OBJECTIVE:To establish a method for content determination of schizantherin E,gomisin J,angeloylgomisin H,schisantherin A,schisantherin B,schisanhenol,anwuligan,schizandrin A,schizandrin B and schizandrin C in Wuzhi tablets.METHODS:RP-HPLC method was adopted.The determination was performed on Symmetry C18 column with acetonitrile-0.1％ phosphoric acid solution (gradient elution) at the flow rate of 1.0 mL/min.The detection wavelength was set 225 rm,and the column temperature was 30 ℃.The sample size was 10 μL.RESULTS:The linear ranges of schizantherin E,gomisin J,angeloylgomisin H,schisantherin A,schisantherin B,schisanhenol,anwuligan,schizandrin A,schizandrin B and schizandrin C were 2.25-67.5ng(r=0.999 6),2.1-63 ng(r=0.999 8),28-840 ng(r=0.999 9),124.6-3 738 ng(r=0.999 9),22.7-681 ng(r=0.999 9),32.7-981 ng(r=0.999 9),47-1 410 ng(r=0.999 9),208-6 240 ng(r=0.999 9),5.36-160.8 rig(r=0.999 9),4.48-134.4 ng(r=0.999 8).The limits of quantitation were 14.17,13.32,9.33,11.37,14.62,19.88,14.66,12.50,16.40,13.55 rg.The limits of detection were 4.62,4.60,3.08,3.76,4.81,6.74,4.93,4.16,5.86,5.03 ng.RSDs of precision,stability and reproducibility tests were less than 3.0％;the recoveries were 96.36％-100.00％(RSD=1.83％,n=6),95.00％-100.00％(RSD=2.07％,n=6),95.00％-98.00％(RSD=1.22％,n=6),95.37％-98.91％ (RSD=1.29％,n=6),95.62 ％-103.71％ (RSD=2.85％,n=6),97.33％-102.67％ (RSD=2.00％,n=6),95.00％-99.33％ (RSD=1.75％,n=6),97.24％-104.93％ (RSD=2.63％,n=6),95.00％-97.50％ (RSD=1.42％,n=6),96.00％-102.00％ (RSD=2.45％,n=6),respectively.CONCLUSIONS:The developed method is accurate,sensitive and reproducible,and it can be used for content determination of 10 lignanoids in Wuzhi tablets.

OBJECTIVEto determine the effects of survivin antisense oligonucleotide (ASODN) on the expression levels of survivin mRNA and human mucoepidermoid carcinoma cell line (highly metastatic Mc3) apoptosis and to explore the feasibility of survivin gene as the mucoepidermoid carcinoma therapeutic targets.

METHODSthe survivin ASODN was designed and synthesized and then respectively transfected into Mc3 cells. The morphological changes of the Mc3 cells were observed 24, 48 and 72 h after transfection by inverted microscope and the apoptosis rate detected by flow cytometry. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the effect of the transfection on cell poliferation, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method for analysis of apoptotic index, and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to detect the expression of survivin.

RESULTSin survivin ASODN transfection group, there was less Mc3 cells than in other groups. The suspended cells dropping from the wall increased and showed typical apoptotic changes and time-dependent. Mc3 cell apoptosis rate in survivin ASODN transfection group transfected for 24, 48, 72 h was 12.96%, 14.43%, 22.69%, respectively, which were significantly higher than in other groups (P < 0.01) and time-dependent (P < 0.05). The inhibitory rate of Mc3 cells in survivin ASODN transfection group was 22.35%, 39.04%, 43.46%, which were significantly higher than other groups (P < 0.01) and time-dependent (P < 0.05). The apoptosis index (AI) of Mc3 cells in survivin ASODN transfection group was 11.038%, 12.172%, 18.900%, significantly higher than other groups (P < 0.01) and time-dependent (P < 0.05). The survivin mRNA levels in Mc3 cells were 0.739 ± 0.008, 0.668 ± 0.007, 0.500 ± 0.006, and the relative inhibition rate in these cells was 18.21%, 26.06%, 44.82%, significantly lower than other groups (P < 0.01) and time-dependent manner (P < 0.01).

CONCLUSIONSsurvivin ASODN could inhibit the proliferation of Mc3 cells and induce the apoptosis of Mc3 cells. It also can inhibit the expression of survivin mRNA. Survivin can be used as a gene therapy targets for mucoepidermoid carcinoma.

Apoptosis ; Carcinoma, Mucoepidermoid ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Gene Targeting ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; Microtubule-Associated Proteins ; Neoplasm Proteins ; Oligonucleotides, Antisense ; RNA, Messenger ; Salivary Gland Neoplasms ; metabolism ; Transfection

This study aimed to report the clinical characteristics and COMP gene mutation of a family with pseudoachondroplasia (PSACH), a relatively rare spinal and epiphyseal dysplasia that is inherited as an autosomal dominant trait. Clinical information on a 5-year-2-month-old PSACH child and his parents was collected and analyzed. Diagnosis was confirmed by PCR amplification and direct sequencing of all the 19 exons and their flanking sequences of COMP gene, and the mutation was further ascertained by cloning analysis of exon 10. The child presented with short and stubby fingers, bow leg, short limb dwarfism and metaphysic broadening in long bone as well as lumbar lordosis. A mutation c.1048_1116del (p.Asn350_Asp372del) in exon 10, inherited from his father who did not demonstrate any phenotypic feature of PSACH, was detected in the child. PSACH was diagnosed definitively by means of COMP mutation analysis, on the basis of the child's clinical and imaging features. The non-penetrance phenomenon of COMP mutation was described for the first time in PSACH.
Achondroplasia ; genetics ; Cartilage Oligomeric Matrix Protein ; genetics ; Child, Preschool ; Cloning, Molecular ; Humans ; Male ; Mutation

Aim To investigate the impact and mechanism of Weichang'an Pill(WCA),its ethanol extract(EE),water extract(WE),and active ingredients on the contraction of isolated rat ileum smooth muscles induced by acetylcholine(ACh). Methods In vitro tissue bath experiment,WCA,EE,WE,or their active ingredients were added under the action of ACh,and then the contraction tension of isolated ileum smooth muscle from rats was recorded. The binding affinity ofthe active ingredients to the muscarinic acetylcholine M3 receptor was explored by molecular docking. Results WCA,EE,and WE were able to considerably inhibit the excitatory contraction of the ileal smooth muscles induced by ACh. Costunolide,dehydrocostus lactone,santalol,muscone,emodin,chrysophanol,physcion,crotonoside,magnolol,and honokiol were also significantly effective against ACh-induced ileal smooth muscle contraction. Conclusions WCA,EE,WE,and their active ingredients may help to promote intestinal smooth muscle relaxation by blocking the binding of the M3 receptor on the membrane of ileal smooth muscle with ACh.

ObjectiveFrom the perspective of energy metabolism， the mechanism of Osteoking （OK） in the treatment of myofascial pain syndrome （MPS） was revealed through systems biology prediction combined with holistic animal experimental validation methods. MethodFirstly， the key targets of MPS and their related molecular mechanisms were predicted by the systems biology method， and the core network targets were screened. Then， the network-predicted targets were verified by animal experiments. Specifically， 60 SD rats were randomly divided into normal group， model group， low， medium， and high dose OK groups （0.66， 1.31， 2.63 mL·kg^-1）， and positive celecoxib group （21 mg·kg^-1）. The MPS model was established by beating combined with a centrifugal exercise method for eight weeks. Except for two days after modeling， the intervention of OK or celecoxib was performed. After the completion of the model， the drug was administered for two weeks. The histopathological changes of trigger point muscle tissue were observed by hematoxylin-eosin staining. The content/activity of Na-K-ATP enzyme （Na⁺-K⁺-ATPase）， Ca²⁺ pump （Ca²⁺ATPase）， Ca²⁺， lactate dehydrogenase （LDH）， glutathione （GSH）， malondialal （MDA）， superoxide dismutase （SOD）， cyclic adenosine phosphate （cAMP）， and protein kinase A （PKA） in serum and/or trigger point muscle tissue in MPS rats was detected by enzyme-linked immunosorbent assay. Protein expression levels of PKA and the peroxisome proliferator-activated receptor γ coactivator 1α （PGC1α） in MPS rats were detected by immunohistochemistry. The protein expression levels of PKA， PGC1α， and mitochondrial transcription factor A （TFAM） in MPS rats were detected by Western blot. ResultThe network prediction results suggest that OK acts on the key target of energy metabolism related to the occurrence and development of MPS and may participate in the activation of the cAMP/PKA/PGC1α signaling pathway. The experimental validation results show that compared with the normal group， contracture nodules and disordered arrangement of muscle fibers appear in the trigger point muscle tissue of MPS rats. Na⁺-K⁺-ATPase， Ca²⁺ATPase， SOD activity， Ca²⁺， and GSH contents in serum and/or trigger point muscle tissue are significantly decreased （P<0.01）. Both LDH activity and MDA contents are significantly increased （P<0.01）， and the protein expression levels of cAMP， PKA， PGC1α， and TFAM are significantly decreased （P<0.01）. Compared with the model group， OK improves the histopathological morphology of trigger point muscle fibers in MPS rats， and after the intervention of OK， Na⁺-K⁺-ATPase， Ca²⁺ATPase， SOD activity， Ca²⁺， and GSH contents in serum and/or trigger point muscle tissue in MPS rats are significantly increased （P<0.05， P<0.01）. LDH activity and MDA contents are significantly reduced （P<0.05， P<0.01）. The protein expression levels of cAMP， PKA， PGC1α， and TFAM are significantly increased （P<0.05， P<0.01）. ConclusionThe mechanism of OK's intervention in MPS rats may be related to its effective activation of the cAMP/PKA/PGC1α signaling pathway， thus promoting mitochondrial energy metabolism and trigger point muscle fiber damage repair in muscle cells.