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Based on Chaihu Biejia Decoction （柴胡鳖甲汤， CBD） created by Professor LIU Duzhou， a newly modified CBD has been formulated. The differentiation and treatment of liver cirrhosis can be divided into three stages， that is， the early stage when liver cirrhosis is about to be， the middle stage when liver cirrhosis is formulated after long accumulations， and the late stage when liver cirrhosis has been transformed. Following the pathogenesis， it is recommended to differentiate the abnormal exuberance of zang-fu （脏腑） organs， qi or blood， deficiency or excess， cold or heat in three stages， and newly modified CBD is taken as the basic formula for further modifications. In early stage of liver cirrhosis， the treatment is mainly to invigorate blood and dissolve stasis， clear dampness and heat， and modifications should be made in accordance with the different causes flexibly. The treatment for the middle stage is to soften hardness and dissipate masses， dissolve stasis and clear heat， while fortifying the spleen and supplementing kidneys is accompanied. In the later stage when the healthy qi declines， and the disease is severe and evil prevails， the treatment is to reinforce healthy qi and supplement deficiency， take mild purgation and dispersion， and medicinals to promote urination， stanch bleeding， direct the turbid downward， or open the orifices can be added in accordance with the syndromes so as to treat the branch.
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Objective:To discuss the differential effects of apolipoprotein E(APOE)gene polymorphism in the neurotoxicity-reactive astrocytes,and to provide the theoretical basis for the study of the pathogenesis of Alzheimer's disease(AD).Methods:The primary cortical astrocytes from the APOE-knockout mice(APOE-/-)were isolated and cultured in vitro,and the purity of the cells was identified by immunofluorescence staining.The human APOE3 and APOE4 recombinant over-expression plasmids were constructed and separately transfected into the primary APOE-/-astrocytes,and the APOE-/-primary cells were regarded as control.Western blotting method was used to detect the expression levels of APOE and glial fibrillary acidic protein(GFAP)proteins in the cells;enzyme-linked immunosorbent assay(ELISA)method was used to detect the APOE level in the cellular culture supernatant.The inflammatory models were prepared with the primary astrocytes transfected with APOE3 and APOE4 and co-stimulated with interleukin-1α(IL-1α),tumor necrosis factor(TNF),and complement C1q.The cells were divided into APOE3+PBS group,APOE4+PBS group,APOE3+IL-1α+TNF+ C1q group,and APOE4+IL-1α+TNF+C1q group.Cell immunofluorescence staining method was used to observe the morphology of the cells in various groups;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of glypican 4(Gpc4),glypican 6(Gpc6),thrombospondin 1(Thbs1),thrombospondin 2(Thbs2),SPARC-like protein 1(Sparcl1)and glial cell line derived neurotrophic factor(GDNF),C3,and S100 calcium binding protein B(S100B)mRNA in the cells in various groups;microsphere phagocytosis assay was used to detect the phagocytic capacities of the cells in various groups;Western blotting was used to detect the protein expression levels of B-cell lymphoma 2(Bcl-2),and cysteinyl aspartate specific protease-3(Caspase-3)proteins in the cells in various groups.Results:Compared with APOE-/-group,the expression levels of APOE and GFAP proteins in the cells and the APOE level in the cellular culture supernatant in transfected APOE3 and transfected APOE4 groups were increased(P<0.01).The fluorescence microscope observation results showed that compared with APOE3+PBS and APOE4+PBS groups,the astrocytic processes in APOE3+IL-1α +TNF+Cq1 group and APOE4+IL-1α+TNF+Cq1 group became shorter and the cell bodies became larger;compared with APOE3+IL-1α +TNF+Cq1 group,the astrocytic processes in APOE4+IL-1α +TNF+Cq1 group were even shorter.Compared with APOE3+PBS and APOE4+PBS groups,the expression levels of Gpc4,Gpc6,Thbs1,Thbs2,and Sparcl1 mRNA in the cells in APOE3+IL-1α +TNF+Cq1 group and APOE4+IL-1α +TNF+Cq1 group were significantly decreased(P<0.01);compared with APOE3+IL-1α +TNF+Cq1 group,the expression levels of Gpc4,Gpc6,Thbs1,Thbs2,and Sparcl1 mRNA in the cells in APOE4+IL-1α +TNF+Cq1 group were significantly decreased(P<0.05 or P<0.01).Compared with APOE3+PBS and APOE4+PBS groups,the expression levels of GDNF mRNA in the cells in APOE3+IL-1α+TNF+Cq1 group and APOE4+ IL-1α +TNF+Cq1 group were decreased(P<0.01),and the expression levels of C3 and S100B mRNA were increased(P<0.01);compared with APOE3+IL-1α +TNF+Cq1 group,the expression level of GDNF mRNA in the cells in APOE4+IL-1α+TNF+Cq1 group was decreased(P<0.05),and the expression levels of C3 and S100B mRNA were increased(P<0.05).Compared with APOE3+ PBS group and APOE4+PBS group,the numbers of hagocytosis of microspheres in the cells in APOE3+ IL-1α +TNF+Cq1 group and APOE4+IL-1α +TNF+Cq1 group were significantly decreased;compared with APOE3+IL-1α+TNF+Cq1 group,the number of hagocytosis of microspheres in the cells in APOE4+IL-1α+TNF+Cq1 group was significantly decreased.Compared with APOE3+PBS group and APOE4+PBS group,the expression levels of Bcl-2 protein in the cells in APOE3+IL-1α+TNF+ Cq1 group and APOE4+IL-1α +TNF+Cq1 group were decreased(P<0.05 or P<0.01)and the expression levels of Caspase-3 protein were significantly increased(P<0.01);compared with APOE3+ IL-1α+TNF+Cq1 group,the expression level of Bcl-2 protein in the cells in APOE4+IL-1α+TNF+ Cq1 group was decreased(P<0.01),and the expression level of Caspase-3 protein was increased(P<0.05).Conclusion:The APOE4 genotype has a stronger ability to induce the inflammatory factors compared with APOE3;it can lead to a neurotoxicity-reactive astrocyte phenotype,increase the neurotoxicity,affect the astrocyte apoptosis,and aggravate the neuron damage.
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Hepatic osteodystrophy （HO） should be treated by stages based on the differentiation of root deficiency and branch excess. The root cause of HO is the insufficiency of the liver， spleen， and kidney， and the branch onset should be differentiated by stages that in the incubation， the pathogenesis is shaoyang （少阳） constraint and block and cardinal disturbance， while in the attack period， it is turbid toxin， static heat， scorching marrow and withering bones. In treatment， attention should be paid to regulating and tonifying the depletion of the liver， spleen， and kidney to cultivate the foundation. In the incubation period， it is suggested to put focus on unblocking cardinal disturbance of shaoyang liver and gallbladder so as to regulate and harmonize qi and blood. In the attack period， the focus should be on dissolving stasis， removing turbidity， and clearing the source to promote gallbladder function and bone strength. In clinical practice， medication should be modified according to individual symptoms， and the root and the branch， the primary and the secondary should be differentiated to closely follow the pathogenesis and be tailored according to the symptoms. At the same time， reasonable and safe medication should be emphasized to protect liver function.
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Aim To explore new ways for developing anticancer drugs by the separation of pigment from Fu-sarium species JN158 ( Fusarium sp JN158 ) , the iden-tification of its structure, the screening of anticancer components and the study of its partial mechanism. Methods Pigment separation was done by HPLC, structural analysis by UV, IR, NMR, the screening of anticancer activity by MTT. Western blot was used to analyze the protein expression of CyclinD1, NF-κB, VEGF in tumor cells. Results The results showed that the pigment from Fusarium produced a total of six different peaks, of which peak Ⅵ was the anthocya-nins. Its molecular weight is about 382, molecular for-mula is C17 H18 O10 . According to investigation, this pig-ment was probably a new compound, which could in-hibit the proliferation of MCF-7 cells markedly ( IC50:0.011mmol·L-1 ,P<0.05;the control medicine ube-nimex IC50:10 mmol · L-1 ) in a concentration-de-pendent manner, and had no effect on human umbilical cord intravenous endotheliocyte ( HUVEC ) . The influ-ence on the gene expression of CyclinD1, NF-κB, VEGF in MCF-7 cells varied with the concentration of this compound. The Western blot results showed that VI pigment compound inhibited CyclinD1, NF-κB, VEGF gene expression (P<0.05 or 0. 01),compared with the control group. Conclusion The Ⅵ pigment compound from Fusarium sp JN158 could inhibit MCF-7 proliferation by inhibiting CyclinD1, NF-κB, VEGF gene expression. The compound may be a promising compound against breast cancer.
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Objective To observe the process of Vibrio vulnificus inducing dendritic cell strain apoptosis.Methods We established the mixed culture model of mouse dendritic cell ( DC 2.4 strain) and Vibrio vulnificus( Vv1.1758 strain ),analyzed morphological characteristics of cell apoptosis by DAPI fluorescence staining,detect DNA fragmentation level of apoptosis cells by DNA Ladder assay,analyze DC2.4 apoptosis rate by Annexin V FITC/PI staining,determine activities of caspase-3 and caspase-8 by means of spectrophotometric method and detect changes of mitochondrial transmembrane potential ( △ Ψm ) by JC-1 fluorescence labeling.Results After Vv1.1758 strain and DC2.4 cell were mixed and cultured for 4 h,DAPI fluorescence staining showed typical apoptosis characteristics-chromatin condensation and marginalization; DNA agarose gel electrophoresis showed apoptosis band; apoptosis rates at 2,4 and 6 h were respectively (37.8±9.8) ％,(54.3 ± 12.7 ) ％ and ( 68.2± 14.6 ) ％ ; Mitochondrial transmembrane potentials (△Ψm) at 1 h,2 h and 4 h reduced by 7.1％,16.1％ and 46.7％ respectively; caspase-8 activity increased at 1.5 h and reached the peak at 2 h [ (2.48±0.19) U/μg],while caspase-3 activity started to increase at 3h and reached the peak at 4 h [ ( 1.91 ±0.16) U/μg ].Conclusion Vibrio vulnificus could induce dendritic cells by two pathways: reducing mitochondrial transmembrane potential and activating caspase-8 promoter and finally activate effector caspase-3 to promote apoptosis.
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Objective To investigate the invasion,internalization and the organelles damage of the cultured dendritic cells ( DC2.4 strain) during Vibrio vulnificus (Vv) infection.Methods The study model was the cultured DCs infected by Vv 1.1758 strain.Electron microscopy was used to observe the localization of bacteria in different time point of infection,cell morphology and the process of organelles changes.The cytoskeleton structure including the microfilaments and the microtubules rearrangement was examined by the fluorescence microscope.Results The Vv were pinocytosed into the DC cells through double-sides,and localized at 1-2 μm of the inner side membrane.It cost 1.27,1.87,3.43 hours reaching the infection ratio of 25％,50％,75％,respectively.Using electron microscopy,the DCs had been observed the phagosome formation within 1h,chromatin activation within 2 h,chromatin aggregation 4 h,and the significant cytoskeleton structure disruption within 6 h.Endoplasmic reticulum,mitochondria and lysosomes became swollen.In DCs,the protruding filaments gradually reduced,and their shape changed from the point-like to the linearlike aggregation at the inner side of the plasma membrane,extended microtubules disappeared,the microtubules at the outside nuclear membrane striking rearranged.Conclusion After DC was infected by Vv,the bacteria were pinocytosed into the inner side of DC membrane,and the microfilaments were observed to move from the cytoplasm to cell membrane.In addition,the microtubules moved from the synapse and the cell membrane to the nuclear membrane.The high lethality of Vv could provoke to the DCs cytoskeleton rearrangements.