Diterbutyl phthalate attenuates osteoarthritis in ACLT mice via suppressing ERK/c-fos/NFATc1 pathway, and subsequently inhibiting subchondral osteoclast fusion.

Osteoarthritis (OA) is the most common arthritis with a rapidly increasing prevalence. Disease progression is irreversible, and there is no curative therapy available. During OA onset, abnormal mechanical loading leads to excessive osteoclastogenesis and bone resorption in subchondral bone, causing a rapid subchondral bone turnover, cyst formation, sclerosis, and finally, articular cartilage degeneration. Moreover, osteoclast-mediated angiogenesis and sensory innervation in subchondral bone result in abnormal vascularization and OA pain. The traditional Chinese medicine Panax notoginseng (PN; Sanqi) has long been used in treatment of bone diseases including osteoporosis, bone fracture, and OA. In this study we established two-dimensional/bone marrow mononuclear cell/cell membrane chromatography/time of flight mass spectrometry (2D/BMMC/CMC/TOFMS) technique and discovered that diterbutyl phthalate (DP) was the active constituent in PN inhibiting osteoclastogenesis. Then we explored the therapeutic effect of DP in an OA mouse model with anterior cruciate ligament transaction (ACLT). After ACLT was conducted, the mice received DP (5 mg·kg-1·d-1, ip) for 8 weeks. Whole knee joint tissues of the right limb were harvested at weeks 2, 4, and 8 for analysis. We showed that DP administration impeded overactivated osteoclastogenesis in subchondral bone and ameliorated articular cartilage deterioration. DP administration blunted aberrant H-type vessel formation in subchondral bone marrow and alleviated OA pain assessed in Von Frey test and thermal plantar test. In RANKL-induced RAW264.7 cells in vitro, DP (20 µM) retarded osteoclastogenesis by suppressing osteoclast fusion through inhibition of the ERK/c-fos/NFATc1 pathway. DP treatment also downregulated the expression of dendritic cell-specific transmembrane protein (DC-STAMP) and d2 isoform of the vacuolar (H+) ATPase V0 domain (Atp6v0d2) in the cells. In conclusion, we demonstrate that DP prevents OA progression by inhibiting abnormal osteoclastogenesis and associated angiogenesis and neurogenesis in subchondral bone.

[Comparative study on appearance characters and internal structure of cultivated and wild Ganoderma lucidum in Huoshan].

Through the comparative study on the appearance characters and internal structure of cultivated and wild Ganoderma lucidum in Huoshan,this paper provides a reference for the further study of G. lucidum. In this study,the similarities and differences between cultivated G. lucidum " Huozhi No. 1" and wild G. lucidum in Huoshan were compared by means of character observation,optical microscopy and scanning electron microscope( SEM). The results showed that the pileus color of " Huozhi No. 1" was yellowish brown and thicker,while that of wild G. lucidum was mainly reddish brown,the context was thinner,and there were gravel and rotten wood at the bottom of the stipe. A clear skeletal hyphae and binding hyphae were observed in cultivated and wild G. lucidum,but there was no significant difference. The shell layer,context layer,mediostratum layer and spores of cultivated and wild G. lucidum were observed by SEM,and the results showed that there was no significant difference. It was found that the mediostratum of " Huozhi No. 1" was thin and irregular,while the mediostratum of wild G. lucidum was neat and compact. There were two types of spores in wild G. lucidum,one of which retained the outer wall of spore type Ⅰ,with tiny pores on the surface. The other is type Ⅱ spores with many spinous processes on the surface,which may be formed by type Ⅰ spores falling off the outwall. In this study,the appearance characters and internal structure of cultivated and wild G. lucidum in Huoshan were systematically observed and compared,which provided theoretical basis and reference for the identification and quality evaluation of cultivated and wild G. lucidum.

[Verification of the theory of "Lieque (LU 7) for the disorders of the head and neck" based on infrared thermography].

OBJECTIVE: To verify that whether or not through the effects of the externally and internally related meridians in treatment, Lieque (LU 7) is adopted specially for the disorders of the head and neck. METHODS: A total of 36 healthy volunteers were collected from the students of Gansu University of CM and were divided into a Lieque group and a Jingqu group according to the random number table, 18 cases in each one. In the Lieque group, Lieque (LU 7) on the unilateral side was punctured in the subjects. In the Jingqu group, Jingqu (LU 8) was taken as the control because it was located close to Lieque (LU 7) and on the same meridian. Before and after acupuncture in the two groups, separately, the infrared thermography was adopted to determine the temperature changes at the acupoints of the lung meridian of hand-taiyin, i.e. Jingqu (LU 8), Lieque (LU 7), Kongzui (LU 6), Chize (LU 5) and Tianfu (LU 3) as well as the acupoints of the large intestine meridian of hand-yangming, i.e., Wenliu (LI 7), Shousanli (LI 10), Quchi (LI 11), Shouwuli (LI 13) and Binao (LI 14). RESULTS: After acupuncture stimulation at Lieque (LU 7), the temperature at the acupoints of the lung meridian of hand-taiyin, i.e. Jingqu (LU 8), Lieque (LU 7), Kongzui (LU 6), Chize (LU 5) and Tianfu (LU 3) and the acupoints of the large intestine meridian of hand-yangming, i.e. Wenliu (LI 7), Shousanli (LI 10), Quchi (LI 11), Shouwuli (LI 13) and Binao (LI 14) was all higher obviously as compared with the temperature before acupuncture stimulation (all P<0.05). After acupuncture stimulation at Jingqu (LU 8), the temperature at the acupoints of the lung meridian of hand-taiyin was all increased obviously as compared with the temperature before acupuncture stimulation (all P<0.05), but there was no significant difference in the temperature at the large intestine meridian of hand-yangming (all P>0.05). CONCLUSION: For the disorders of the head and neck, acupuncture at Lieque (LU 7) achieves the stimulation and communication of both the lung meridian and the large intestine meridians, so that it is applicable for the disorders of the externally and internally related meridians.

Assessment of arsenic trioxide toxicity on cock muscular tissue: alterations of oxidative damage parameters, inflammatory cytokines and heat shock proteins.

To evaluate the toxicity of arsenic trioxide (As2O3) in the muscular tissues (wing, thigh and pectoral) of birds, 72 one-day-old Hy-line cocks were selected and randomly divided into four groups. They were fed either a commercial diet or an arsenic-supplemented diet containing 7.5, 15 or 30 mg/kg As2O3. The experiment lasted for 90 days and the samples of muscular tissues were collected at 30, 60 and 90 days. The results showed that As2O3 exposure significantly lowered the activities of antioxidant enzymes (catalase (CAT), glutathione peroxidase (GSH-Px)) and inhibition ability of hydroxyl radicals (OH) and increased the malondialdehyde (MDA) contents. Furthermore, the mRNA levels of inflammatory cytokines (tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS), prostaglandin E synthase (PTGEs)) and heat shock proteins (HSPs) in muscular tissue were significantly upregulated in the As2O3 exposure groups. The results indicated that As2O3 exposure resulted in oxidative damage, induced the inflammatory response, and influenced the mRNA levels of HSPs in muscular tissue of cocks. Additionally, the results suggested that HSPs possibly resisted due to the As2O3 exposure-induced oxidative stress and inflammatory response, which provided a favorable environment and played protective roles in the muscular tissues of cocks. The information presented in this study is helpful to understand the mechanism of As2O3 toxicity in bird muscular tissues.

[Cloning and prokaryotic expression analysis of DNA methyltransferas 1(MET1) in Ganoderma lucidum].

DNA methyltransferase is the key enzyme in the process of DNA methylation, playing an important role in regulation of gene expression in vivo. According to the Ganoderma lucidum transcriptome data, a full-length cDNA sequence of MET1 from G. lucidum was cloned for the first time, the GenBank registration number is KU239998, and we conducted a comprehensive bioinformatics analysis of the genetic characteristics and spatial structure. The prokaryotic expression analysis showed that E.coli[pET28a(+)-GlMET1] in BL21(DE3) could induce objective protein, shaking the culture at 16 ℃ until the host bacterium(A600) was approximately 0.8, and added IPTG to finally concentration of 0.2 mmol•L⁻¹, and then the optimal expression of GlMET1 recombinant protein was accumulated for the induction time of 20 h. The real-time PCR results showed that the expression levels of GlMET1 had obvious differences among varieties of G. lucidum. During the maturity stage, the expression levels of GlMET1 were lower than that in juvenile stage, the results showed that with the growth of G. lucidum, the expression levels of GlMET1 were on the decline. The research provided an important basis for studying the mechanism of DNA methyltransferase thoroughly.

[Based on evidence to establish reporting guidelines for TCM-featured acupuncture systematic reviews/Meta-analysis: a methodology discussion].

Existing reporting guidelines for systematic reviews/Meta-analysis (SRs/MAs) cannot meet the requirements of clinical practice and scientific research, so based on evidence, methods and thoughts on establishing reporting guidelines for TCM-featured acupuncture systematic reviews/Meta-analysis were proposed. Through literature analysis, according to evidence-based principle, preliminary ideas on methodology of establishing reporting guidelines for TCM-featured acupuncture systematic reviews /Meta-analysis were proposed. With consensus as one main research method, it was proposed that in the consensus that was selected and established by experts, the pro- portion of Chinese scholars should be increased to fully declare the opinions of Chinese acupuncturists, and by verification of practice and application, the reporting guidelines for TCM-featured acupuncture systematic reviews/Meta-analysis can be finally made.

[Cloning and bioinformatics analysis of SmNAC1 from Salvia miltiorrhiza hairy root].

In order to study function of NAC transcription in development, hormone regulation and the stress response of Salvia miltiorrhiza, the NAC transcription was cloned and analyzed. By retrieving cDNA database of S. miltiorrhiza hairy root one NAC unigene was found, then a full length of cDNA was cloned by designing specific primers and PCR amplifying. Using ORF finder it was found that the cDNA containing a NAC-AB conserved domain in N-terminal, so the cDNA was a NAC transcription factor, named as SmNAC1 (kF006346). Bioinformatics analysis showed that SmNAC1 had an open reading frame (ORF) of 591 bp encoding 196 amino acids. The calculated protein had isoelectric point (pI) of 4.36 with molecular weight about 21.66 kDa. The transcription level of SmNAC1 after dealing with yeast extract (YE) and silver ion (Ag+) in S. miltiorrhiza hairy root was markedly stimulated up regulating. It was 1.4 fold compared with the control after induction 2 h, and maintained 2.0 fold on 4-12 h after induction. SmNAC1 may participate in regulation of stress response of YE + Ag+.

[Cloning and bioinformatics analysis of chorismate mutase gene from Salvia miltiorrhiza].

Chorismate mutase catalyzes the conversion of chorismate to prephenate that is the first committed step in the biosynthesis of the aromatic amino acids phenylalanine and tyrosine. A chorismate mutase gene, designated SmCM1, was isolated from Salvia miltiorrhiza by using RT-PCR. The full length of SmCM1 cDNA consists of 948 nucleotides and has an open reading frame of 765 bp. The deduced amino acid sequence of SmCM1 has 255 amino acid residues which forms a 36.0 kD polypeptide with calculated pI of 6.41 as expected. The putative polypeptide contains a CM_2 super family function domain. Blast W results showed that SmCM1 had 70% of the similarity with Petunia x hybrid CM, 72% of the similarity with Arabidopsis thaliana CM, and 64% of similarity with Populus trichocarpa CM. The transcription level of SmCM1 in root, stem and leaf was analysed by realtime quantitative PCR. The results showed the expression level of the SmCM1 in leaf was highest, and lowest in root. Yeast extract and silver ion joint induction could markedly stimulate the increase of mRNA expression of SmCM1 and its upstream 3-deoxy-7- phosphoheptulonate synthase (DAHPS) and chorismate synthase (CS). It was 7.9, 5.5 and 9.8 times of control on 8 h after induction, respectively.

Effects of a dark-septate endophytic isolate LBF-2 on the medicinal plant Lycium barbarum L.

Dark septate endophytes (DSE) are ubiquitous root associated fungi; however, our understanding of their ecological function remains unclear. Here, we investigated the positive effect of a DSE fungus on its host plant Lycium barbarum L. A DSE isolate, LBF-2, isolated from the roots of L. barbarum, was inoculated onto the roots of plants, which were grown under greenhouse conditions for five weeks. The result of molecular analyses of internal transcribed spacer regions indicated that LBF-2 was 96% similar to Paraphoma chrysanthemicola. Melanized septate hyphae were observed in the root cortical cells of L. barbarum using a light microscope. Inoculation with LBF-2 increased the total biomass by 39.2% and also enhanced chlorophyll fluorescence. Inoculation increased the concentration of total chlorophyll by 22.8% and of chlorophyll a by 21.3%, relative to uninoculated controls. These data indicate that the LBF-2 isolate might be used to facilitate the cultivation of L. barbarum, which has medicinal applications.

[Explanatory clinical trials and pragmatic clinical trials on clinical effective evaluation of acupuncture].

The concepts and relations of explanatory clinical trials and pragmatic clinical trials on randomized controlled trials of acupuncture are stated. The explanatory clinical trials and pragmatic clinical trials are different in the purpose of study, the controlled method, the treatment method and the solution problems. The explanatory clinical trials may enhance the understanding of the mechanism of a treatment; the findings of pragmatic clinical trials may be applied immediately in clinical practice. This paper explains that these clinical trials are of great importance to the methodology and practices of clinical effective evaluation of acupuncture based on TCM theory.

[Study on key problems in the positive control design methods of acupuncture and moxibustion ].

To probe into the design method of the clinical studies with positive control of acupuncture and moxibustion, the several key problems are analyzed, which include the definition of positive control, form of the control, statistical and design types, proper application of positive drugs, and properly use placebo control method to counteract weaknesses of studies, reasonably select observation indexes so as to show characteristics of Chinese medicine. The authors indicate that only fully thinking these key problems, can provide clinical evidence of acupuncture and moxibustion with high quality and convincingness.

[Study on apoptosis of BEL-7402 cells induced by galangin].

OBJECTIVE: To investigate apoptosis of BEL-7402 cells induced by Galangin. METHODS: Cytotoxicity and cell viability were assayed by MT methods. Morphologic assessment of apoptosis was performed by fluorescence microscope, cell cycle and mitochondrial membrane potential were analyzed by flow cytometry, Caspases Activities were detected by chromogenic substrate assay. RESULTS: BEL-7402 cells treated with Galangin showed apparently cytotoxicity, IC50 of Galangin was 30.15 mg/L The growth curve showed that the ratio of growth decreased with the increasing of concentration of Galangin. The apoptosis appeared 24 hours after treated with 20-80 mg/L Galangin, morphologic changes included nuclear chromatin condensation and fluorescence strength was observed with fluorescence microscope. The activity of Caspase-9 attained to peak value at 6 h, while Caspase-6 activity was up to peak value at 12h and Caspase-3 activity attained to peak value at 18 h. CONCLUSION: Galangin may induce BEL-7402 cells apoptosis by mitochondrial pathway.

[Characterization of chemical components of essential oil from cupitulum of Chrysanthemum morifolium cultivated in Tongxiang city].

OBJECTIVE: To analyze the chemical components of the essential oil extracted from five cultivars' Hangjuhua (Zaoxiaoyangju, Chixiaoyangju, Dayangju, Yizhongdabaiju and Xiaotangju), which were breed came from the Chrysanthemum morifolium Ramat, and provide scientific basis for quality control. METHOD: The essential oil of five cultivars' Hangjuhua were extracted by water-steam distillation and separated by GC capillary column chromatography. The components were quantitatively determined with normalization method, and identified by GC-MS. RESULT: From the Zaoxiaoyangju, Chixiaoyangju, Dayangju, Yizhongdabaiju and Xiaotangju, 179, 167, 209, 211 and 270 chromatographic peaks were detected, among them 54, 56, 60, 55 and 65 components were identified, which were composed of 73.40%, 64.14%, 65.19%, 53.55% and 62.89% of the total essential oil, respectively. CONCLUSION: The common chemical constituents were juniper camphor, 1, 3, 3-trimethylcyclohex-1-ene-4-carboxaldehyde, borneol, alpha-curcumene, alpha-bisabolol, cis-caryophyllene, benzyl benzoate, methyl beta, beta-dimethylbenzenepropanoic acid ester, 2, 4-decadienal and heneicosane in the essential oil of the five cultivars' Hangjuhua. Among them, juniper camphor is most which were composed of 11.96%, 10.51%, 10.95%, 13.28% and 10.77% of the total essential oil, and methyl beta, beta-dimethylbenzenepropanoic acid ester is second, which were composed of 1.51%, 1.86%, 2.52%, 4.89% and 2.51% of the total essential oil, respectively. There are marked differences in the compounds contained in the five samples. The juniper camphor is a characteristic constituent in the essential oil of Hangjuhua. The resultant data provided the evidences to establish Hangjuhua' cultivars and scientific basis for quality control of Hangjuhua.

[Studies on chemical constituents in Huangjuhua (flowers of Chrysanthemum morifolium)].

OBJECTIVE: To study the chemical constituents in Huangjuhua (flowers of Chrysanthemum morifolium). METHOD: The chemical constituents were isolated by various column chromatographic methods and structurally elucidated by NMR and MS evidences. RESULT: Eleven compounds were obtained and identified as luteolin (1), quercetin (2), acacetin 7-O-beta-D-(3"-acetyl) -glucopyranoside (3), luteolin 7-O-beta-D-(6"-acetyl)-glucopyranoside (4), hesperetin 7-O-beta-D-glucopyranoside (5), acacetin 7-O-beta-D-glucopyranoside (6), diosmetin 7-O-beta-D-glucopyranoside (7), apigenin 7-O-beta-D-glucopyranoside (8), hesperidin (9), linarin (10), and luteolin 7-O-beta-D-glucopyranoside (11). CONCLUSION: Compounds 4, 5 were isolated from the plant for the first time.

[Characterization of chemical components of essential oil from flowers of Chrysanthemum morifolium produced in Anhui province].

OBJECTIVE: To analyze the chemical components of the essential oil extracted from six cultivars' Juhua (Zaogongju, Wangongju, Huangyaoju, Chuju, Xiaoboju and Daboju) produced in Anhui province of China, which were breed from Chrysanthemum morifolium, and to provide scientific basis for quality control. METHOD: The essential oils of the six cultivars'Juhua were extracted by water-steam distillation and separated by GC capillary column chromatography. The components were quantitatively determined with normalization method, and identified by GC-MS. RESULT: From Zaogongju, Wangongju, Huangyaoju, Chuju, Xiaoboju and Daboju, 247, 226, 246, 182, 216 and 122 chromatographic peaks were detected, among them 75, 54, 78, 50, 53 and 43 components were identified,which were composed of 85.67%, 82.80%, 81.38%, 73.22%, 71.51% and 72.87% of the total essential oil, respectively. CONCLUSION: Monoterpenoid compounds were higher more than sesquiterpenoid compounds in the five juhua except for Chuju. There was no difference in the constituents of essential oil of Zaogongju and Wangongju. And verbenyl acetate was the main chemical constituent and composed of 32.10% and 37.85% of the total essential oil, respectively. (1R)-Camphor and bisabolol oxide A were the main chemical constituents in Huangyaoju, which were composed of 28.70% and 12.58% of the total essential oil, respectively. beta-Selinene and borneol were the main chemical constituents in Chuju, which were composed of 17.85% and 12.84% of the total essential oil, respectively. Eucalyptol was the main chemical constituent in Xiaoboju, which were composed of 21.33%. Verbene oxides and chrysanthenone were composed of 25.32% and 8.26% of the total essential oil, respectively, in the Daboju. The common chemical constituents were camphene, borneol, bornyl aceate, (1R)-camphor, (-)-4-terpineol, alpha-terpineol, eucalyptol, cis-caryophyllene, caryophyllene oxide, juniper camphor, beta-sesquiphellandrene, alpha-curcumene and beta-farnesene in the six cultivars' Juhua produced in Anhui province of China. The resultant data provided the further evidences to distinguish Juhua' cultivars and scientific basis for quality control of Juhua.

[GC-MS analysis of essential oil from Xiaoboju processed by aeration-desiccation and sulfur-burnin fumigation].

OBJECTIVE: Analysis of the constituents of the essential oil extracted from the Xiaoboju, one of commercial breed came from the flowers of the Chrysanthemum morifolium, processed by the aeration-desiccation and desiccation after the sulfur-bumin fumigation, and to provide scientific basis for quality control. METHOD: The essential oil was extracted by water-steam distillation and separated by GC capillary column chromatography. The components were quantitatively determined with normalization method, and identified by GC-MS. RESULT: From the aeration dried sample and the dried sample after sulfur-burnin fumigation, 216 and 211 components were detected, among them fifty and six-five components were identified, which were composed of 73.21% and 82.32% of the total essential oil, respectively. CONCLUSION: The yield of the essential oil extracted from the aeration dried sample was 3.50%, and that from the dried sample after sulfur-bumin fumigation was 4.22%. The latter is 1.2 times higher than the former. The components of the essential oil of both samples are mostly monoterpenoids and secondly sesquiterpenoids compounds, but there are marked differences between the compounds contained in the two samples. Therefore, the processing of flowers of the C. morifolium should be strictly controlled and standardized.