Yinchen Linggui Zhugan Decoction Ameliorates Nonalcoholic Fatty Liver Disease in Rats by Regulating the Nrf2/ARE Signaling Pathway.

Yinchen Linggui Zhugan Decoction (YCLGZGD) is the combination of Linggui Zhugan (LGZGD) and Yinchenhao (YCHD) decoctions, two famous traditional Chinese medicine prescriptions. In previous studies, we found that Yinchen Linggui Zhugan Decoction (YCLGZGD) could regulate lipid metabolism disorder and attenuate inflammation in pathological process of nonalcoholic fatty liver disease (NAFLD). However, the exact underlying mechanism remains unknown. The aim of this study was to explore the effect of Yinchen Linggui Zhugan Decoction on experimental NAFLD and its mechanism in rats with high-fat diet (HFD) which was established by 8-week administration of HFD. YCLGZGD, LGZGD, and YCHD were administered daily for 4 weeks, after which the rats were euthanized. The level of blood lipid, liver enzymes, H&E, and Oil Red O staining were determined to evaluate NAFLD severity. Western blotting and real-time polymerase chain reaction were, respectively, used to determine hepatic protein and gene expression of Keap1, Nrf2, NQO1, and HO-1. Oral YCLGZGD ameliorated HFD-induced NAFLD. Furthermore, YCLGZGD increased the protein and gene expression of Nrf2, NQO1, and HO-1 without changing Keap1. Overall, these results suggest that YCLGZGD ameliorates HFD-induced NAFLD in rats by upregulating the Nrf2/ARE signaling pathway.

An ErChen and YinChen Decoction Ameliorates High-Fat-Induced Nonalcoholic Steatohepatitis in Rats by Regulating JNK1 Signaling Pathway.

ErChen and YinChen decoction (ECYCD) is an effective traditional Chinese medicine and has been widely used in traditional Chinese medicine to treat nonalcoholic steatohepatitis (NASH), with good curative effects. However, the specific mechanisms underlying these effects are unclear. In this study, we determined the efficacy of ECYCD in a high-fat diet-induced NASH rat model, established by 8-week administration of a high-fat diet. ECYCD was administered daily for 4 weeks, after which the rats were euthanized. The results demonstrated that ECYCD ameliorated high-fat diet-induced NASH, as evidenced by decreased liver indexes, reduced hepatic lipid deposition and liver injury, lower serum biochemistry markers (including low-density lipoprotein), and reduced HOMA-IR scores. Moreover, levels of free fatty acids, tumor necrosis factor, and malondialdehyde were decreased, whereas glutathione was increased in the liver. Serum high-density lipoprotein was also increased in the liver, and ECYCD regulated the c-Jun N-terminal kinase 1 (JNK1) signaling pathway by decreasing the levels of JNK1 protein, JNK1 mRNA, activator protein- (AP-) 1 protein, AP-1 mRNA, and phospho-insulin receptor substrate- (IRS-) 1ser307 and increasing phopsho-PKBser473 levels. These results suggested that ECYCD could ameliorate high-fat diet-induced NASH in rats through JNK1 signaling. ECYCD may be a safe therapeutic option for the treatment of NASH.

Qingchang Wenzhong Decoction Ameliorates Dextran Sulphate Sodium-Induced Ulcerative Colitis in Rats by Downregulating the IP10/CXCR3 Axis-Mediated Inflammatory Response.

Qingchang Wenzhong Decoction (QCWZD) is an effective traditional Chinese medicine prescription. Our previous studies have shown that QCWZD has significant efficacy in patients with mild-to-moderate ulcerative colitis (UC) and in colonic mucosa repair in UC rat models. However, the exact underlying mechanism remains unknown. Thus, this study was conducted to determine QCWZD's efficacy and mechanism in dextran sulphate sodium- (DSS-) induced UC rat models, which were established by 7-day administration of 4.5% DSS solution. QCWZD was administered daily for 7 days, after which the rats were euthanized. Disease activity index (DAI), histological score (HS), and myeloperoxidase (MPO) level were determined to evaluate UC severity. Serum interferon gamma-induced protein 10 (IP10) levels were determined using ELISA kits. Western blotting and real-time polymerase chain reaction were, respectively, used to determine colonic protein and gene expression of IP10, chemokine (cys-x-cys motif) receptor (CXCR)3, and nuclear factor- (NF-) κB p65. Intragastric QCWZD administration ameliorated DSS-induced UC, as evidenced by decreased DAI, HS, and MPO levels. Furthermore, QCWZD decreased the protein and gene expression of IP10, CXCR3, and NF-κB p65. Overall, these results suggest that QCWZD ameliorates DSS-induced UC in rats by downregulating the IP10/CXCR3 axis-mediated inflammatory response and may be a novel UC therapy.

Dopaminergic neuron-like cells derived from bone marrow mesenchymal stem cells by Lmx1α and neurturin overexpression for autologous cytotherapy in hemiparkinsonian rhesus monkeys.

Bone marrow-derived mesenchymal stem cells hold great potential for cytotherapeutics of neurodegenerative disorders, including Parkinson's disease. The neurotrophic factor neurturin can rescue dopaminergic neurons damaged during the disease process. Lmx1α can promote mesencephalic dopaminergic differentiation during embryogenesis. In this study, we tested a cytotherapeutic strategy combining NTN/Lmx1α gene therapy and cell transplantation to ameliorate disease progression in hemiparkinsonian rhesus. Rhesus BMSCs were prepared for autologous grafting by transfection with recombinant adenoviral vectors expressing secreted NTN and Lmx1α,and cultured in the presence of induce factors, particularly the Lmx1α regulatory factor sonic hedgehog, to guide dopaminergic differentiation. These induced rh-BMSCs exhibited gene/protein expression phenotypes resembling nigral dopaminergic neurons. They survived and retained dopaminergic function following stereotaxic injection into the MPTP-lesioned right-side substantia nigra as indicated by SPECT measurement of DAT activity. Injected cells preserved and supplemented the remaining endogenous population of dopamine neurons (TH-positive cell ipsilateral/contralateral ratio was 56.81% ± 7.28% vs. 3.86%±1.22% in vehicle-injected controls; p<0.05). Cell injection also partially restored motor function and reduce apomorphine-evoked rotation (p<0.05). Moreover, function recovery occurred earlier than in previous studies on injected BMSCs. Our findings demonstrate a promising strategy for restoration of PD-associated motor dysfunction by transplantation of autologous BMSCs overexpressing NTN/Lmx1α.

[Recombinant adenovirus-mediated expression of tyrosine hydroxylase in 293 cells and activity assay of the expressed protein in vitro by capillary electrophoresis].

OBJECTIVE: To construct a recombinant adenovirus for carry tyrosine hydroxylase (TH) gene and expressing bioactive TH protein in the animal model of Parkinson disease. METHODS: The TH gene was inserted into the shuttle plasmid, which was transformed into E.coli BJ-5183 for homologous recombination with the adenovirus genome. 293 cells were transfected with the recombinant adenovirus genome to obtain the recombinant virus, and the transcription and expression of TH were determined by RT-PCR and immunofluorescence assay, respectively. The production of L-DOPA in the in vitro reaction system was determined using capillary electrophoresis. RESULTS: We have successfully constructed the recombinant adenovirus. The TH mRNA and the corresponding protein were detected by RT-PCR and immunofluoresence assay in 293 cells. L-DOPA was also detected in the reaction system. CONCLUSION: The adenovirus constructed allows efficient expression of bioactive TH protein in vitro, which provides a basis for future study of gene therapy of Parkinson disease in animal models.

[Construction of a recombinant human adenovirus expressing the ORF2 antigen of HEV and immunization of mice by mucosal system].

OBJECTIVE: To construct a replication-defective recombinant adenovirus expressing the ORF2 (112-660aa) antigen of hepatitis E virus (HEV) and evaluate its immunization effect in BALB/c mice by mucosal inoculation. METHODS: The HEV ORF2 gene encoding for 112-660aa was amplified from plasmid pUC-HEV and inserted into the transfer vector pTrack-CMV. The recombinant plasmid and adenoviral backbone plasmid pAdEasy-1 were co-transformed into E. coli strain BJ5183. Taking the advantage of the high efficient homologous recombination machinery presented in bacteria, the recombinant adenovirus backbone plasmid was generated in BJ5183, and then was transfected into 293 cells. Recombinant Adenoviruses were propagated in 293 cells with high titers. 8-week-old BALB/c mice were inoculated intraperitoneally and intranasally with 10(7) pfu recombinant adenovirus each on weeks 0, 3, 5, 7, 10. RESULTS: Both groups of mice induced humoral IgG immune response with the highest titers 1:1,000 and 1:10,000 each. Only the group inoculated intranasally could induce mucosal IgA immune response. CONCLUSIONS: The adenoviral recombinant can stimulate specific humoral and mucosal immune response in mice and is potentially to be used as a candidate vaccine for the treatment of HEV infection.

[Vaccination of rhesus monkeys with recombinant antigen fragments and protection from hepatitis E virus infection].

OBJECTIVE: To observe anti-HEV IgG response to vaccination of recombinant antigen fragments and evaluate its protection from Hepatitis E Virus infection in rhesus monkeys (Macaca mulatta). METHODS: Twelve monkeys were divided into three groups and immunized respectively with three different recombinant antigens: namely Ag1 (carboxyl terminal 431 amino acids of ORF2), Ag2 (128aa fragment at the carboxyl terminal of ORF2), and Ag3 (full length ORF3 ligated with two ORF2 fragments encoded by 6743-7126nt and 6287-6404nt). The monkeys were challenged intravenously with fecal suspension from experimentally infected rhesus monkeys, and the other three monkeys served as the placebo group for challenge with HEV. The dynamic changes of the levels of ALT and anti-HEV IgG were examined. Pathological changes of liver tissue were observed by light microscope. Excretion of virus was detected by RT-nPCR. RESULTS: Hepatic histopathology of two monkeys in the placebo group was consistent with acute viral hepatitis, and ALT was elevated 3-4 weeks after inoculated with virus, up to 10-20 times higher than normal level. The liver tissue of monkeys immunized with antigen kept normal, ALT in several monkeys elevated mildly, and anti-HEV IgG conversation occurred at 1-2 weeks after vaccination, with the titer reaching 1:12,800. The virus RNA could be detected by RT-nPCR from days 7 to 50 in monkeys of control group, and from days 7 to 21 in vaccinated monkeys after challenged with virus. CONCLUSIONS: The recombinant antigens could induce the production of anti-HEV IgG, which protected rhesus monkeys from acute Hepatitis symptoms related to HEV infection.