ABSTRACT
Objective:To investigate the function, mechanism and therapeutic potential of macrophages in non-alcoholic steatohepatitis (NASH).Methods:Eight-week-old male foz/ foz (Alms mutant) mice were fed with a high fat diet (HFD) for 6, 8 and 10 weeks and 8-week-old male C57BL/6 mice were fed with a methionine and choline-deficient (MCD) diet for 7 d, 3 weeks and 4 weeks to establish NASH models. The mice of control group were fed with normal diet or MCD control diet. The expression of F4/80 mRNA level in the livers of mice of NASH model group and control group was detected by fluorescence quantitative polymerase chain reaction. Macrophages in the livers of mice of NASH group and control group were determined by immunofluorescence staining. After transgenic lysM-Cre/DTR mice were fed with MCD diet for 5 weeks, they were divided into transgenic experimental group (ablation of macrophages induced by diphtheria-toxin (DTox) injection) and transgenic control group (phosphate buffer saline injection). The levels of triglyceride and lipid peroxide in the livers of transgenic experimental group and transgenic control group were detected, and the inflammation of the livers of the mice was scored. The mechanism of macrophages regulating inflammation in NASH was investigated by cytokine profiliny analysis and Western blotting. The interaction between hepatocytes and macrophages were determined by co-culturing the conditional medium of hepatocytes AML-12 and macrophages RAW264.7. Macrophages of mice of control group and NASH model group were depleted by liposomal clodronate to confirm its value in NASH prevention. Independent sample t-test was used for statistical analysis. Results:F4/80 mRNA level in the livers of NASH model foz/ foz mice fed with HFD for 6 weeks, 8 weeks and 10 weeks was higher than that of control group (1.49±0.19, 1.70±0.15 and 1.93±0.04 vs.1.05±0.22), and the differences were statistically significant ( t=3.06, 4.92 and 7.92, all P<0.05). The expression of F4/80 mRNA level of the livers of NASH model mice fed with MCD for 7 d and 3 weeks was higher than that of control group (2.70±0.99 and 3.08±1.71 vs.1.00±0.83), and the differences were statistically significant ( t=3.43 and 3.54, both P<0.01). The results of immunofluorescence demonstrated that compared with that of control group, the number of F4/80 + inducible nitric oxide synthase (iNOS) + M1 macrophages were significantly increased, while F4/80 + CD206 + M2 macrophages were significantly decreased in the livers of NASH model mice fed with MCD for 4 weeks. After macrophages depletion, the inflammation score, the levels of triglyceride and lipid peroxide in the liver of transgenic experimental mice were all lower than those of transgenic control mice (0.69±0.32 vs. 1.95±0.74, (43.97±13.24) g/mg vs. (63.09±14.85) g/mg, (24.84±6.21) nmol/mg vs.(37.91±8.91) nmol/mg), and the differences were statistically significant ( t =3.14, 2.72 and 2.41, all P<0.05). The results of cytokine profiling analysis showed that macrophage depletion could lower the levels of interleukin (IL)-12 and macrophages inflammatory protein-1α (the difference between multiples: -3.98, -2.74, both P<0.05). CCAAT/enhancer binding protein β was defected in the nuclear of transgenetic experimental mice. In vitro study showed that RAW264.7 macrophages conditional medium could promote lipid accumulation in AML-12 hepatocytes, while conditional medium from MCD medium-treated AML-12 hepatocytes could promote RAW264.7 macrophages to M1 polarization. After treated with liposomal clodronate, the levels of triglyceride and lipid peroxidation in the liver of control mice were both lower than those of MCD-induced NASH model mice((45.33±14.59) g/mg vs. (63.10±16.02) g/mg, (2.11±0.48) nmol/mg vs. (2.73±0.17) nmol/mg), and the differences were statistically significant ( t=2.84 and 2.73, both P<0.05). The results of Western blotting indicated that after treating with liposomal clodronate, the relative content of phosphorylated protein kinase R-like endoplasmic reticulum kinase, inositol requiring enzyme-1α, protein disulfide isomerase, glucose regulatory protein 78, phosphorylated eukaryotic initiation factor 2α in the liver of NASH model mice were all lower than those of NASH model mice without liposomal clodronate treatment (1.84±0.36 vs. 3.05±0.83, 1.50±0.84 vs. 6.65±1.47, 0.87±0.12 vs. 2.28±0.52, 1.68±0.43 vs. 4.76±1.13, 1.42±0.19 vs. 2.75±0.79), and the differences were statistically significant( t=2.32, 5.28, 4.56, 4.41 and 2.85, all P<0.05). Conclusions:Macrophages are polarized into M1 phenotype in NASH. M1 macrophages contributed to NASH progression by interacting with hepatocyets to promote the secretion of inflammatory cytokines, up-regulation of lipogenic factors, oxidative stress and endoplasmic reticulum stress, resulting in the progression of NASH. Macrophages depletion by liposomal clodronate is a potential noval approach for NASH prevention.
ABSTRACT
Objective:To explore the Chinese medicine to reduce red blood cell transfusion mechanism, anemia treatment and provide a theoretical basis for patients with severe hypoxic ischemic symptoms.Methods: in our hospital from 2012 May to 2013 May in our hospital were severe anemia in 96 cases, according to the order of admission, were randomly divided into study group(48 cases) and control group(48 cases), study group was treated with Chinese medicine Yiqi Buxue cream treatment, the control group using sugar iron injection treatment, treatment effects were compared between the two groups.Results: the treatment group the total effective rate was 97.9% in treatment group, the total effective rate was 87.5%, and there was significant difference between two groups(P<0.05). Study group Hb after treatment was (106.43±13.08) g/L, the control group after treatment Hb(98.69±14.25)g/L, there was significant difference between two groups(t=13.26,P<0.05). After treatment, study group MCV, MCH, MCHC improvement, compared with the control group with statistical significance; the two groups in the improvement of red blood cell morphology were obvious effect, and the study group was better than control group. Statistical significance of group SF after treatment and the control group at the same time difference(t=11.28,P<0.05). The control group had 10 cases of gastrointestinal discomfort, study group 1 cases of no adverse reaction.Conclusion: TCM symptoms of Chinese medicine Yiqi Buxue cream treatment can significantly improve the syndrome of blood deficiency of iron deficiency anemia, reduce the number and the number of red blood cell transfusion, clinical curative effect, less adverse reaction.
ABSTRACT
Objective To evaluate the replication, encapsulation and expression of the recombinant HBV vector with the truncated C gene. Methods C gene truncated HBV vectors were constructed by technologies of molecular clone and PCR based site-directed mutagenesis in vitro. The expression of GFP was observed with the fluorescence microscope after transfection of the recombinant HBV vector plasmids into HepG2 cells by using the liposome method. The capability of HBV vector replication, encapsulation and progeny virus production were examined with semi nested-PCR, native agarose gel electrophoresis blot, quantitative southern blot analysis and the routine PCR assays. Results The HBV vectors with truncated C gene were constructed successfully. The gene was expressed effectively after transfection. The C gene truncated HBV vectors could be replicated and encapsulated in hepatocytes with the helper virus. The encapsulated efficiency were as 4～8 folders as the non C gene truncated HBV vectors by quantitative analysis. In addition, the mature HBV particles carrying the interesting gene of GFP could secrete out from hepatocytes by the help of adjunctive vector. Conclusions The truncation of C gene could improve the encapsulation efficiency of HBV vectors with no effect on the replication and expression of the intact HBV vectors.