Lycium barbarum polysaccharides attenuate kidney injury in septic rats by regulating Keap1-Nrf2/ARE pathway.

Lycium barbarum polysaccharides (LBP) are derived from Wolfberry and have antioxidant activities. This study aimed to evaluate the efficacy of LBP for kidney injury in a rat model of sepsis. Male rats were divided randomly to control group (Con), LPS group (LPS), ulinastatin group (ULI), low dose LBP group (LBP-1), middle dose LBP group (LBP-2) and high dose LBP group (LBP-3). After intraperitoneal injection of LPS (5 mg/kg) to make sepsis model (LPS group), 10,000 U/kg ulinastatin were given in ULI group, and 200, 400 and 800 mg/kg LBP was given in LBP-1, -2, -3 group, respectively. Serum IL-1ß, IL-6, IL-8, TNF-α and NF-κB levels were measured by ELISA. Nrf2, Keap1, NF-κB, HO-1 and NQO1 expression levels were detected by PCR and Western blot analysis. We found that LBP decreased the levels of NF-κB and pro-inflammatory cytokines while attenuated kidney injury. In addition, LBP regulated Keap1-Nrf2/ARE signaling pathway in the kidney. In conclusion, LBP attenuates inflammation injury in the kidney via possible regulation of Keap1-Nrf2/ARE signaling.

Effects of ginsenoside Rg1 on the expression of toll-like receptor 3, 4 and their signalling transduction factors in the NG108-15 murine neuroglial cell line.

As one of the most important components of Panax ginseng, ginsenoside Rg1 has certain anti-aging effects, improving the activity of learning and memory. Studies have showed that ginsenoside Rg1 improves the memory impairment associated with Alzheimer's disease (AD). In this study, the effects of ginsenoside Rg1 were investigated through the activity of toll-like receptor (TLR) 3, TLR4 and their signaling transduction pathways in amyloid ß peptide 25-35 (Aß25-35) induced AD cell model. Thus we investigated several critical components of the TLR pathway. The neuroglial cell line NG108-15 was stimulated with or without Aß25-35, while different concentrations of ginsenoside Rg1 were administered. After 24 h, tumor necrosis factor-α (TNF-α), interferon-ß (IFN-ß) in cell supernatant and inducible nitric oxide synthase (iNOS) in cell lysate supernatant were measured with enzyme-linked immunosorbent assays (ELISAs). The mRNA and protein expression of TLR3, TLR4, nuclear factor kappa B (NF-κB) and tumor necrosis factor receptor-associated factor-6 (TRAF-6) were detected by real-time PCR and western blot methods, respectively. The experimental results showed that Aß25-35 could markedly raise the level of TNF-α, IFN-ß and iNOS, and increase the expressions of mRNA and TLR3, TLR4, NF-κB and TRAF-6 protein in the NG108-15 cells. At the same time, the ginsenoside Rg1 significantly reduced the expressions of proteins and mRNA of TLR3, TLR4, NF-κB and TRAF-6, and down-regulated the levels of TNF-α, IFN-ß of cell supernatant and iNOS of cell lysate supernatant in a concentration-dependent manner. In conclusion, ginsenoside Rg1 has good activity for suppressing the signaling transduction pathway of TLR3 and TLR4, and decreasing the inflammation factors induced by Aß25-35 in NG108-15 cells, and this may be the mechanism of ginsenoside Rg1 action in AD treatment, but more studies are needed to identify its specificity.

[Exploration of property theory of Tibetan medicine].

Tibetan Herbal medicine has its own complete theory based on five sources doctrine. And the theories of "Liuwei", "Baxing" and "Shiqi Gongxiao" formed the basic core components of the property theory of Tibetan medicine. However, books and literature of Tibetan medicine have never been systematically expounded and discussed about it specially which thus will limit the further development of Tibetan medicine theory. In this thesis, we firstly introduced three basic core components of the property theory-the "Liu Wei", "Baxing", and "Shiqi Gongxiao" and their interactions as well. At the same time, the links and similarities between the theory of Tibetan medicine and Chinese medicine theory were compared. The job of the thesis done above is to lay the foundation for further systematic reveal and development of Tibetan medicine theory.

[Accurate identification of Psammosilene tunicoides and its confused species by systematic identification method].

OBJECTIVE: To develop an effective identification method for accurately discriminating Psammosilene tunicoides and its confused species by the combined method of microscopic identification and molecular identification, so-called systematic identification of Chinese materia medica (SICMM). METHOD: P. tunicoides and its confused species were accurately discriminated by SICMM method, which was established by comprehensively use of microscopic identification and DNA identification method. The DNA identification included the following analysis: the BLAST alignment, specific bases and N-J phylogenetic tree analysis. RESULT: The cluster crystals were not observed in P. tunicoides, but great deals of them were found in Silene viscidula. Further more, big differences of ITS sequence were observed and analyzed between P. tunicoides and its confused specie of S. viscidula. CONCLUSION: The system method is a scientific and accurate method for the identification of P. tunicoides and its counterfeit species.

[A accurate identification method for Chinese materia medica--systematic identification of Chinese materia medica].

This paper put forward a more accurate identification method for identification of Chinese materia medica (CMM), the systematic identification of Chinese materia medica (SICMM) , which might solve difficulties in CMM identification used the ordinary traditional ways. Concepts, mechanisms and methods of SICMM were systematically introduced and possibility was proved by experiments. The establishment of SICMM will solve problems in identification of Chinese materia medica not only in phenotypic characters like the mnorphous, microstructure, chemical constituents, but also further discovery evolution and classification of species, subspecies and population in medical plants. The establishment of SICMM will improve the development of identification of CMM and create a more extensive study space.

[Researches on influence of squalene synthase gene polymorphism on catalytic efficiency of its encode enzyme in Glycyrrhiza uralensis].

OBJECTIVE: To analyse the polymorphism of squalene synthase gene and reveal the influence of squalene synthase (SQS) gene polymorphism on the catalytic efficiency of its encode enzyme in Glycyrrhiza uralensi. METHOD: The total RNA was extracted. PCR was used to amplify the coding sequences of squalene synthase gene, which were sequenced and analysed. The expression vectors containing different SQS gene sequences, including SQS1C, SQS1F, SQS2A, SQS2B, were constructed and transformed into Escherichia coli BL21. The fusion protein was induced to express by IPTG, then was isolated, purified and used to carry out the enzymatic reaction in vitro. GC-MS was used to analyse the production. RESULT: There were three kinds of gene polymorphism existing in SQS1 gene of G. uralensis, including single nucleotide polymorphism (SNPs), insertion/deletion length polymorphism (InDels) and level of amino acid, the proportion of conservative replace of SQS1 was 53.94%, and there were 2 mutational sites in structural domains. The proportion of conservative replace of SQS2 was 60%, and there was 1 mutational site in structural domains. The production squalene could be detected by GC-MS in all the 4 kinds of enzymatic reactions. The capacity of accumulating squalene of SQS1F was higher than other SQS genes. CONCLUSION: The polymorphism of SQS gene was quite abundant in G. uralensis, which maybe the molecular foundation of the formation of high-quality liquorice.

[Researches on the influence of 3-hydroxy-3-methylglutary-coenzyme A reductase gene polymorphism on catalytic efficiency of its encode enzyme in Glycyrrhiza uralensis].

OBJECTIVE: To analyse the effect of expression proteins containing different escherichia coli of 3-hydroxy-3-methylglutary-coenzyme A reductase(HMGR) genic mutation on the conversion efficiency of MVA with GC-MS method, in order to lay a foundation for revealing the function of HMGR gene polymorphism of Glycyrrhiza uralensis in the production of high-quality G. uralensis medicines. METHOD: The expression carrier was established from four HMGR genic mutation types cloned from G. uralensis and transformed into Escherichia coli BL21. The protein was induced to express, detected and purified. The purified protein was adopted for in vitro enzymatic reaction. TLC and GC-MS were used for qualitative and quantitative analysis on reaction products. RESULT: The catalytic activity of L/V genotype(-HSL and -HSV) was similar, and so was the catalytic activity of the genotype with GA insertion (GALLV and GALSV), but the catalytic activity of the latter was around 2 times higher than that of the former. CONCLUSION: The functional gene polymorphism of G. uralensis may be the molecular foundation for the production of high-quality G. uralensi medicines.

[Analysis on correlation between 3-hydroxy-3-methylglutary-coenzyme A reductase gene polymorphism of Glycyrrhiza uralensis and content of glycyrrhizic acid].

OBJECTIVE: To reveal the 3-hydroxy-3-methylglutary-coenzyme A reductase (HMGR) gene polymorphism of Glycyrrhiza uralensis, and the correlation between HMGR gene polymorphism and the content of glycyrrhizic acid. METHOD: Liquorice plants containing different content of glycyrrhizic acid were used as materials. RT-PCR was used to amplify their HMGR gene sequences, which were connected with vector pMD19-T for clone sequencing. Multiple alignments were performed to analyse HMGR gene polymorphism of G. uralensis. Then the correlation between HMGR gene polymorphism and the content of glycyrrhizic acid was revealed. RESULT: HMGR gene sequences polymorphism included codon mutation, base substitution mutation, copy number polymorphism and allele heterozygosity. There were 4 types of mutations in HMGR gene coding amino acid sequences, namely -HSL, -HSV, GALLV, GALSV. Among them, -HSV type was common in liquorice plants, -HSL type only existed in liquorice plants with low content of glycyrrhizic acid, and GALSV type only existed in liquorice plants with high content of glycyrrhizic acid. CONCLUSION: HMGR gene sequences of G. uralensis are highly polymorphic and related to the content of glycyrrhizic acid.

[Molecular cloning and SNP analysis of a acetyl-CoA C-acetyltransferase gene (SmAACT) from Salvia miltiorrhiza].

Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpene synthesis pathway, catalyzed two units of acetyl-CoA to acetoacetyl-CoA. In order to study the tanshinone biosynthesis in Salvia miltiorrhiza, a novel AACT gene, SmAACT, was cloned using cDNA microarray and RACE strategy. The full length cDNA of SmAACT is 1 623 bp (accession No. EF635969), which contained a 1 200 bp open reading frame (ORF) encoding a 399 amino acid protein. Nine introns were found in the genomic sequence. SmAACT was upregulated by YE and Ag+ elicitors both with cDNA microarray and quantitative RT-PCR analyses along with the accumulation of tanshinones. Sequence homology comparison and phylogenetic analysis all suggested that SmAACT belonged to the class of acetyl-CoA C-acetyltransferase. The transcription level of SmAACT was relatively higher in root than that in stem and leaf tissues. SNP analysis revealed that SmAACT was highly variable in the region of 6 to 9 introns with 33 SNPs in the 600 bp region, there are 5 SNPs in the cDNA region while they are all synonymous cSNPs. Some special genotypes were found in Salvia miltiorrhiza from different areas. SmAACT will be an useful gene for further analyze the mechanism of gene regulation among the tanshinones biosynthesis.

[A full length cDNA of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase cloning and analysis of introduced gene expression in Salvia miltiorrhiza].

This paper firstly introduced the acquired full length cDNA of 4-(cytidine 5'-diphospho)-2-C-methyl-D-erythritol kinase from hairy roots of Salvia miltiorrhiza (Abbr: SmCMK, GenBank number: EF534309). Results of KEGG analysis showed that SmCMK was belong to the upstream of nonemevalonate pathway, the only one kinase of the pathway. The full-length cDNA was deduced as encoding 4-(cytidine 5'-diphospho)-2-C-methylerythritol kinase (designated as SmCMK), and the sequence had a 1493 bp including 5' UTR 71 bp and 3' UTR 232 bp, an open reading frame (ORF) encoding a protein of 396 amino acid residues. The deduced protein had isoelectric point (pI) of 6.78 and a calculated molecular weight about 43 kDa, similar to cloned diterpene of CMK from other species of plants such as Mentha piperita and Lycopersicon esculentum reported previously. Real time PCR results indicated that elicitors of MJ stimulated the increase of mRNA expression of SmCMK. At the same time, results of high performance liquid chromatography (HPLC), used to examine the accumulation of diterpenoid tanshinones in hairy roots, showed that the contents of diterpenoid tanshinones in hairy root of Salvia miltiorrhiza were increased dramatically after treated with methyl jasmonate (MJ). This result showed a positive correlation between the levels of mRNA expression and tanshinones accumulation in Salvia miltiorrhiza stimulated by MJ. It proved primarily that the increased expression level of mRNA of SmCMK helps to enhance tanshinones' accumulation, which will be the basis for further study on the mechanism of gene regulation of secondary metabolism of tanshinones.

[Functional genomics studies of Salvia miltiorrhiza III. analyze of metallothionein (MT-2) genes].

OBJECTIVE: To study the metallothionein genes of Salvia miltiorrhiza through bioinformatics and characterization of tissue expression in regenerated shoots. METHOD: Metallothionein genes were obtained by cDNA microarray analyze. BLAST was used for align, ORF finder software was used to find open reading frame, Prosite database was used to analyze the protein. Semi-quantitative RT-PCR method was used to detect the gene expression level. RESULT: Two metallothionein genes were obtained which were contained a deduced amino acid sequence of 80 and 79 residues, named as SmMT-2a and SmMT-2b, they had a homology of 71.25%. Semiquantitative RT-PCR indicated that metallothionein genes were expressed in all tissues such as root, stem and leaf in regenerated shoots, while the expression level was higher in leaf than in root and stem. CONCLUSION: It was the first time that metallothionein genes were obtained from S. miltiorrhiza. It provides a good basis for further functional study of S. miltiorrhiza.

[Functional genomics studies of Salvia miltiorrhiza I. Establish cDNA microarray of S. miltiorrhiza].

OBJECTIVE: Establishing cDNA microarray, in order to study functional genomics of Salvia miltiorrhiza. METHOD: Total RNA samples were prepared from S. miltiorrhiza roots using a modified CTAB method. mRNA was isolated by Quichprep Micro mRNA Purification Kit from Pharmacia. Then cDNA was synthesized and cloned into the EcoRI-XhoI sites of the ZAP Express vector using a cDNA synthesis kit, and the ligation mixture was packaged using a ZAP-cDNA Gigapack Gold III cloning kit (Stratagene). The single phage was isolated for PCR amplification, Aliquots of the PCR reactions were analyzed in a 1.5% agarose gel to verify the quality of PCR. The remaining cDNA was purified by Multiscreen filter plates (Millipore) and aliquots were analyzed by agarose gel again to verify the quality of purification. Clones passed verification was resuspended in 15 microL 50% DMSO for arraying. An actin gene from S. miltiorrhiza was used for positive control. PloyA and 50% DMSO was used for negative controls. RESULT: Bacterial colonies containing cNDAs of S. miltiorrhiza were inserted with average insert size of 0. 5 kb to 2. 5 kb. Total 4 354 genes were singled out from the first 8 736 PCR product and used for cDNA microarray manufacture. Single color fluorescence hybridization showed that all positive controls had signals while negative controls had no signals. CONCLUSION: It was the first cDNA microarray about traditional Chinese herbs especially for geoherbs. It could be a powerful tool for studying functional genomics of S. miltiorrhiza.

[Effects of methyl jasmonat on accumulation and release of tanshinones in suspension cultures of Salvia miltiorrhiza hairy root].

OBJECTIVE: To study the effects of methyl jasmonate (MJ) on the accumulation and release of tanshinones in suspension cultures of Salvia miltiorrhiza hairy roots. METHOD: After 18 day's suspension culture of S. miltiorrhiza hairy roots induced by Agrobacterium rhizogenes ATCC15834, the chemical elicitor--methyl jasmonat was added into 6-7V suspension cultures and at the same time, tanshinones contents (including cryptotanshinone and tanshinone II(A)) on the day 2, 6 and 9, after dealing with MJ, was quantified by HPLC. RESULT: After dealing with MJ on the day 2, 6 and 9, the concentration of cryptotanshinone reached to 0.039, 0.204, 0.571 mg x g(-1) respectively,and tanshinone II(A) reached 0.251, 0.601 and 1.563 mg x g(-1) respectively. After 9 day's treatment by MJ, the maximum increase of cryptotanshinone and tanshinone II(A) were 23.8 fold and 6.2 fold higher than that of the control respectively. CONCLUSION: MJ could stimulate the accumulation of tanshinones in S. miltiorrhiza and have released them into the culture medium.

[Effects and mechanism of total saponins of Psammosilene tunicoids against rheumatoid arthritis].

OBJECTIVE: To observe the antiarthritic effects and the possible mechanism of total saponins of Psammruosilene tunicoids (TSPT) against rheumatoid arthritis (RA). METHOD: After establishing AA rat model, the TSPT'S antiarthritic effects and mechanism against RA were studied through observing the changes of ankle swelling, arthritis index and levels of IL-1beta and TNF-alpha after medication. RESULT: TSPT could effectively inhibits articular swelling, decrease arthritis index and regulate down the content of IL-1beta and TNF-alpha in the inflammatory tissue soak of AA rats. CONCLUSION: TSPT has good antiarthritic effects and the possible mechanism may be related to its down-regulation of IL-1beta and TNF-alpha.