AIM:To observe the effects of Huazhuo granules on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/Akt)/glycogen synthase kinase-3β(GSK-3β)and PI3K/Akt/forkhead box protein O1(FOXO1)signaling pathways in the liver tissue of type 2 diabetes mellitus(T2DM)rats,and to explore its mechanism of action in regulating glycogen synthesis and gluconeogenesis.METHODS:Thirty 8-week-old SPF-grade male SD rats were chosen,with 5 ran-domly selected ones as the blank control group.The remaining 25 rats were fed to a high-sugar and high-fat diet for 4 weeks,followed by a single intraperitoneal injection of streptozotocin to establish a T2DM rat model.After successful mod-eling,these rats were randomly divided into 5 additional groups:the model group,a positive control group treated with met-formin,and low,medium,and high-dose Huazhuo granule groups,resulting in a total of 6 groups with 5 rats in each.Rats in the treatment groups were gavaged with the corresponding doses of medication,while those in the blank control and model groups were gavaged with an equal volume of saline once daily for 4 consecutive weeks.After 24-hour fasting with free access to water,the rats were euthanized to measure blood glucose,serum insulin levels,and serum triglyceride(TG),total cholesterol(T-CHO),high-density lipoprotein cholesterol(HDL-C),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels.Liver pathological morphology was assessed by hematoxylin-eosin staining,and liver glycogen storage was detected by periodic acid-Schiff staining.Immunohistochemistry and Western blot analysis were employed to detect the protein levels of p-PI3K,p-Akt,p-FOXO1 and p-GSK-3β in the liver of rats in each group.The mRNA levels of phosphoenolpyruvate carboxykinase(PEPCK),glucose-6-phosphatase(G6Pase),GSK-3β,FOXO1,PI3K and Akt in liver tissues were detected by RT-qPCR.RESULTS:Compared with the model group,the rats in the high-dose Huazhuo Granule group and the metformin group exhibited decreased blood glucose and insulin levels(P<0.01),reduced AST,ALT,TG,and T-CHO concentrations(P<0.01),and increased HDL-C concentrations(P<0.01).The phosphorylation levels of PI3K,Akt,FOXO1,and GSK-3β proteins were up-regulated(P<0.01).In con-trast,the gene expressions of G6Pase,PERCK,FOXO1,and GSK-3β were all down-regulated(P<0.01).Conversely,the gene expressions of PI3K and Akt were upregulated(P<0.01).Pathological morphology of the liver tissue improved,accompanied by increased glycogen deposition in hepatocytes.CONCLUSION:Huazhuo granule manifests effects in lowering blood glucose and serum insulin levels,ameliorating blood lipids,and enhancing liver function in rats.These ef-fects are revealed to increase glycogen synthesis by activating the PI3K/Akt/GSK-3β signaling pathway and reduce gluco-neogenesis by activating the PI3K/Akt/FOXO1 signaling pathway.

AIM:To observe the effects of Huazhuo granules on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/Akt)/glycogen synthase kinase-3β(GSK-3β)and PI3K/Akt/forkhead box protein O1(FOXO1)signaling pathways in the liver tissue of type 2 diabetes mellitus(T2DM)rats,and to explore its mechanism of action in regulating glycogen synthesis and gluconeogenesis.METHODS:Thirty 8-week-old SPF-grade male SD rats were chosen,with 5 ran-domly selected ones as the blank control group.The remaining 25 rats were fed to a high-sugar and high-fat diet for 4 weeks,followed by a single intraperitoneal injection of streptozotocin to establish a T2DM rat model.After successful mod-eling,these rats were randomly divided into 5 additional groups:the model group,a positive control group treated with met-formin,and low,medium,and high-dose Huazhuo granule groups,resulting in a total of 6 groups with 5 rats in each.Rats in the treatment groups were gavaged with the corresponding doses of medication,while those in the blank control and model groups were gavaged with an equal volume of saline once daily for 4 consecutive weeks.After 24-hour fasting with free access to water,the rats were euthanized to measure blood glucose,serum insulin levels,and serum triglyceride(TG),total cholesterol(T-CHO),high-density lipoprotein cholesterol(HDL-C),aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels.Liver pathological morphology was assessed by hematoxylin-eosin staining,and liver glycogen storage was detected by periodic acid-Schiff staining.Immunohistochemistry and Western blot analysis were employed to detect the protein levels of p-PI3K,p-Akt,p-FOXO1 and p-GSK-3β in the liver of rats in each group.The mRNA levels of phosphoenolpyruvate carboxykinase(PEPCK),glucose-6-phosphatase(G6Pase),GSK-3β,FOXO1,PI3K and Akt in liver tissues were detected by RT-qPCR.RESULTS:Compared with the model group,the rats in the high-dose Huazhuo Granule group and the metformin group exhibited decreased blood glucose and insulin levels(P<0.01),reduced AST,ALT,TG,and T-CHO concentrations(P<0.01),and increased HDL-C concentrations(P<0.01).The phosphorylation levels of PI3K,Akt,FOXO1,and GSK-3β proteins were up-regulated(P<0.01).In con-trast,the gene expressions of G6Pase,PERCK,FOXO1,and GSK-3β were all down-regulated(P<0.01).Conversely,the gene expressions of PI3K and Akt were upregulated(P<0.01).Pathological morphology of the liver tissue improved,accompanied by increased glycogen deposition in hepatocytes.CONCLUSION:Huazhuo granule manifests effects in lowering blood glucose and serum insulin levels,ameliorating blood lipids,and enhancing liver function in rats.These ef-fects are revealed to increase glycogen synthesis by activating the PI3K/Akt/GSK-3β signaling pathway and reduce gluco-neogenesis by activating the PI3K/Akt/FOXO1 signaling pathway.

Objective To establish F0 generation chimeric rabbits with FOXN1 gene knockout and explore method for the in vivo conservation of immunodeficient rabbits in a conventional housing environment.Methods Initially,CRISPR/Cas9 technology was employed to inject constructed sgRNA and Cas9 protein into a single cell from rabbit two-cell stage embryos to obtain chimeric embryos with FOXN1 gene editing.The embryos were subsequently transferred into surrogate does.Finally,the F0 generation offsprings were genotyped using PCR and Sanger sequencing,and their growth and development in a conventional housing environment were observed.Results The PCR and Sanger sequencing result confirmed the successful establishment of himeric rabbits with FOXN1 gene knockout.On observation,the chimeras exhibited normal growth and development in a conventional environment without any immunodeficient phenotypes.Conclusions This study established a preliminary chimeric rabbit model with FOXN1 gene knockout that grows and develops normally in standard laboratory environments.This lays the foundation for the further breeding of FOXN1 immunodeficient rabbits in the future.

Klebsiella pneumoniae (KP) is a common conditional pathogen, and also one of the common pathogens causing infection in immunocompromised patients, with its infection rate increasing year by year. Carbapenem antibiotics are effective drugs to control KP infection. But with the widespread use of carbapenem antibiotics, carbapenemresistant Klebsiella pneumoniae (CRKP) appears and increases year by year. Organ transplant recipients are at high risk of CRKP infection due to the suppressed immune system. Once drug-resistant bacteria infection occurs, it is often difficult to control and the survival rate of transplant organs is reduced, which brings great challenges to clinical treatment. In this article, the current status and treatment progress of CRKP infection in organ transplantation are summarized.

Objective To investigate and analyze the characteristics of blood physiological and biochemical parameters of Tibetan chickens bred in Guangzhou. Methods Blood samples of Tibetan chickens bred in Guangzhou were collected,and the physiological and biochemical parameters were measured. Results (1)The blood RBC,PLT,PDW, RDW-SD and P-LCR were not significantly different in the males than females(P > 0.05).(2)HCT(P < 0.05), MCHC(P< 0.05),MPV(P< 0.05),HGB(P< 0.01),MCV(P< 0.01)and MCH(P< 0.01)were significantly higher between the males and females.(3)RDW-CV was significantly lower in the blood physiological parameters of males than females.(4)AST,TRIG,ALKP,ALT,Ca,CHOL,CREA,GLU,PHOS and TBIL were not remarkably different in the blood of males than females(P > 0.05).(5)The blood AMYL(P < 0.05)and TP(P < 0.01)were significantly higher in the males than females.(6)The blood ALB(P< 0.01),UREA(P< 0.05), and GLOB(P<0.01)were significantly lower in the males than females. Conclusions The essential data of blood physiological and biochemical indexes of Tibetan chickens bred in Guangzhou are obtained.

Objective To provide original reference data for oral ecosystem research, Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats were selected to study their respective characteristics of oral microbial mmunities and compared with normal data of humans.Methods Total DNA was extracted from the specimens of oral microbial communities of Tibet minipigs, beagle dogs, rhesus monkey, New Zealand white rabbits and Wistar rats, and used to amplify 16S rRNA V4 fragments with labeled universal primers.The diversity and structure of microbial communities from those animals were compared with that of humans using BIPES and QIIME analysis after Illumina sequencing of 16S rRNA V4 fragments.Results The richness of the oral microbial communities of humans and the five species of laboratory animals was significantly different (P <0.05).Different species of animals have their own unique oral flora, among which the oral flora of the monkey is the most similar to that of humans.Conclusions Among the five species of laboratory animals, the oral microbial communities of rhesus monkeys and humans have highest similarity. Specifically, the Fusobacterium and Porphyromonas levels of rhesus monkeys is most similar to those of humans.Our findings indicate that rhesus monkeys may be suitable animal model for studies of human oral microbial communities.Tibet minipigs may be suitable animal model for Proteobacteria studies, while beagle dogs may be appropriate for modeling of diseases related to Spirochaetes.

Objective To observe the therapeutic effect of acupuncture therapy for the rat model of type 2 diabetes mellitus (T2DM) combined with renal hypertension and try to explore its mechanism. Methods We randomly select 10 Wistar rats as the blank group and 40 rats were used to make the model groups, which were divided into simple diabetes group, simple renal hypertension group, the compound group with electroacupuncture and the compound group without elec?troacupuncture, with 10 rats in each group. After a high fat and sugar diet for 4 weeks, the Wistar rats were given strepto?zotocin i. p. injection to establish models of type 2 diabetes mellitus. Renal hypertension was developed by the“2K1C” im?proved method to make unilateral renal artery ligation?induced renal artery stenosis. Then, electroacupuncture treatment was performed on the rats for 2 weeks except the compound group without electroacupuncture. The changes of values of BP, FBG, Cr, BUN, glycated hemoglobin, renin and Ang II were recorded and analyzed. Results The values of BP, FBG, Cr, BUN, glycated hemoglobin, renin and Ang II in the compound group with electroacupuncture showed a significant re?duction compared with the compound group without electroacupuncture after 2 weeks (P<0?01), but there was no obvious changes in the values of Cr and BUN(P>0?05). Conclusions The blood glucose and blood pressure in the rat model of compound group can be reduced to a normal level with continuous electroacupuncture at bilateral acupoints Zusanli, and it can also be kept at a stable level after single electroacupuncture for 2-3 days. The acupuncture therapy is more suitable for early clinical treatment and can be used in basic research with advantages of economic, safe, no side effect and so on.

Objective To detect and analyze the HBV?related indexes in the urine of HBV transgenic mice and further understand the biological characteristics of transgenic mice, and to clarify the tissue sources of HBV?related indexes. Methods HBV?related indexes in the urine of transgenic mice were tested using enzyme?linked immune sorbent assay ( ELISA ) and fluorescence quantitative PCR ( real?time RCR ) . The tissue sources were confirmed by several experiments, i. e. hydrodynamic transfection of mice, RNA interference to inhibit HBV?expression in the transgenic mice, and to infect normal mice with HBV?positive serum from patients. Results Expression of HBsAg, HBeAg and HBV?DNA was present in the urine of transgenic mice, of which the HBsAg expression level was high (6674 ± 619?8 IU/mL), but lower than that in the serum (16470 ± 2704 IU/mL). The level of HBsAg expression in the urine of male mice was higher than that in female mice. The level of HBeAg expression in the urine was lower and the HBeAg positive rate of urine was higher than that of blood, and the levels of HBeAg expression showed significant inter?individual and inter?sexual differences. HBV?DNA level reached 103 -105 copy/mL in the urine, but no related antibody expression was detected. The experiments such as hydrodynamic infection test indicated that the HBV?related indexes in the urine are derived from replication in the kidneys rather than secreted from the liver, entered into the blood circulation, and discharged from the urine. The kidneys are an independent expression site of HBV. Conclusions The expression of HBV?related indexes is present in the urine of transgenic mice and it is a long?term expression along with the age in months, of which the expression levels of HBsAg and HBV?DNA are rather high and stable. HBsAg titer in the urine of the male mice is higher than that of female mice. HBeAg expression level in the male mice is more stable compared with that in female mice. No expressions of various kinds of antibodies have been found in the urine. The kidneys are an independent expression site of HBV.

Objective To transfect EGFP gene to porcine embryonic fibroblasts ( PEFs) of Tibetan miniature pigs by Lonza Nucleofector II machine and compare the tansfection efficiency between this method and the lipofection method. Method A plasmid carrying green fluorescent protein ( GFP) was transfected into PEFs of Tibetan miniature pigs via the Lonza Nucleofector II machine ( program U020) and by Lipofectamine 2000.Results 5 hours after nucleofection, green fluorescence was observed, indicating 80%transfecting efficiency in the nucleofection group, which is significantly higher than the lipofection group. Conclusion Nucleofector II machine can efficiently transfect PEFs, provides a reliable method for efficiently generate transgenic Tibetan minipigs.

Objective To evaluate the efficacy, safety and compatibility of a new type of atrial septal defect ( ASD) occluder in atrial spetal defect mini-pigs model.Methods Five Tibet mini-pigs were selected as the ASD models which were established by the combination of atrial septal puncture and balloon dilation.Then the new type occluder was implanted for the therapy of ASD.Transthoracic echocardiography with color Doppler was used in all animals during closure and in follow up examinations.The animals were killed at 3 months after occlusion for electron microscopical observation and microscopic examination.Result The ASD models had been created in five piglets successfully without complication, all of whom were implanted successfully with the new device without shunts.Postmortem and microscopic examination of the 5 specimens 3 months after placement showed complete.Conclusion Transcatheter ASD occlusion with new type ASD occluder is safe,feasible and effective.This occlusion can repair the atrial septal defect successfully.