Effect on biosynthesis of terpenoid active components through RNA interference with MCT gene in Tripterygium wilfordii / 药学学报

MCT is an important key enzyme in the terpenoid biosynthesis in MEP pathway. In this study, Gateway technology was used to construct RNAi vector of TwMCT, and a vector fragment with a size of 484 bp was obtained. The TwMCT RNAi vector was transferred into the suspension cells of Tripterygium wilfordii by gene gun. Accumulation of terpenoids was assayed by UPLC, and the result showed that the content of triptolide and celastrol in cells decreased by 23.4% and 42.8%, respectively, compared with the control group pK7GWIWG2D. Moreover, the gene expression of TwMCT and major genes in terpenoid biosynthesis pathway was detected by qRT-PCR, which demonstrated that the expression of TwMCT reduced by 29.2% relative to that of the control group pK7GWIWG2D, and the relative expression of TwDXR, TwGGPS, TwHMGR and TwHMGS diminished by 36.3%, 31.3%, 62.2%, and 29.1%, respectively, but the expression of TwDXS was up-regulated by 114.2%, and there was no significant change in TwFPS. Thus, it was verified in vivo that interference with TwMCT expression significantly inhibited the accumulation of triptolide and celastrol in Tripterygium wilfordii, laying a foundation for further exploring the regulation mechanism of MCT gene on the terpenoid biosynthesis in Tripterygium wilfordii.

Clinical Observation of Tiao Shen Jie Jing Needling in Treating Post-stroke Upper-limb Spastic Palsy / 上海针灸杂志

Objective To observe the clinical efficacy of Tiao Shen Jie Jing needling (regulating the mind and release spasm) in treating post-stroke upper-limb spastic palsy. Method Ninety patients with post-stroke upper-limb spastic palsy were randomized into an observation group and a control group, 45 cases each. The observation group was intervened by Tiao Shen Jie Jing needling, while the control group was given ordinary acupuncture. The Modified Ashworth Scale (MAS), short-form Fugl-Meyer Assessment (SFMA) and Barthel Index (BI) were evaluated and recorded prior to the treatment, after 2-week and 4-week treatment and 3 months later after the intervention; the serumγ-aminobutyric acid (GABA) was detected before and after the treatment. Result The total effective rate was 83.3% in the observation group versus 61.0% in the control group, and the efficacy of the observation group was superior to that of the control group (P<0.05). The scores of each scale showed improvements after the treatment in both groups (P<0.05), and the improvements in the observation group were more significant (P < 0.05). The follow-up showed continuing improvements in the scales in the observation group, which were superior to those in the control group (P<0.05); after the treatment, the content of GABA in the observation group was significantly higher than that in the control group (P<0.05). Conclusion Tiao Shen Jie Jing needling can effectively ameliorate the upper-limb spasm,as well as the motor function of the upper extremities, improve the activities of daily living and maintain a long-term efficacy, better than ordinary acupuncture. The action mechanism is related to the regulation of GABA content in serum.

Cloning and protein expression of sterol-C-24-methyl transferase 2 in Tripterygium wilfordii / 药学学报

24-Alkyl sterols are the major players in the control of membrane component and plant growth. In this paper, we cloned an important rate-limiting enzyme:sterol-C-24-methyl transferase (SMT) in the sterol biosynthetic pathway according to the transcriptome data of Tripterygium wilfordii. suspension cells, whose full-length cDNA was 1 631 bp with an open reading frame of 1 080 bp, encoding a protein of 359 amino acids. It was estimated that theoretical isoelectric point (pI) was 6.43 and the molecular mass was 40.0 kDa. Bioinformatics analysis attributed the SMT gene to SMT2 family. The expression vector was constructed as the pMAL-c2x-TwSMT2 plasmid and the recombinant protein was expressed in E. coil BL21 (DE3) competent cells. After methyl jasmonate treatment, the relative expression level of TwSMT2 has improved significantly in 24 h. SDS-PAGE electrophoresis and Western Blot showed that protein of TwSMT2 in BL21 (DE3) strain was expressed after induction by IPTG. In this study, TwSMT2 was cloned for the first time and the recombinant protein was expressed, which lay the foundation for elucidation of the sterol biosynthetic pathway of Tripterygium wilfordii in the future.

Cloning and expression analysis of squalene synthase gene in Tripterygium wilfordii / 药学学报

In this paper, we cloned the full-length cDNA of TwSQS from Tripterygium wilfordii suspension cells (GenBank:KR401220) and performed the bioinformation and mRNA expression analysis. The expression after methyl jasmonate (MJ) treatment of the gene was detected by RT-PCR. The full-length cDNA of TwSQS was 1800 bp containing a 1242 bp open reading frame (ORF) encoding a polypeptide of 413 amino acids. The theoretical isoelectric point (pI) was 7.94 and the calculate molecular weight was about 47.20 kD. The relative expression level of TwSQS was deduced by MJ and reached the highest at 4 h after the treatment. The gene information we got in this study enriched the biosynthesis pathway of triterpenoids in Tripterygium wilfordii and laid foundation for further studies.

Tongxinluo Reverses the Hypoxia-suppressed Claudin-9 in Cardiac Microvascular Endothelial Cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Claudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported.</p><p><b>METHODS</b>In this study, HCMECs were stimulated with CoCl2to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs.</p><p><b>RESULTS</b>We found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription.</p><p><b>CONCLUSIONS</b>The results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.</p>

Cloning and bioinformatics analysis of geranylgeranyl diphosphate synthase gene of Tripterygium wilfordii / 中国中药杂志

A full-length cDNA of GGPPS gene from Tripterygium wilfordii suspension cells was obtained by use of RACE strategy (GeneBank: KM978333), and then analyzed by bioinformatics approaches. TwGGPPS cDNA has 1857 nucleotides and an open reading frame (ORF) encoding a protein of 514 amino acid residues. The deduced protein has isoelectric point (pI) of 7.85, a calculated molecular weight about 57.13 kD, 5 conserved domains and 2 functional domains. PSORT Prediction showed it was located at plasma membrane. Phylogenetic analysis demonstrated that TwGGPPS1 was similar to GGPPS from other species of plants. For the first time the cloning of geranylgeranyl diphosphate synthase gene from T. wilfordii was reported, it lays the foundation for further research of diterpenoids biosynthetic pathway.

Cloning and expression analysis of 4- (cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase gene in Tripterygium wilfordii / 中国中药杂志

4-(Cytidine-5-diphospho) -2-C-methyl-D-erythritol kinase is a key enzyme in the biosynthesis pathway of terpenoids. According to the transcriptome database, the specific primers were designed and used in PCR. The bioinformatic analysis of the sequenced TwCMK gene was performed in several bioinformatics software. The Real-time fluorescence quantification polymerase chain reaction (RT-qPCR) were used to detect the expression levels of TwCMK from T. wilfordii after elicitor MeJA supplied. The results showed that the full length of TwCMK cDNA was 1 732 bp encoding 387 amino acids. The theoretical isoelectric point of the putative TwCMK protein was 5.79 and the molecular weight was about 42.85 kDa. MeJA stimulated the rising of TwCMK expression in suspension cell and signally impacted at 24 h. The research provides a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Cloning and expression analysis of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene in Tripterygium wilfordii / 中国中药杂志

To clone the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (TwMCT) full length cDNA from Tripterygium wilfordii, the specific primers were designed according to the transcriptome data and the LCPCR were carried out. After a series of bioinformatics analysis on the TwMCT, the MeJA induced expression content were investigated by real-time fluorescence quantification polymerase chain reaction (RT-qPCR). The result showed that the full of TwMCTcDNA was 1 318 bp nucleotides encoding 311 amino acids. The molecular weight of the deduced TwMCT protein was about 34.14 kDa and the theoretical isoelectric point was 8.65. Result of the RT-qPCR analysis indicated that the content of TwMCT mRNA expression in T. wilfordii suspension cell was rising after treating with MeJA and reached the maximum in 24 h. Cloning and analyzing TwMCT gene from T. wilfordii provided gene element for studying the function and expression regulation of secondary metabolites.

Tongxinluo inhibits cyclooxygenase-2, inducible nitric oxide synthase, hypoxia-inducible factor-2α/vascular endothelial growth factor to antagonize injury in hypoxia-stimulated cardiac microvascular endothelial cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endothelial dysfunction is considered as the initiating process and pathological basis of cardiovascular disease. Cyclooxygenase-2 (COX-2) and prostacyclin synthase (PGIS), inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) are key enzymes with opposing actions in inflammation and oxidative stress, which are believed to be the major driver of endothelial dysfunction. And in hypoxia (Hx), Hx-inducible factor (HIF)-1α and HIF-2α are predominantly induced to activate vascular endothelial growth factor (VEGF), resulting in abnormal proliferation. Whether and how Tongxinluo (TXL) modulates COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF in Hx-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been clarified.</p><p><b>METHODS</b>HCMEC were treated with CoCl 2 to mimic Hx and the mRNA expressions of COX-2, PGIS, iNOS, eNOS, HIF-1α, HIF-2α, and VEGF were first confirmed, and then their mRNA expression and protein content as well as the cell pathological alterations were evaluated for TXL treatment with different concentrations. In addition, the effector molecular of inflammation prostaglandin E 2 (PGE 2 ) and the oxidative marker nitrotyrosine (NT) was adopted to reflect HCMEC injury.</p><p><b>RESULTS</b>Hx could induce time-dependent increase of COX-2, iNOS, HIF-2α, and VEGF in HCMEC. Based on the Hx-induced increase, TXL could mainly decrease COX-2, iNOS, HIF-2α, and VEGF in a concentration-dependent manner, with limited effect on the increase of PGIS and eNOS. Their protein contents verified the mRNA expression changes, which was consistent with the cell morphological alterations. Furthermore, high dose TXL could inhibit the Hx-induced increase of PGE 2 and NT contents, attenuating the inflammatory and oxidative injury.</p><p><b>CONCLUSIONS</b>TXL could inhibit inflammation-related COX-2, oxidative stress-related iNOS, and HIF-2α/VEGF to antagonize Hx-induced HCMEC injury.</p>

Effect of Huanshuai Recipe Oral Liquid (characters: see text) on renal dysfunction progression in patients with atherosclerotic renal artery stenosis / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of Huanshuai Recipe Oral Liquid ([characters: see text], HSR) on retarding the progression of renal dysfunction in patients with atherosclerotic renal artery stenosis (ARAS).</p><p><b>METHODS</b>A total of 52 ARAS patients with the Chinese medicine (CM) syndrome of qi deficiency and blood stasis, phlegm and dampness retention were recruited and randomly assigned into the treatment group (36 cases) and the control group (16 cases). Both groups received a basic treatment (high-quality low-protein diet, blood pressure control, lipid-lowering, correcting the acidosis, etc.). In addition, the treatment group received 20 mL HSR and the control group received placebo, 3 times a day for 6 months. Renal function (serum creatinine, blood urea nitrogen and uric acid) and blood lipids (cholesterol, triglycerides and low density lipoprotein) were examined monthly. The estimated glomerular filtration rate (eGFR) and CM syndrome score were compared between groups.</p><p><b>RESULTS</b>After treatment, compared with the control group, the serum creatinine level, uric acid level and CM syndrome score of the treatment group were significantly decreased (P<0.05 or P<0.01), and the eGFR in the treatment group were significantly increased (P<0.05).</p><p><b>CONCLUSION</b>HSR can effectively improve the renal function and clinical symptoms of ARAS patients.</p>

Medicinal plant hairy roots generating and their applications / 中国中药杂志

As a kind of the plant tissue cultures, hairy root culture is characterized by rapid growth without exogenous hormones source and high yield of secondary metabolites, which attracted the attention of scholars in resent years. This work systematically summarized the research of medicinal plant hairy roots, including the mechanism, current situation of medicinal plant hairy roots, and their applications.

Study on effect of combination of Epimedii Folium and Ligustri Lucidi Fructus on osteoporosis rats induced by retinoic acid / 中国中药杂志

<p><b>OBJECTIVE</b>To explore the effect of combination of Epimedii Folium and Ligustri Lucidi Fructus on osteoporosis rats induced by retinoic acid.</p><p><b>METHOD</b>Sixty three-month-old male Wistar rats were randomly divided into the normal control group, the model group, the Epimedii Folium group, the Ligustri Lucidi Fructus group, the combination group of Epimedii Folium and Ligustri Lucidi Fructus and the raloxifene group. The osteoporosis model was established through oral administration with retinoic acid for two weeks. Meanwhile, all of treatment groups were administered with corresponding drugs for three weeks. The contents of serum calcium (Ca), phosphorus (P), alkaline phosphatase (AKP) and tartrate-resistant acid phosphatase (StrACP) were detected, and the pathomorphological changes of femurs were observed.</p><p><b>RESULT</b>The model control group showed much lower contents of serum Ca and P than the normal control group, but with significantly higher AKP and StrACP activity than the normal control group. The femoral head area showed reduced, narrow and sparse trabecular bones, with typical osteoporosis-like changes. Compared with the model control group, all of treated groups showed significant increase in Ca and P contents in serum, and down-regulate AKP and StrACP levels, while trabecular bones became more and wider, and densely interweaved as a reticular formation. Among them, the combination group showed the most significant effect.</p><p><b>CONCLUSION</b>Epimedii Folium and Ligustri Lucidi Fructus could effectively correct the abnormal bone metabolism and improve pathological conditions of bone tissues, so as to show the anti-osteoporosis effect. The combined application of the two drugs showed a better efficacy.</p>

The body weight loss during acute exposure to high-altitude hypoxia in sea level residents / 生理学报

Weight loss is frequently observed after acute exposure to high altitude. However, the magnitude and rate of weight loss during acute exposure to high altitude has not been clarified in a controlled prospective study. The present study was performed to evaluate weight loss at high altitude. A group of 120 male subjects [aged (32±6) years] who worked on the construction of the Golmud-Lhasa Railway at Kunlun Mountain (altitude of 4 678 m) served as volunteer subjects for this study. Eighty-five workers normally resided at sea level (sea level group) and 35 normally resided at an altitude of 2 200 m (moderate altitude group). Body weight, body mass index (BMI), and waist circumference were measured in all subjects after a 7-day stay at Golmud (altitude of 2 800 m, baseline measurements). Measurements were repeated after 33-day working on Kunlun Mountain. In order to examine the daily rate of weight loss at high altitude, body weight was measured in 20 subjects from the sea level group (sea level subset group) each morning before breakfast for 33 d at Kunlun Mountain. According to guidelines established by the Lake Louise acute mountain sickness (AMS) consensus report, each subject completed an AMS self-report questionnaire two days after arriving at Kunlun Mountain. After 33-day stay at an altitude of 4 678 m, the average weight loss for the sea level group was 10.4% (range 6.5% to 29%), while the average for the moderate altitude group was 2.2% (-2% to 9.1%). The degree of weight loss (Δ weight loss) after a 33-day stay at an altitude of 4 678 m was significantly correlated with baseline body weight in the sea level group (r=0.677, P<0.01), while the correlation was absent in the moderate altitude group (r=0.296, P>0.05). In the sea level subset group, a significant weight loss was observed within 20 d, but the weight remained stable thereafter. AMS-score at high altitude was significantly higher in the sea level group (4.69±2.48) than that in the moderate altitude group (2.97±1.38), and was significantly correlated with baseline body weight. These results indicate that (1) the person with higher body weight during stay at high altitude loses more weight, and this is more pronounced in sea level natives when compared with that in moderate altitude natives; (2) heavier individuals are more likely to develop AMS than leaner individuals during exposure to high-altitude hypoxia.

CT Findings of an Ectopic Pancreas in the Anterior Mediastinum

We report here on a rare case of an ectopic pancreatic tissue in the anterior mediastinum. A 32-year-old woman without any symptoms was transferred to our hospital because of an abnormal large mediastinal shadow on her chest radiograph during a checkup. The computed tomography (CT) scan revealed a giant cystic-solid mass that measured 16 x 13 x 8 cm and it was located in the center of the anterior mediastinum and it symmetrically grew to two sides. On enhanced CT scans, the solid component of the mass showed marked enhancement. We performed total surgical resection of the mass and complete pancreatic tissues were verified on the pathological examination.

Ecological Significance and Processes in Research of the Viable but Nonculturable State in Bacteria / 微生物学通报

The viable but noncuturable (VBNC) state is a survival strategy when bacteria are exposed to en- vironment stress. The VBNC state is part of the life cycle of non-differentiating bacteria, and it has a far-reaching impact on traditional bacteriology. Cells in the VBNC state fail to grow on the routine bacterio- logical media, here its significance in human health and environment science are detailed. Cells entering the VBNC state exhibit dwarfing and a number of metabolic changes in respiration rates and macromolecular synthesis. This paper summarized the variations in DNA and protein comparing to the culturable cells. We also discussed the ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form. Some new methods for monitoring the VBNC state were listed. Finally the future was suggested.

Evaluation of CYRTA21-1, CEA and SPECT in diagnosis of bone metastasis in lung cancer / 肿瘤研究与临床

Objective To evaluate the value of CYRTA21-1,CEA and SPECT in diagnosis of bone metastasis in lung cancer.Methods 216 patients with lung cancer were examined by radionuclide bone imaging and serum CYFRA21-1,CEA.20 patients with benign lung disease were also studied.Results 121 of the 216 lung cancer patients were confirmed bone metastasis including 101 multi-metastasis and 20 singu- lar-metastasis. The positive rate was 56.02 %.The serum CYFR21-1,CEA level and positive rate of lung cancer patients with bone metastasis were obviously higher than those of patients without bone metastasis or with benign lung disease.The level of serum CYFR21-1,CEA had significant difference(P

Measurement of Serum Interleukin-16,-10 and Immunoglobulin E in Asthmatic Children and Its Significance / 实用儿科临床杂志

Objective To discuss the level and its relativity analysis of IL-16 and IgE in asthmatic children .Methods Compare and analyze the level of IL-16,IL-10 and IgE between 18 asthmatic children and 14 healthy children.Results The serum level of IL-16 and IgE in asthmatic children was much higher than the contrast, P