Objective To investigate the therapeutic effect of Huangqi Xuming Decoction for Alzheimer's disease(AD)patients with spleen and kidney deficiency syndrome and to explore its possible mechanism.Methods A total of 134 patients with AD of spleen and kidney deficiency type were randomly divided into observation group and control group,with 67 cases in each group.The control group was treated with routine western medicine,and the observation group was treated with Huangqi Xuming Decoction on the basis of treatment for the control group.The course of treatment for the two groups covered 12 weeks.The changes of traditional Chinese medicine(TCM)syndrome score,Mini-Mental State Examination(MMSE)score,Alzheimer's Disease Assessment Scale-Cognitive Subscale(ADAS-Cog)score and serum levels of 5-hydroxytryptamine(5-HT),norepinephrine(NE),brain-derived neurotrophic factor(BDNF),superoxide dismutase(SOD),interleukin-6(IL-6)and hypersensitive C-reactive protein(Hs-CRP)in the two groups before and after treatment were observed.After treatment,the clinical efficacy of the two groups was evaluated.Results(1)After 12 weeks of treatment,the total effective rate of the observation group was 89.55％(60/67)and that of the control group was 71.64％(48/67),and the intergroup comparison(tested by chi-square test)showed that the efficacy of the observation group was significantly superior to that of the control group(P＜0.05).(2)After treatment,the MMSE scores of the patients in the two groups were higher(P＜0.05)and the TCM syndrome scores and ADAS-Cog scores were lower than those before treatment(P＜0.05).The intergroup comparison showed that the increase of the MMSE scores and the decrease of the TCM syndrome scores and ADAS-Cog scores in the observation group were significantly superior to those in the control group(P＜0.05).(3)After treatment,the serum 5-HT,NE,BDNF,and SOD levels of the two groups were higher(P＜0.05)and serum IL-6 and Hs-CRP levels were lower than those before treatment(P＜0.05).The intergroup comparison showed that the increase of serum 5-HT,NE,BDNF,and SOD levels and the decrease of serum IL-6 and Hs-CRP levels in the observation group were significantly superior to those in the control group(P＜0.05).Conclusion For patients with AD of spleen and kidney deficiency type,the application of Huangqi Xuming Decoction based on the conventional western medicine treatment is helpful to relieving the inflammatory response,regulating the imbalance of oxidative stress,promoting the improvement of the patients'abilities of daily living,and enhancing the clinical efficacy.

Abstract：Objective To predict the structure and function of sterol O⁃acyltransferase 1（SOAT1）related to hepatocellular carcinoma（HCC）by using bioinformatics tools，in order to understand its mechanism as the marker and therapeutic target of S⁃Ⅲ subtype. Methods The structure，function and protein interaction of SOAT1 were predicted and analyzed by using databases or softwares such as NCBI，STRING，Protscale，SignalP，TMHMM，PSORT，SOPMA，SWISS ⁃ MODEL， NetNGlyc，NetOGlyc，Netphos and ProtParam. Results The protein encoded by SOAT1 was a hydrophobic protein with good stability，which was a nonclassical pathway protein with 8 transmembrane regions，mainly distributed among the cell membrane. SOAT1 was expressed in many tissues，while most of them in the adrenal gland，which showed multiple phosphorylation sites and was mainly involved in the synthesis and catabolism of cholesterol. Conclusion Bioinformatics analysis of structure and function of SOAT1 showed that SOAT1 lipid synthesis and catabolism pathways played an important role，and lipid expression was closely related to the development of cancer，indicating that the treatment of HCC may be achieved by regulating the expression of SOAT1 gene.

Pulmonary fibrosis is a severe lung interstitial disease characterized by the destruction of lung tissue structure, excessive activation and proliferation of fibroblasts, secretion and accumulation of a large amount of extracellular matrix (ECM), and impaired lung function. Due to the complexity of the disease, a suitable animal model to mimic human pulmonary fibrosis has not yet been established. Precision-cut lung slice (PCLS) has been a widely used in vitro method to study lung physiology and pathogenesis in recent years. This method is an in vitro culture technology at the level between organs and cells, because it can preserve the lung tissue structure and various types of airway cells in the lung tissue, simulate the in vivo lung environment, and conduct the observation of various interactions between cells and ECM. Therefore, PCLS can compensate for the limitations of other models such as cell culture. In order to explore the role of discoidin domain receptor 2 (DDR2) in pulmonary fibrosis, Ddr2^flox/flox mice were successfully constructed. The Cre-LoxP system and PCLS technology were used to verify the deletion or knockdown of DDR2 in mouse PCLS. Transforming growth factor β1 (TGF-β1) can induce fibrosis of mouse PCLS in vitro, which can simulate the in vivo environment of pulmonary fibrosis. In the DDR2 knock down-PCLS in vitro model, the expression of various fibrosis-related factors induced by TGF-β1 was significantly reduced, suggesting that knocking down DDR2 can inhibit the formation of pulmonary fibrosis. The results provide a new perspective for the clinical study of DDR2 as a therapeutic target in pulmonary fibrosis.
Animals ; Humans ; Mice ; Discoidin Domain Receptor 2/metabolism* ; Fibroblasts/pathology* ; Fibrosis ; Lung/pathology* ; Pulmonary Fibrosis/metabolism* ; Transforming Growth Factor beta1/metabolism*

To give full play to the therapeutic advantages of traditional Chinese medicine （TCM） in sepsis， clarify the entry point of integrated TCM and western medicine， further standardize the clinical treatment of TCM， develop a recognized and integrated treatment protocol of TCM and western medicine， and improve the clinical efficacy on sepsis，the Chinese Association of Chinese Medicine organized TCM and western medicine experts specialized in sepsis treatment to conduct in-depth discussions on the advantages of TCM and integrated TCM and western medicine in the treatment of sepsis based on the TCM etiology and pathogenesis of sepsis， a representative acute and critical disease. They emphasized the pathogenesis characteristics of asthenia of healthy Qi and sthenia of pathogenic factors and summarized the roles of Chinese medicine in correcting the imbalance of inflammatory response， improving blood coagulation dysfunction， and relieving organ damage. Furthermore， they proposed the treatment protocol with integrated TCM and western medicine， which is expected to provide references for actual clinical treatment and scientific research.

OBJECTIVES: To estimate the system efficiency of uncle-nephew relationship identification by increasing STR markers and adding reference samples based on the test results of simulated data and real samples, so as to provide references for selecting the appropriate number of STRs and reference samples for uncle-nephew relationship identification. METHODS: Five common models of uncle-nephew relationship identification were constructed by adding different reference samples. In each model, the likelihood ratio (LR) for 10 000 pairs of uncle-nephew relationships and 10 000 pairs of unrelated individuals were simulated by detecting 19, 39 or 55 STRs, and the system efficiency at different thresholds was simulated. The samples of the Han population in Zhejiang were collected, and 55 autosomal STRs were obtained by using SiFaSTR^TM 23plex kit, Goldeneye^® DNA ID 22NC kit and AGCU 21+1 PCR amplification kit. When 19, 39 and 55 STRs were detected, the LR of each model and system efficiency under different thresholds were calculated and compared with the simulation results. RESULTS: Under the same detection system, the calculated results of simulated data and corresponding true samples were basically consistent. In the same model, there was a positive correlation between the system efficiency of uncle-nephew relationship identification and the number of STRs detected. Moreover, the system efficiency of introducing relatives was higher than identifying only two individuals. The order of preference for the introduction of relatives was the full sibling (or mother) of the uncle and the full sibling (or mother) of the nephew. CONCLUSIONS The system efficiency of uncle-nephew relationship identification could be improved by increasing the number of STRs and introducing known relatives, which would provide the basis for selecting the most appropriate detection system and reference individuals in actual cases.
DNA ; DNA Fingerprinting ; Humans ; Microsatellite Repeats ; Polymerase Chain Reaction ; Siblings

OBJECTIVE: To study insulin sensitivity and the serum level of adiponectin in infants with intrauterine growth retardation (IUGR) and the effect of breastfeeding on the insulin sensitivity through a follow-up study. METHODS: A total of 106 full-term IUGR infants who were hospitalized from October 2014 to October 2018 were enrolled as the IUGR group, and 90 full-term appropriate for gestational age (AGA) infants who were born during the same period of time were enrolled as the AGA group. Birth weight and body length were recorded. Serum levels of fasting blood glucose (FBG), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), insulin, and adiponectin were measured on day 7 after birth. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. According to the feeding pattern, the IUGR group was further divided into a breastfeeding group with 37 infants and a formula feeding group with 42 infants. The above serum indices and growth indices were also measured at the age of 3 and 6 months. RESULTS: Compared with the AGA group, the IUGR group had significantly increased levels in serum insulin and HOMA-IR and a significantly decreased level of adiponectin (P<0.05). There were no significant differences between the breastfeeding and formula feeding groups in growth indices and serum levels of FBG, TG, LDL, and HDL on day 7 after birth and at the ages of 3 and 6 months (P>0.05). In the breastfeeding group, serum insulin and HOMA-IR decreased and adiponectin level increased over the time of breastfeeding (P<0.05). CONCLUSIONS Insulin sensitivity decreases in the early stage after birth in IUGR infants, and breastfeeding can improve insulin sensitivity.
Adiponectin ; Fetal Growth Retardation ; Follow-Up Studies ; Humans ; Infant ; Infant, Newborn ; Insulin ; Insulin Resistance

Objective: To explore the expression of amphiregulin (AREG) in nasal polyps patients with different degrees of eosinophil infiltration, and to analyze the correlation between AREG and tissue remodeling. Methods: Forty-eight patients underwent endoscopic sinus surgery in the Department of Otorhinolaryngology Head and Neck Surgery, Remin Hospital, Wuhan University from July 2017 to August 2018 were recruited, including 40 males and 8 females, aged from 16 to 60 years old. The subjects were divided into three groups: control group (n=14), eosinophilic chronic sinusitis with nasal polyps (ECRSwNP) group (n=19) and noneosinophilic chronic rhinosinusitis with nasal polyps (non-ECRSwNP) group (n=15). The relative expression of AREG in nasal mucosa was detected by Western blot assay and immunohistochemical staining. Tissue remodeling was detected by HE staining, AB-PAS staining and Masson staining. Kruskal-Wallis test was used for comparison among multiple groups, and Spearman correlation analysis was conducted between the expression level of AREG and the related indexes of tissue remodeling. Results: The expression of AREG in ECRSwNP group was significantly higher than that in non-ECRSwNP group and control group (median protein expression of Western blot was 1.592 vs 0.617 vs0.582, all P<0.05). The degree of epithelial injury and goblet cell metaplasia in ECRSwNP group was significantly higher than that in control group (all P<0.05), the percentage of collagen fibrosis area in ECRSwNP group was significantly lower than that in control group (P=0.01). In chronic rhinosinusitis with nasal polyps (CRSwNP) patients, the area of mucous glands was negatively correlated with the expression of AREG (r=-0.616, P<0.05), and the percentage of collagen fibrosis area was negatively correlated with the expression of AREG (r=-0.738, P<0.05). Conclusion The expression of AREG is higher in ECRSwNP patients, which is related to the process of tissue remodeling.

Objective　CARD9 can activate several pathways involved in immunity, such as NF-ΚB, MAPK, etc. However the mechanism of this process has not yet been elucidated. For conducting relevant experiments in vitro, a prokaryotic expression vector of CARD9-MBP fusion protein has to been construct, and the fusion protein need to be expressed and purified.　Methods　The coding sequence of CARD9 and MBP genes were amplified by PCR and the recombinant plasmid was correctly inserted into the pET-30a(+) vector. The recombinant plasmid was transformed into E.coli DH5α competent cells and proceeded PCR identification, restriction analysis and gene sequencing. The correct recombinant plasmid was transformed into E.coli BL21(DE3) competent cells. The target protein was induced to express by IPTG under different conditions. Relative molecular weight of the target protein was detected by SDS-PAGE electrophoresis. The CARD9-MBP fusion protein was purified by MBP maltose chromatography column and gel filtration chromatography column, and identificated by MALDI-TOF mass spectrometry after MBP-tag to be removed by HRV3C enzyme.　Results　The CARD9-MBP fusion protein was successfully constructed and confirmed by PCR and restriction analysis. The result of gene sequencing was consistent with the target sequence. The SDS-PAGE electrophoresis showed that the target protein with molecular mass (MR) about 105 000 was successfully induced to express in E.coli BL21 (DE3). A quite pure CARD9-MBP fusion protein was obtained by purification of MBP maltose chromatography column. Identification by MALDI-TOF mass spectrometry demonstrated that the target protein after MBP-tag to be removed by HRV3C enzyme is CARD9 protein. In the later stage, gel filtration chromatography column was used to obtain further pure CARD9-MBP fusion protein.　Conclusion　The prokaryotic expression vector of CARD9-MBP fusion protein was successfully constructed and a large number of soluble protein expressed. The purified target protein can be obtained by purification with MBP maltose chromatography column and gel filtration chromatography column.

Objective To construct a human renal epithelial cell line HEK293T by CRISPR-Cas9-based site-directed knock-in of vascular endothelial growth factor 165 (VEGF165) gene, and avoid the off-target effect caused by lentivirus infection. Methods The VEGF165 expression vector with homologous arm (pUCm-T-VEGF165 plasmid) and the sgRNA expression vector [pSpCas9(BB)-2A-Puro-sgRNA plasmid] were designed and constructed based on the DNA sequence of the EZH2 gene, and then co-transfected into HEK293T cells. The expression of VEGF165 mRNA was detected by qPCR and the expressions of VEGF165 proteins were detected by Western Blot. Results The qPCR and Western Blot results showed that, comparing with the control, the pUCm-T-VEGF165 plasmid and pSpCas9(BB)-2A-Puro-sgRNA plasmid, the expression of the co-transfection plasmid were significantly increased, i.e. 3.42±0.30 vs. 1.02±0.21, 1.13±0.16 and 0.98±0.18 for the VEGF165 mRNA level (all P<0.01), and 1.13±0.16 vs. 1.02±0.06, 0.88±0.03 and 0.80±0.05 for the VEGF165 protein level (all P<0.01), respectively. Besides, the expression of EZH2 was significantly down-regulated, i.e. 0.14±0.06 vs. 1.08±0.11, 1.02±0.12 and 1.13±0.16 for the EZH2 mRNA level (all P<0.01), and 0.23±0.03 vs. 1.05±0.13, 0.91±0.04 and 0.81±0.06 for the EZH2 protein level (all P<0.01), respectively. This result showed that the VEGF165 was successfully inserted into the EZH2 genome, interfering the EZH2 expression. Conclusions VEGF165 gene can be successfully knocked into HEK293T cells by CRISPR/Cas9 system.

Macrophages show significant heterogeneity in function and phenotype, which could shift into different populations of cells in response to exposure to various micro-environmental signals. These changes, also termed as macrophage polarization, of which play an important role in the pathogenesis of many diseases. Numerous studies have proved that Hesperidin (HDN), a traditional Chinese medicine, extracted from fruit peels of the genus citrus, play key roles in anti-inflammation, anti-tumor, anti-oxidant and so on. However, the role of HDN in macrophage polarization has never been reported. Additional, because of its poor water solubility and bioavailability. Our laboratory had synthesized many hesperidin derivatives. Among them, hesperidin derivatives-12 (HDND-12) has better water solubility and bioavailability. So, we evaluated the role of HDND-12 in macrophage polarization in the present study. The results showed that the expression of Arginase-1 (Arg-1), interleukin-10 (IL-10), transforming growth factor β (TGF-β) were up-regulated by HDND-12, whereas the expression of inducible Nitric Oxide Synthase (iNOS) was down-regulated in LPS- and IFN-γ-treated (M1) RAW264.7 cells. Moreover, the expression of p-JAK2 and p-STAT3 were significantly decreased after stimulation with HDND-12 in M1-like macrophages. More importantly, when we taken AG490 (inhibitor of JAK2/STAT3 signaling), the protein levels of iNOS were significantly reduced in AG490 stimulation group compare with control in LPS, IFN-γ and HDND-12 stimulation cells. Taken together, these findings indicated that HDND-12 could prevent polarization toward M1-like macrophages, at least in part, through modulating JAK2/STAT3 pathway.
Animals ; Cytokines ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; Gene Expression Regulation ; drug effects ; Hesperidin ; chemistry ; pharmacology ; Inflammation ; genetics ; metabolism ; Janus Kinase 2 ; antagonists & inhibitors ; metabolism ; Macrophages ; drug effects ; immunology ; metabolism ; Medicine, Chinese Traditional ; Mice ; Molecular Structure ; Phosphorylation ; drug effects ; RAW 264.7 Cells ; STAT3 Transcription Factor ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects