Nontargeted metabolomics and enzyme inhibitory and antioxidant activities for chemical and biological characterization of jujube (Ziziphus jujuba) extracts.

The chemical and biologically active characterization of jujube samples (fruits, cores, and leaves) were carried out by the integrated nontargeted metabolomics and bioassay. Firstly, collision cross-section values of active compounds in jujubes were determined by ultrahigh-performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry. Then, a multidimensional statistical analysis that contained principal component analysis, partial least squares-discriminant analysis and hierarchical clustering analysis was employed to effectively cluster different tissues and types of jujubes, making identification more scientific. Furthermore, angiotensin-converting enzyme (ACE) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were used to evaluate the quality of jujubes from a double activity dimension. The analytical results obtained by using ACE and DPPH to evaluate the quality of jujube were different from multivariate statistics, providing a reference for the application of jujube. Therefore, integrating chemical and biological perspectives to evaluate the quality of jujube provided a more comprehensive evaluation and effective reference for clinical needs.

[Application of "Sancai principle" for needle-knife treatment of bi syndrome of neck region].

Under the guidance of the "Sancai principle", based on the understanding of the etiology and pathogenesis of the imbalance of muscles and bones in bi syndrome of neck region, holistic treatment should be used. The needle-knife release therapy is applied at corresponding acupoints in the three parts i.e. head, neck and back including Tiancai points (Naohu [GV 17] and Naokong [GB 19]), Rencai points (neck Jiaji [EX-B 2]), and Dicai points (Dazhui [GV 14], Quyuan [SI 13] and Tianzong [SI 11]). According to the layers of the lesion's meridians and muscles, the needle-knife is inserted into skin, muscle and bone to relax the tendons and treat bone disorders, and restore the normal mechanical balance of neck.

Transcriptome sequencing leads to an improved understanding of the infection mechanism of Alternaria solani in potato.

BACKGROUND: Alternaria solani (A. solani), the main pathogen of potato early blight, causes serious yield reductions every year. The application of fungicides is the most common and effective method of controlling Alternaria-caused diseases. The differentially expressed transcripts of A. solani infecting potato were identified, revealing a group of valuable candidate genes for a systematic analysis to increase the understanding of the molecular pathogenesis of A. solani, and providing scientific data for formulating additional measures to prevent and control potato early blight. In this study, a deep RNA-sequencing approach was applied to gain insights into A. solani pathogenesis. At 3, 4, and 5 days post inoculation (dpi), RNA samples from the susceptible potato cultivar Favorita infected with A. solani strain HWC-168, were sequenced and utilized for transcriptome analysis, and compared to the transcriptome obtained 0 dpi. RESULTS: A total of 4430 (2167 upregulated, 2263 downregulated), 4736 (2312 upregulated, 2424 downregulated), and 5043 (2411 upregulated, 2632 downregulated) genes were differentially expressed 3, 4 and 5 dpi, respectively, compared with genes analysed at 0 dpi. KEGG enrichment analysis showed that genes involved in the pathways of amino acid metabolism, glucose metabolism, and enzyme activity were significantly differentially expressed at the late infection stage. Correspondingly, symptoms developed rapidly during the late stage of A. solani infection. In addition, a short time-series expression miner (STEM) assay was performed to analyse the gene expression patterns of A. solani and Profile 17 and 19 showed significant change trends 3, 4 and 5 dpi. Both profiles, but especially Profile 17, included enzymes, including transferases, oxidoreductases, hydrolases and carbohydrate-active enzymes (CAZYmes), which may play important roles in late fungal infection. Furthermore, possible candidate effectors were identified through the adopted pipelines, with 137 differentially expressed small secreted proteins identified, including some enzymes and proteins with unknown functions. CONCLUSIONS: Collectively, the data presented in this study show that amino acid metabolism, and glucose metabolism pathways, and specific pathway-related enzymes may be key putative pathogenic factors, and play important roles in late stage A. solani infection. These results contribute to a broader base of knowledge of A. solani pathogenesis in potato, as indicated by the transcriptional level analysis, and provide clues for determining the effectors of A. solani infection.

[Cloning and expression analysis of LncRNA-RgAGT2 responding to consecutive monoculture in Rehmannia glutinosa].

The consecutive monoculture obstacle is a major problem in the field of Rehmannia glutinosa( R. glutinosa),has severely declined the yield and quality of R. glutinosa. Here,using hi TAIL-PCR and RACE techniques,we have cloned the full-length transcript( 1 573 bp) of Unigene 29334＿All screened by DGE as a consecutive monoculture obstacle response gene of R. glutinosa. Based on ORF Finder prediction,all ORFs detected in the full-length transcript were less than 300 nt,which suggested that the above transcript was confirmed to be a long non-coding RNA( LncRNA). With alignment in R. glutinosa transcriptome,this LncRNA was partially homologous to alanine glyoxylate transaminase 2 gene( Rg AGT2),which was named LncRNA-RgATG2. To further explore the function of LncRNA-RgAGT2,we have examined expression patterns of LncRNA-RgAGT2 and Rg AGT2 at five critical development stages( seedling,elongation,pre-expanding,mid-expanding,late-expanding) in the first and second year replanting of R. glutinosa,respectively. The results indicated that LncRNA-RgAGT2,as a potential regulator,is possible to play a vital role in Rg AGT2 expression regulation. Meanwhile,LncRNA-RgAGT2 has presented significant variation in all development stages of R. glutinosa,which could be used as a " diagnostic label" to assess consecutive monoculture obstacle. This study,for the first time,showed that LncRNA was responsible for the response and regulation of consecutive monoculture obstacle,which would be a powerful supplement to reveal the molecular mechanisms of consecutive monoculture obstacle of R. glutinosa.

Therapeutic evaluation of acupoint stimulation with needle-scapelon on rat model of degenerative cervical intervertebral discs.

Cervical spondylosis (CS), which is resulted from degeneration of cervical intervertebral disc, is a common disease seriously threatening human health and quality of life. However, there is still no effective clinic strategies for the treatment of this disease. The acupoint stimulation with needle-scalpel is a widely used approach to treat orthopedic diseases. In the present study, we evaluated the therapeutic effects of acupoint stimulation around neck with needle-scalpel on delaying the degeneration of cervical intervertebral discs and hopefully provided an approach for the precaution and early intervention of CS. We firstly established a rat model of CS by cervical static-dynamic imbalance to mimics disc degeneration and then stimulated the acupoints around neck with needle-scalpel. The cervical intervertebral disc samples were collected to measure type I and II collagen by quantitative PCR (qPCR), immunohistochemistry, and western blot. The changes in micro-structure and ultra-structure of nucleus pulposus were analyzed under the optical microscope and electron microscope respectively. Acupoint stimulation with needle-scapelon increased type I collagen production and decreased type II collagen production, and improved the micro-structure and ultra-structure of nucleus pulposus. Our results suggest that acupoint stimulation around neck with needle-scapelon could inhibit intervertebral disc degeneration through modulating the extracellular matrix collagen system and improving the changed structure of nucleus pulposus.

[Effect of Acupotomy Lysis at Cervical Acupoints on Expression of Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinase 1 and Changes of Pulpiform Nucleus Ultrastructure in Rats with Degenerated Cervical Intervertebral Discs].

OBJECTIVE: To observe the effect of acupotomy lysis at acupoints around the neck on expression of matrix metalloproteinase 1 (MMP-1), MMP-2 and tissue inhibitor of metalloproteinase 1 (TIMP-1) genes and ultrastructure of pulpiform nucleus in cervical intervertebral disc degeneration (IVDD) rats, so as to explore its mechanism underlying easing IVDD. METHODS: SD rats were randomly allocated to control (n = 15), model (n = 14), Jiaji (EX-B 2, n = 13), cervico-acupoint (n = 14) and medication groups (n = 14). The cervical IVDD model was established by using static-dynamic imbalance method. For rats of the Jiaji (EX-B 2) and cervico-acupoint groups, EX-B 2-points of the cervical 2-7 segments, and peri-cervical acupoints: bilateral "Naokong" (GB 19) , "Naohu" (GV 17), "Dazhui" (GV 14), bilateral "Quyuan" (SI 13) and bilateral "Tianzong" (SI 11) were separately punctured with a needle-knife, once every 5 days for 3 times, and for rats of the medication group, Brufen Capsules (15 mg · kg(-1) · d(-1)) and Jingfukang Granule (0.5 mg · kg(-1) · d(-1)) were given by intragastric administration, once daily for 10 days. The expression levels of MMP-1, MMP-3 and TIMP-1 genes in the pulpiform nucleus of cervical intervertebral discs were detected by RT-PCR and changes of the ultrastructure of the pulpiform nucleus observed under transmission electron microscope. RESULTS: Compared to the control group, the expression levels of MMP-1 mRNA and MMP-3 mRNA of the cervical intervertebral disc tissues were significantly up-regulated in the model group (P < 0.05), and that of TIMP-1 mRNA was obviously down-regulated in the model group (P < 0.05). After the treatment, the increased expression of MMP-1 mRNA and MMP-3 mRNA and the decreased expression of TIMP-1 mRNA were reversed by acupotomy lysis and medication (P < 0.05) except TIMP-1 mRNA in the medication group (P > 0.05). No significant differences were found between the Jiaji (EX-B 2) and cervico-acupoint groups in down-regulating MMP-1 mRNA and MMP-3 mRNA expression and up-regulating TIMP-1 mRNA expression (P > 0.05). Results of electron microscope examinations showed that the ultrastructural injury changes of cells of the pulpiform nucleus were relatively milder in the Jiaji (EX-B 2) and cervico-acupoint groups, followed by the medication group in comparison with those of the model group. CONCLUSION: Acupotomy lysis at acupoints around the neck can improve the ultrastructural changes of cells of the pulpiform nucleus of cervical intervertebral discs in IVDD rats, which is possibly by regulating the expression of MMP-1, MMP-3 and TIMP-1 genes.

[Effect of Acupotome Relaxing on Expression of Type I and II Collagens of Degenerative Cervical Inter- vertebral Discs in Rats].

OBJECTIVE: To observe the effect of acupotome relaxing at cervical acupoints on type I and II collagens of degenerated cervical intervertebral discs in rats, so as to explore its potential mechanism underlying anti-degeneration of intervertebral discs. METHODS: Rats were randomly divided into control, model, Jiaji acupoints, cervical acupoints and medication groups (n = 15 in each group). The rat model of cervical intervertebral disc degeneration due to static-dynamic imbalance was made as previously specified. The Jiaji acupoints were those located along the cervical vertebra 2-7. The cervical acupoints included bilateral "Naokong"(GB 19) , "Naohu" (GV 17) , "Dazhui"(GV 14) , bilateral "Quyuan" (SI 13) and bilateral "Tianzong" (SI 11). Acupoints were treated according to the procedures of acupotome for 3 times in ten days with five days' break between every two treatment sessions. Rats of the medication group were intragastrically administered with Jing Fu Kang Granules and ibuprofen daily for ten days. Twenty days after the end of treatment, all rats were sacrificed for further examination of morphological changes of the intervertebral disc tissue. Immunoactivity of protein and mRNA expression levels of collagen type I and II of the intervertebral discs were measured by means of immunohistochemistry and RT-PCR, respectively. RESULTS: In comparison with the control group, the immunoactivity and mRNA expression levels of collagen type I and II of the intervertebral discs were significantly elevated or reduced in rats of the model group, respectively (P < 0.05). After acupotome intervention and medication, the increased and decreased expression levels of type I and II collagen proteins and genes were markedly reversed (P < 0.05). The effects of acupotome relaxing of both cervical and Jiaji acupoints were significantly superior to those of medication in down-regulating expression of type I collagen protein and mRNA, and in up-regulating that of type II collagen protein and mRNA (P < 0.05). No significant differences were found between the cervical acupoints and Jiaji acupoints groups in the above- mentioned outcomes (P > 0.05) . The degree of severity of the degenerated intervertebral discs was the worst in the model group, followed by the medication group, then the Jiaji acupoints group and cervical acupoints group, and the control group the least. CONCLUSION: Acupotome at neck acupoints can regulate the extracellular matrix of the intervertebral disc via inhibiting the transformation between type I and type II collagens, which may contribute to its effect in delaying the degenerative process of the cervical intervertebral discs.

[Influence of acupuncture intervention on neurologic deficits, cerebrocortical cell apoptosis and Protein kinase A expression in rats with focal cerebral ischemia].

OBJECTIVE: To observe the influence of acupuncture intervention on expression of protein kinase A (PKAP in the cerebrocortex of rats with focal cerebral ischemia-reperfusion injury(CI/RI), so as to explore its underlying neuroprotection mechanism on cerebral ischemia. METHODS: Ninety male SD rats were randomly and evenly divided into sham-operation (sham), model and acupuncture groups which were further randomized into 3, 7 and 14-days (d) subgroups (10 rats/subgroup). CI/RI model was established by occlusion of the left middle cerebral artery for 2 hours and reperfusion. Manual acupuncture stimulation was applied to "Baihui" (GV 20) and "Shuigou" (GV 26), as well as the right "Quchi" (LI 11), "Hegu" (LI 4), "Neig (PC 6), "Zusanli" (ST 36),"Sanyinjiao"(SP 6) and "Taichong" (LR 3) for 30 min, once daily for 3, 7 and 14 d respectively. Rats of the sham and model groups were restrained for 30 min each day. Neurological defects were assessed by ethologic scoring according to Bederson's neurologic assessment scales. Cellular apoptosis in the ischemic cortex was detected by flow cytometry and PKA expression determined by immunohistochemistry for calculating its PKA immunoreaction (IR)-positive cell rate, respectively. RESULTS: Compared with the sham group, the neurologic scores, cortical cellular apoptosis rates and PKA IR-positive cell rates of the model group were significantly increased at the time-points of day 3, 7 and 14 post-treatment (P<0. 05). In comparison with the model group, the neurologic scores and cortical cellular apoptosis rates of the acupuncture group at the time-points of day 3, 7 and 14 post-treatment were considerably down-regulated (P<0. 05), and the cortical PKA IR-positive cell rates of the acupuncture group were remarkably increased (P<0. 05). In addition, along with the increase of acupuncture treatment sessions, lower apoptosis rates and more PKA IR-positive cells were found, suggesting a cumulative effect. CONCLUSION: Acupuncture intervention can lower cellular apoptosis rate of the ischemic cerebrocortex and up-regulate cortical PKA expression level in CI/RI rats, which may be responsible for its effect in improving neurologic deficits.