Effects of extracts of Dragon's blood on fibroblast proliferation and extracellular matrix hyaluronic acid / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of Dragon' s blood extract on proliferation and secret extracellular matrix function of fibroblasts in vitro.</p><p><b>METHODS</b>Dragon' s blood was extracted by chloroform, acetoacetic ester, alcohol. Human fibroblast were cultured in vitro in media containing gradient dilutions of Dragon' s blood extracts (0.002, 0.02, 0.2, 2, 20 mg/ml) , which was followed by cell proliferation assessed with MTT assay on 0, 12, 24, 36, 48, 60, 72 h. Under the optimal concentration, the cell growth curves were drawn and the flow cytometry (FCM) was used to determine the changes of cell cycle. On 0, 12, 24, 36, 48, 60, 72 h, the concentration of hyaluronic acid in the supernatant of fibroblast culture was measured by radioimmunoassay.</p><p><b>RESULTS</b>0.2-2 mg/ml Dragon' s blood extracts enhanced the proliferation of fibroblasts in a dose-dependent manner. 2 mg/ml was the optimal dilution of Dragon's blood extract, and it increased the ratio of S cells in cell cycle [(25.80 ± 3.10)%] than control group [(7.50 ± 0.70)%, P < 0.01]. From 12 h to 72 h, in 2 mg/ml Dragon's blood group, concentration of Hyaluronic acid secreted by fibroblasts gradually increased, but were less than control (P < 0.01).</p><p><b>CONCLUSIONS</b>Dragon's blood acetoacetic ester extract improved the proliferation of cultured human fibroblasts in vitro, might be beneficial to promote wound healing.</p>

Effects of Dragon’s blood extracts on fibroblast proliferation and procollagen type III / 中国组织工程研究

BACKGROUND:Dragon’s blood is the main ingredient of traditional medicine prescription for promoting granulation, which has been used in clinical treatment of a variety of refractory wounds and achieved the exact effects. But the Dragon’s blood effect on colagen secretion from normal fibroblasts has not been reported. OBJECTIVE: To investigate the effects of Dragon’s blood extract on the proliferation and secret function of fibroblastsin vitro. METHODS: Dragon’s blood was extracted by extracts chloroform, acetoacetic ester, and alcohol in turn. Normal human fibroblasts were respectively cultured in Dragon’s blood extracts of chloroform, acetoacetic ester, and alcohol, DMEM containing 1% dimethyl sulfoxide, and normal culture medium. Then, the fibroblasts were cultured in vitro in different media containing gradient dilutions of Dragon’s blood extracts (0.002, 0.02, 0.2, 2, 20 g/L), which was folowed by cel proliferation determination assessed with MTT assay. Under the optimal concentration, the cel growth curves were drawn and the flow cytometry was used to determine the changes of cel cycle. The concentration of procolagen type III in the supernatant of the fibroblast culture systems was measured by radioimmunoassay. RESULTS AND CONCLUSION:0.2 g/L-2 g/L dilution of Dragon’s blood extracted by acetoacetic ester enhanced the proliferation of fibroblasts in a dose-dependent manner. The 2 g/L was the optimal dilution of Dragon’s blood extracted by acetoacetic ester, and it increased the ratio of S cels in cel cycle than control group and decreased procolagen type III. These findings indicate that Dragon’s blood acetoacetic ester extract can improve the proliferation of cultured human fibroblastsin vitro, and decrease the secretion of procolagen type III of fibroblasts, and it can be beneficial to improve wound healing and inhibit hypertrophic scar.

Influence of hepatic oval cell on the expression of ERK and P38MAPK signaling pathway protein in liver tissue of murine hepatofibrosis / 中华普通外科杂志

Objective To observe the influence of hepatic oval cell (HOC) on the expression ERK and P38MAPK signaling pathway protein in liver tissue of murine experimental hepatofibrosis (HF).Method SD rats were fed with 10% ethanol and food with high-fat and low-protein, and were injected subcutaneously with carbontetrachloride once every four days for 8 weeks to establish hepatic fibrosis. HOGs were isolated from male HF rats by collagenase porfusion of the liver. HF rats at 8th week were transplanted with 0. 5 ml HOC suspension medium at a density of 1 × 109 cell /ml via portal vein, and the rats were sacrificed at 8th, 15th, 30th day respectively. Histopathologic changes of liver tissues were observed by HE and Masson. The expression of ERK and P38MAPK signaling pathway protein were determined by Western blotting. Result Hepatofibrosis was reversed and the degree of hyperplasia fibrilcollagen in hepatic fibrosis rats decreased significantly by HOC transplantion. HOC down-regulated the protein expression of Ras, ERK,p-ERK, c-fos, c-jun, STAT3, ALB, FGF-3, PCNA ( F = 91.88,36.28,54.66,93.07,64.76,58.49,52.63,20.45 ,27.03, all P ＜ 0.05 ), up-regulated the protein expression level of HNF-α1, PDGF-Rβ significantly in liver tissues(F = 18.63,25.99,P ＜0.05). Conclusions HOC improves the degree of hepatofibrosis through inhibiting hyperplasia of collagen fibril in liver tissue of hepatofibrosis rats. With the presence of HOC the expression of c-fos,c-jun,STAT3,5 was not activated by p-P38MAPK. The expression of c-kit and HNF-1α increased and that liver tissue injury alleviated, and hepatofibrosis was improved.

A genome sequence of novel SARS-CoV isolates: the genotype, GD-Ins29, leads to a hypothesis of viral transmission in South China / 基因组蛋白质组与生物信息学报·英文版

We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.

The M protein of SARS-CoV: basic structural and immunological properties / 基因组蛋白质组与生物信息学报·英文版

We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To investigate its immunogenicity, we synthesized eight oligopeptides covering 69.2% of the entire ORF and screened them by using ELISA (enzyme-linked immunosorbent assay) with sera from SARS patients. The results confirmed our predictions on antigenic sites.

The E protein is a multifunctional membrane protein of SARS-CoV / 基因组蛋白质组与生物信息学报·英文版

The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and alpha-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin.

The structure analysis and antigenicity study of the N protein of SARS-CoV / 基因组蛋白质组与生物信息学报·英文版

The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.

Complete genome sequences of the SARS-CoV: the BJ Group (Isolates BJ01-BJ04) / 基因组蛋白质组与生物信息学报·英文版

Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.

The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity / 基因组蛋白质组与生物信息学报·英文版

In order to develop clinical diagnostic tools for rapid detection of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development.

Genome organization of the SARS-CoV / 基因组蛋白质组与生物信息学报·英文版

Annotation of the genome sequence of the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) is indispensable to understand its evolution and pathogenesis. We have performed a full annotation of the SARS-CoV genome sequences by using annotation programs publicly available or developed by ourselves. Totally, 21 open reading frames (ORFs) of genes or putative uncharacterized proteins (PUPs) were predicted. Seven PUPs had not been reported previously, and two of them were predicted to contain transmembrane regions. Eight ORFs partially overlapped with or embedded into those of known genes, revealing that the SARS-CoV genome is a small and compact one with overlapped coding regions. The most striking discovery is that an ORF locates on the minus strand. We have also annotated non-coding regions and identified the transcription regulating sequences (TRS) in the intergenic regions. The analysis of TRS supports the minus strand extending transcription mechanism of coronavirus. The SNP analysis of different isolates reveals that mutations of the sequences do not affect the prediction results of ORFs.

Effects of Tanshinone ⅡA on LSP-induced hepatocyte injury and cytokines-secreting of by kupffer cells / 中国药理学通报

Aim To investigate the influence of Tanshinone ⅡA on cytokine secreted by kuppfer cells via LPS stimulation,and explore its mechanism of therapying chronic hepatic disease. Methods The liver cell and kupffer cells were isolated and the model of liver cell injuries was made induced by LPS and D-GlaN, and the effects of TanshinoneⅡA was observed on the injuries of liver cell induced by lipopoly-saccharide(LPS) and D-Galactosamine(D-GlaN). Cytokine were released secreted by kupffer cells through LPS stimulation and the contents of TNF-?,IL-6,IL-8 were determined by radio-immunoassay. The hepatocyte morphological character was observed by HE stain,the expressions of TNF-?、CD14、iNOS、eNOS in kupffer cell were explored by immunohistochemical, double-decker chamber was used for observing the damage of hepatocyte caused by cytokine secreted by kupffer via LPS stimulation. Results Tanshinone ⅡA could restore the injury of liver cell induced by D-GlaN and LPS, the levels of ALT、MAO、LDH-L、GSH-S and contents of MDA were significantly reduce.It could inhibit TNF-? IL-8 secreted by kupffer cells through LPS stimulation ,and the expressions of TNF-?、CD14 Inos、eNOS in kupffer cell through LPS stimulation were inhibited. Blocking injuries of the inflammatory cytokine release excessive on liver cell, it hadn′t the protect action on liver cell damage suffer from inflammatory cytokine.Conclusion The mechanism of Tanshinon ⅡA of blocking liver cell injuries induced by D-GlaN and LPS may be correlation with inhibiting the excessive cytokine by kupffer cells

Effect of Wuling Capsules on IL - 2 and IL - 6 in Healthy Mice and Mice With Immunological Liver Damage / 中国药房

OBJECTIVE: To investigate the effect of Wuling capsules on the activities of IL-2 and IL -6 in healthy mice and mice with immunological liver damage .METHODSBALB/C mice were treated with LPS(experimental group)or were not treated (control group) .12 days after giving Wuling capsule ig, the suspension of spleen cells of mice was cultured for 48h and 72h and the supernant fluid was collected for test.The IL - 2 was detected with MTT and IL - 6 with radio - immunoas-say. RESULTS: Wuling capsules could promote Th cells to secret IL- 2 and markedly elevate the liver - damage - induced low level of IL- 2.Wuling capsules did not effect the levels of IL-6 significantly in both healthy mice and mice with liver damage. CONCLUSION: Wuling capsules can promote the secretion of IL-2 and have immunoregulatory actions.

Experimental Study on Bupleuri in Combination With Saliva Miltiorrhiza in Treatment of Hepatic Fibrosis in Rats / 中国药房

OBJECTIVE:To investigate the effects of Bupleuri plus Saliva miltiorrhiza on preventing the formation of hepatic fibrosis in rats METHODS:To establish an animal model of hepatic fibrosis by feeding high fat diet and 10% alcohol and subcutaneously injecting CCl4 and to detect the zymogram and biochemical parameters and observe the pathological changes of liver tissue RESULTS:Bupleuri and Saliva miltiorrhiza could significantly depress the activities of ALT,AST,MAO,ALP and had no influence on the ?-GT in serum,the serum TG was decreased and TG in tissue was obviously raised and serum GSH was elevated in hepatic fibrosis rat The extract could significantly decreased the levels of PC Ⅰ and PC Ⅲ in hepatic fibrosis rat The histopathological examination showed that the extract could diminish liver degeneration and necrosis,and block the formation of fiber diazoma,the degree of collagen proliferation was significantly lower than that of the control group CONCLUSION:Bupleuri and Saliva miltiorrhea extract could effectively protect the liver cell,and could retard the formation of hepatic fibrosis as well

Effects of Ganoderma lucidum and it combined with Radix Salvia e Miltiorrhizae, Radix Bupleuri, and Fructus Schisandrae Chinensis respectivel y on experimental hepatic injuries in mouse / 第三军医大学学报

Objective　To investigate the protective effects of alcoholic extracts of Ganoderma lucidum (GL) and its combination with Rad ix Salviae Miltiorrhizae (RSM), Radix Bupleuri (RB), and Fructus Schisandrae Chi nensis (FSC) respectively on experimental hepatic injuries. Methods　Hepatic injury models were established with the injections of carbon tetrac hloride (CCl4) or D-galactosamine(D-GlaN) into mouse, and then the therapeut ic agents at same dose were given respectively. The activity of cholinesterase(C hE) and alaline aminotransferase(ALT) in the serum and hepatic homogenate, the c ontents of malonyl dialdehyde (MDA), alkaline phosphatase (ALP) and reduced glut athione (GSH) in hepatic homogenate from all mouse were determined. The morpholo gic changes of the livers were also observed with VG staining. Results 　In CCl4 treated mice, serum ALT activity was increased markedly wh ile ChE acti vity decreased significantly. GL and GL+RB, GL +FSC could relieve these changes ; GL combined with all other agents could inhibit the GSH accumulation, but only GL+FSC had a suppressive effect on MDA increase and made it return to normal. For D-GlaN treated mice, all agent groups had little effect on the activity of ChE; Only GL dramatically reduced the increased activity of ALT; GL +RSM could e liminate the GSH, but not significant; GL and all GL combinations could decrease the increased MDA (P<0.01). Pathological observation showed that hepatocyte damage in GL group and GL+FSC group incline d to recover. Conclusion　The results indicate that Ganoderma lucidum is an effective agent against hepatic injuries, especially combined wi th F ructus Schisandrae Chinensis, which may be associated with its anti-hepatocyte oxidation.