the tumor, and the change intumor volume was calculated. The animals were terminated on 15 days after tumor inoculation, the tumor tissue wasremoved and weighed, and the pathological changes of the tumor tissue were detected by HE staining. Results Comparedwith the Lung cancer group, the weight of the 75% Matrigel group increased significantly from the second day of modeling( P < 0. 05 or P < 0. 01). The weight of mice in the 100% Matrigel group increased significantly from the 10th day ofmodeling ( P < 0. 05 or P < 0. 01). The average diet of mice in each group decreased eight days after modeling, and thevolume of drinking water showed an upward trend. The tumor formation rate in each group was 100%. Mice with Matrigelhad earlier tumor formation time, faster tumor growth rate, and a larger tumor than those without. Among them, the 75%Matrigel group had the fastest tumor volume growth and the greatest tumor weight, (1358. 88 ± 388. 14) mg, comparedwith the Lung cancer group ( P < 0. 05). The tumor volume growth and tumor weight in the 100% Matrigel group were thesecond greatest, and the tumor weight was (1142. 37 ± 423. 08) mg, which was significantly different from that in theLung cancer group ( P < 0. 05). The tumor volume growth and tumor weight in the 50% Matrigel group were the smallest,(808. 83 ± 393. 41) mg. The result of HE staining show that, compared with the Lung cancer group, the tumor cells withMatrigel group had vigorous growth, a clear outline, large nuclear volume, and significantly more vascular components inthe stroma of the tumor tissue. Conclusions Matrigel can stabilize the tumor formation rate, accelerate tumor growth, andincrease the tumor formation volume. Under the condition with 75% Matrigel, the tumor growth was the fastest, the tumorvolume was the largest, and the tumor was uniform.

Abstract:Objective To observe the expression of Toll-like receptor 4 ( TLR4) and nucleotide bindingoligomerization domain protein 2 (NOD2) in the colon of a ulcerative colitis ( UC) rat model, and to explore thetherapeutic mechanism of Sishen pills in the treatment of UC from the perspective of repairing the intestinal mucosal barrier. Methods A UC rat model was prepared by administration of a trinitrobenzenesulfonic acid/ ethanol solution enema. Fortyrats were randomly divided into four groups, a blank group, model group, salazopyrimidine group(0. 36 g/ (kg·d)), andSishen pills group(5 g/ (kg·d)) The mRNA and protein expressions of TLR4 and NOD2 in colon tissue were detected byHE staining, RT-qPCR, SP immunohistochemistry and Western Blot. Results Compared with the blank group, thecolonic mucosal injury score of the model group was significantly greater ( P < 0. 01), and the expressions of TLR4, NOD2mRNA and protein were significantly greater ( P < 0. 01, P < 0. 05); Compared with the model group, the scores ofcolonic mucosal injury in each treatment group were significantly less ( P < 0. 01, v < 0. 05), and the expressions ofTLR4, NOD2 mRNA and protein were significantly less ( P < 0. 01, P < 0. 05). Conclusions Sishen pills can improvethe immune function of rats and inhibit intestinal inflammatory reactions for UC treatment.

Abstract:Objective CatSper1 is a sperm-specific voltage-gated Ca2+ channel protein that plays an essential rolein spermatogenesis and fertilization. We analyzed the expression regulation pattern, sequence characteristics, and potentialbiological function of CatSper1 gene of Banna mini-pig inbred (BMI) line. Methods The testes of adult BMI boars wereused for RNA sequencing (RNA-seq), and the complete coding sequence of CatSper1 was cloned using reverse-transcriptionpolymerase chain reaction. The sequence, structural characteristics, and interacting proteins of CatSper1 wereanalyzed. The lncRNA and miRNA regulatory network of CatSper1 was constructed, and gene ontology function annotationswere carried out using RNA-seq data. Results The average expression level and transcripts per million of CatSper1obtained by RNA-seq was 2817. 5 and 33. 6, respectively. The full-length coding sequence of CatSper1 was 2166 bp (GenBank accession number: OK042306), encoding 721 amino acids. CatSper1 protein contained an Ion_trans conserveddomain of 233 amino acid residues and a conserved transmembrane helix structure related to spermatogenesis. CatSper1protein interacted with 10 proteins related to male reproduction, especially CatSper2-4 of the CatSper family. Ten miRNA sregulated CatSper1 gene by a targeted mode, and 16 and 14 lncRNAs completed with CatSper1 for binding to ssc-miR-1343and ssc-miR-744, respectively. The gene ontology annotation result indicated that the CatSper1 gene plays important rolesin molecular functions, biological processes, and cellular components. Conclusions This study reported the expression CatSper1 in BMI testis, the molecular structure characteristics and expression regulatory network. The fundings will laygroundwork for further research of CatSper1 function in important biological processes such as spermatogenesis, spermcapacitation and acrosome reaction in BMI line.

Abstract:Objective The level of skeletal muscle glucose metabolism is an important factor affecting aerobicexercise capacity, and is regulated by the biological clock. Chrono is a newly discovered circadian clock gene. It isinvolvedin the feedback inhibition of transcription of the core circadian clock transcription factor BMAL1 to its downstream circadianclock target genes, and is considered a transcription repressor of BMAL1. Studies have shown that BMAL1 is involved inthe regulation of skeletal muscle glucose metabolism. However, CHRONO is a transcription inhibitor of BMAL1, and theeffect of the CHRONO-BMAL1 pathway on exercise capacity and glucose tolerance is currently unknown. Therefore, thisstudy employed skeletal muscle-specific Chrono overexpression mice and wild-type mice to explore the effect of theCHRONO-BMAL1 pathway on glucose tolerance and exercise capacity. Our findings provide a theoretical basis forelucidating new mechanisms affecting skeletal muscle health. Methods In total, 20 healthy 8-week-old C57BL/6N wildtype(WT) mice and 20 skeletal muscle-specific Chrono overexpression (Chrono-TG) mice (50:50, male: female) wereused in this study. Body weight and food intake were recorded. The body composition test, autonomous activity test,incremental load exercise ability test, glucose tolerance test, and grip test were performed. Skeletal muscle was weighedand the mRNA expression levels of the Chrono, MyhcI, MyhcIIa, MyhcIIb, MyhcIIx, and Pdha1 genes were detected byReal-time PCR. The content of muscle glycogen was detected using an appropriate kit. Results (1) Compared with WTmice, the autonomous mobility and exercise ability of Chrono-TG mice were obviously reduced ( P < 0. 05 or P < 0. 01);(2) Compared with WT mice, the weights of the gastrocnemius, quadriceps, soleus, extensor digitorum longus, and tibialanterior muscles of TG mice were significantly increased ( P < 0. 01), while the weights of the gastrocnemius andquadriceps femoris of female TG mice were lower than for male TG mice ( P < 0. 01); (3) Compared with WT mice, thestrength of the front paw of male TG mice was significantly reduced ( P < 0. 05), and MyhcIIs mRNA expression in skeletalmuscle was also decreased or showed a downward trend. The forepaw grip and MyhcIIs mRNA expression in female TG micewere clearly higher than in male TG mice ( P < 0. 05 or P < 0. 01); (4) Compared with WT mice, the blood glucose valueand the area under the curve at each point in the glucose tolerance test of the TG mice were increased ( P < 0. 01), whilefemale TG mice showed lower levels than male TG mice ( P < 0. 01); (5) Compared with WT mice, the content of muscleglycogen in TG mice was increased ( P < 0. 05), and the expression of Pdha1 mRNA was decreased or showed a decreasingtrend, while the muscle glycogen content and Pdha1 mRNA expression in female TG mice were significantly higher than inmale TG mice ( P < 0. 05 or P < 0. 01). Conclusions Overexpression of the Chrono gene in skeletal muscle can reduceautonomic mobility, impair glucose tolerance, and affect the aerobic exercise capacity of mice.

Abstract:Objective To investigate the myocardial protective effect and possible regulatory mechanism ofHonokiol (HKL) on Acute Myocardial Infarction (AMI) in vivo . Methods Eighty male C57BL/6J mice were randomlydivided into the following groups: Sham (Sham) group, Myocardial Infarction model and Vehicle (MI + V) group, Myocardial Infarction model and HKL treatment (MI + HKL) group, Myocardial Infarction model, HKL treatment andSirtuin-1(SIRT1) inhibitor (selisistat, EX527)(MI + HKL + EX) group, with twenty mice in each group. The mortalityof the mice during modeling stage was recorded after the operation. The echocardiogram and serum samples of the mice were gathered on the 28th day after the operation. The inflammatory indexes in the serum were detected by enzyme linkedimmunosorbent assay (ELISA). Besides, dihydroethidium staining(DHE) was utilized to display the intensity of reactiveoxygen species in myocardial tissue. Apoptosis ratio was evaluated by detection of terminal-deoxynucleoitidyl transferasemediated nick end labeling(TUNEL)and the expression of other target molecules was detected by Western Blot. Results Compared with the model group, the heart function of MI mice treated with oral HKL was significantly improved, the levelsof inflammatory factors in serum were decreased. Additionally, cardiomyocyte apoptosis rate and reactive oxygen species inmyocardial tissue were reduced. Simultaneously, the expression of SIRT1 was significantly up-regulated while theexpression of Ac-Foxo1 protein was down-regulated, which were reversed by SIRT1 inhibitor (EX527) ( P < 0. 05). Conclusions Oral HKL attenuate myocardial damage induced by myocardial infarction and significantly improvemyocardial function, which may be regulated by the SIRT1/ Ac-Foxo1 signal.

Abstract:Objective To illustrates the effect of resveratrol on motor dysfunction and peripheral immunity in α-synuclein A53T mice. Methods Eight-month-old A53T α-syn transgenic mice were randomly divided into four groups:model group (PD), model treatment group (PD + RES), control group (WT) and control treatment group (WT + RES).The treatment groups were given Resveratrol (RES)by gavage every 3 days for 11 months, while the control and modelgroups were given an equal volume of normal saline. The motoric function of the animals was analyzed with a forelimb gripstrength test, Rotarod test, and Pole climbing test. The proportion of T lymphocyte subsets in different groups of mice wasdetected by flow cytometry. IL-6, TNF-α, IL-18 and TGF-β in each group were detected by an ELISA kit. Results themotor function scores in the PD group were worse than those in the WT group. Compared with the PD group, the motorfunction score of the PD + RES group was significantly improved. The flow cytometry result showed that compared with theWT group, the T lymphocyte ratio, CD4⁺ T cell ratio, and CD4⁺ / CD8⁺ ratio were less in the PD group, while the CD8⁺ Tcell ratio was not significantly different. After RES treatment, T lymphocytes, CD4⁺ cells, and the CD4⁺ / CD8⁺ ratio weresignificantly greater in the PD + RES group than the PD group. The ELISA result showed that compared with the WTgroup, the IL-6 and IL-18 levels in the PD group were greater, the TGF-β level were less, and the TNF-α level showed nosignificant statistical difference. After RES treatment, compared with the PD group, the IL-6 and IL-18 levels in the PD +RES group were significantly less, while the TGF-β level was greater; however, the result showed no statistical difference. Conclusions Resveratrol can reduce motor disorders, regulate peripheral immune function, and effectively reduceneuroinflammation.

Abstract:Objective The purpose of this study was to analyze the immune responses induced by Brucellamelitensis(B. melitensis) outer membrane protein OMP25. Methods The omp25 gene from B. melitensis 16M was amplifiedby PCR. The fragment was cloned into pET-32a vector plasmid. The constructed recombinant plasmid pET32a-OMP25 wastransformed to E. coli BL21 (DE3) and was induced to express the fusion protein. Then the protein was purified using aNi2+ column purification kit. Mice were immunized with rOMP25 and current vaccine Rev. 1, and IFN-γ, IL-2 insplenocytes and IgG antibody and anti-inflammatory factor IL-10 levels in the serum were detected by ELISA. Thereactionogenicity was detected by Western Blot. Results (1) The full length of the omp25 gene was 642 bp, encoding 214amino acids. rOMP25 was approximately 40. 8 × 10³ as measured by SDS-PAGE. There was a single band after purification. (2) The t-test method showed that mice were immunized with rOMP25 and Rev. 1, the levels of IFN-γ andIL-2 in splenocytes, IgG antibody and IL-10 levels in serum in the rOMP25 group were similar to those in the Rev. 1 group,and significantly greater than those in the control ( P < 0. 01). (3) rOMP25 had good reactivity as observed in the WestemBlot. These result confirmed that rOMP25 could induce the body to produce high levels of cellular and humoral immunity,with good reactivity. Conclusions This study provides technical support for further study of the function of OMP25 proteinfor B. melitensis related vaccine development.

Abstract:Objective In this experiment, a mouse model of nephropathy induced by lead acetate was created tostudy the mechanism of detoxification and protection of grub peptide extract on mice, which provided experimental evidencefor the prevention and treatment of lead-induced nephropathy. Methods Mice were randomly divided into a Control group,Model group, Positive drug group, and Grub peptide groups. The Grub peptide groups were given different doses (80,160, 320 mg/ kg) of grub peptide. All mice, except the Control group, were injected intraperitoneally with 20 mg/ kg leadacetate every other day, for 15 consecutive days. The mice in the Control group and Model group were then fed normalsaline, while the mice in the Positive drug group were fed a dimercaptosuccinic acid (DMSA 70 mg/ kg) suspension. Themice in the Grub peptide groups were fed different doses of grub peptide extract. The state of the kidney tissue was observedby HE staining once a day for 15 days. The renal function indexes (BUN, Cr), antioxidant enzyme levels (SOD, GSHPx),and peroxide (MDA) content in renal tissue were detected. RT-PCR and Western Blot techniques were used todetect and analyze the gene and protein expression levels of phase II detoxification enzyme (NQO1), an antioxidant enzyme(HO-1), and a signaling molecule (Nrf2). Results The weight of mice in the Grub peptide groups was greater than thosein the Model group; however, they were less than those in the Control group, and the morphology of the kidney tissue wassignificantly better. Compared with the control group, the morphology of the kidney tissue of mice in the Grub peptidegroups was significantly improved, the level of BUN and Cr in the serum was significantly less ( P < 0. 05), the level ofantioxidant enzymes (SOD, GSH-Px) in renal tissue was significantly less ( P < 0. 05), and the content of MDA wassignificantly less. The phase II detoxification enzyme gene (NQO1), antioxidant enzyme (HO-1), and signal molecule(Nrf2) mRNA, and protein expression levels were significantly up-regulated ( P < 0. 01). Conclusions Grub peptidescan activate Nrf2-ARE signaling pathway to enhance antioxidant function and increase the gene expression of detoxificationenzymes in lead-poisoned mice. The protective detoxification effect can then be extracted.

Abstract:Objective To investigate the protective effect of paeoniflorin on nerve injury in rats with acute cerebral infarction by activating the liver kinase B1 ( LKB1) /5’-amp activated protein kinase (AMPK) signaling pathway. Methods An acute cerebral infarction model was established by occlusion of the middle cerebral artery in rats using sutures, and the rats were randomly divided into a Model group, Paeoniflorin (10 mg/ kg) group, AMPK inhibitor Compound C (CC) (0. 2 mg/ kg) group, and Paeoniflorin (10 mg/ kg) + CC (0. 2 mg/ kg) group, with 12 animals per group. In addition, 12 rats were separated from the common carotid artery and external carotid artery without the suture plug and used as the Sham group. After group administration, a Morris water maze test was used to evaluate the cognitive function of the rats. Triphenyltetrazolium chloride (TTC) staining was used to detect the cerebral infarction of rats in each group. TUNEL staining was used to determine the hippocampal neuron apoptosis rate of rats in each group. Kits were used to measure the levels of serum inflammatory factor inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β) levels, brain catalase (CAT), reactive oxygen species (ROS), and malondialdehyde (MDA) in the brain tissues of the rats. Western blotting was used to detect the expression of apoptosis-related proteins B-cell lymphoma-2 (Bcl-2), BCL2-associated X protein (Bax), and LKB1/ AMPK pathway related proteins (p-LKB1/ LKB1, p-LKB1/ LKB1, p-AMPK/ AMPK) in the brain tissues. Results Compared with the Sham operation group, the number of times crossing the original platform, residence time in the original platform quadrant, brain tissue CAT content, p-LKB1/ LKB1, p-AMPK/ AMPK, and Bcl-2 expression levels in the Model group were significantly less ( P < 0. 05), and the cerebral infarction area, hippocampal neuron apoptosis rate, serum iNOS and IL-1β levels, brain tissue ROS and MDA content, and Bax expression level were significantly greater ( P < 0. 05). Compared with the Model group, the number of times crossing the original platform, residence time in the original platform quadrant, brain tissue CAT content, p-LKB1/ LKB1, p-AMPK/ AMPK, and Bcl-2 expression levels in the Paeoniflorin group were significantly greater ( P < 0. 05), the cerebral infarction area, hippocampal neuron apoptosis rate, serum iNOS and IL-1β levels, brain tissue ROS and MDA contents, and Bax expression level were significantly less ( P < 0. 05). CC can reverse the protective effect of paeoniflorin in a rat cerebral infarction. Conclusions Paeoniflorin may activate the LKB1/ AMPK signal to prevent the occurrence and development of inflammation, reduce the oxidative stress level, reduce brain tissue infarction and hippocampal neuronal apoptosis in rats, improve cognitive function, and play a role in protecting nerves. CC can reduce the protective effect of paeoniflorin in a rat cerebral infarction. 【Keywords】　paeoniflorin; liver kinase B1/5’-amp activated protein kinase; acute cerebral infarction; nerve