Objective To verify the performance of quantitative detection of interleukin-6 by using IMMUNITE1000 chemilumi-nescence analyzer.Methods According to the requirements of International Organization for Standardization(ISO)1 5 189,serum specimen were collected and levels of IL-6 were detected.The precision,accuracy,analytical measurement range,reportable range amd normal reference range of quantitative detection of interleukin-6 by using IMMUNITE1000 chemiluminescence analyzer were verified,and its performance was evaluated.Results The coefficient variation(CV)of between-day precision of high and low value was 6.42% and 1.97% respectively,and that of within-run precision was 3.40% and 3.82% respectively.Compared the test re-sults with the target values,the bias % was 0.91%.The regression equation:Y =0.986X - 7.1 (r 2 = 0.999,P < 0.05 ).With 27 times diluted,the recovery rate was from 97% to 100%,and the clinical reportable range was 2 to 27 000 pg/mL.The 95% refer-ence interval ranged from 0 to 5.3 pg/mL.Conclusion The performance of this system meets the manufacturer′s declaration,and could satisfy the quality requirements of clinical laboratory.

Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of β-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.

AIM:To investigate whether angiotensin-(1-7) [Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) acti-vation and necroptosis .METHODS:The expression levels of receptor-interacting protein 3 ( RIP3;an indicator of necrop-tosis) and TLR4 were determined by Western blot .Cell viability was measured by CCK-8 assay.The activity of lactate de-hydrogenase ( LDH) in the culture medium was measured with a commercial kit .The releases of interleukin-1β( IL-1β) and tumor necrosis factor-α( TNF-α) were measured by ELISA .The intracellular level of reactive oxygen species ( ROS) was analyzed by 2 ’ , 7 ’-dichlorfluorescein-diacetate ( DCFH-DA ) stating followed by photofluorography .Mitochondrial membrane potential ( MMP) was examined by rhodamine 123 staining followed by photofluorography .RESULTS:After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased . Co-treatment of the cells with 30μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG.Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100μmol/L necrostatin-1 ( Nec-1;a specific inhibitor of necroptosis ) and HG for 24 h attenua-ted the up-regulation of TLR4 expression induced by HG .Moreover, 1μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG.On the other hand, co-treatment of the cells with 1μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response , leading to the increase in the cell viability , and the decreases in the activity of LDH , ROS generation , MMP loss as well as the releases of IL-1βand TNF-α.CONCLUSION:Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR 4 activation and necroptosis .

Traditional processes are mostly adopted in traditional Chinese medicine (TCM) preparation production and the quality of products is mostly controlled by terminal. Potential problems of the production in the process are unpredictable and is relied on experience in most cases. Therefore, it is hard to find the key points affecting the preparation process and quality control. A pattern of research and development of traditional Chinese medicine preparation process based on the idea of Quality by Design (QbD) was proposed after introducing the latest research achievement. Basic theories of micromeritics and rheology were used to characterize the physical property of TCM raw material. TCM preparation process was designed in a more scientific and rational way by studying the correlation among enhancing physical property of raw material, preparation process and product quality of preparation. So factors affecting the quality of TCM production would be found out and problems that might occur in the pilot process could be predicted. It would be a foundation for the R&D and production of TCM preparation as well as support for the "process control" of TCMIs gradually realized in the future.
Drug Compounding ; methods ; standards ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; standards ; Humans ; Medicine, Chinese Traditional ; standards ; trends ; Quality Control ; Research ; standards ; trends ; Research Design ; standards ; Technology, Pharmaceutical ; instrumentation ; methods ; standards

Taxol is one of the most potent anti-cancer agents, which is extracted from the plants of Taxus species. Isopentenyl diphosphate isomerase (IPI) catalyzes the reversible transformation between IPP and DMAPP, both of which are the general 5-carbon precursors for taxol biosynthesis. In the present study, a new gene encoding IPI was cloned from Taxus media (namely TmIPI with the GenBank Accession Number KP970677) for the first time. The full-length cDNA of TmIPI was 1 232 bps encoding a polypeptide with 233 amino acids, in which the conserved domain Nudix was found. Bioinformatic analysis indicated that the sequence of TmIPI was highly similar to those of other plant IPI proteins, and the phylogenetic analysis showed that there were two clades of plant IPI proteins, including IPIs of angiosperm plants and IPIs of gymnosperm plants. TmIPI belonged to the clade of gymnosperm plant IPIs, and this was consistent with the fact that Taxus media is a plant species of gymnosperm. Southern blotting analysis demonstrated that there was a gene family of IPI in Taxus media. Finally, functional verification was applied to identify the function of TmIPI. The results showed that biosynthesis of β-carotenoid was enhanced by overexpressing TmIPI in the engineered E. coli strain, and this suggested that TmIPI might be a key gene involved in isoprenoid/terpenoid biosynthesis.
Amino Acid Sequence ; Carbon-Carbon Double Bond Isomerases ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; Paclitaxel ; biosynthesis ; Phylogeny ; Plant Proteins ; genetics ; Taxus ; enzymology ; genetics

Objective:To compare the modified Qjng Long Bai Wei needling method and ordinary acupuncture method in the effects of improving the levels of interleukin (IL)-1β,IL-6 and tumor necrosis factor (TNF)-α in the treatment of knee osteoarthritis (KOA),and to determine the advantage of the modified Qing Long Bai Wei needling method for KOA.Methods:One hundred KOA patients were randomized into a treatment group and a control group by using the random number table,with 50 cases in each group.The treatment group was intervened by the modified Qing Long Bai Wei needling method,and the control group was given ordinary acupuncture.The two groups were observed before and after the treatment to determine the changes in the levels of IL-1β,IL-6 and TNF-α in synovial fluid,and the clinical efficacies were compared between the two groups.Results:The total effective rate and clinical recovery rate were 97.9％ and 52.1％ respectively in the treatment group,versus 85.1％ and 25.5％ in the control group,and the between-group differences were statistically significant (both P＜0.01).After the treatment,the levels of Ib1β,IL-6 and TNF-cα in synovial fluid changed significantly in both groups (all P＜0.01);there were significant differences in the levels of IL-1β,IL-6 and TNF-α in synovial fluid between the two groups (all P＜0.01).Conclusion:The modified Qjng Long Bai Wei needling is an effective method for KOA and it can significantly improve the levels of IL-1β,IL-6 and TNF-α in synovial fluid.

Background Our previous study demonstrated that epigallocateehin-gallate(EGCG),an active ingredient of green tea,has protective effect on optical nerve after optic nerve crush.Astrocyte was proved to play key role in the repair of nerve tissue,but the influence of EGCG on astrocyte is unclear.Glial flbrillary acidic protein (GFAP) is a special marker for astrocyte. Objective The aim of this study was to investigate the effect of EGCG on the expression of GFAP in optic nerve tissue after optic nerve crush. Methods Seventy-two clean Wistar rats were randomly divided into normal control group,sham+EGCG group,optic nerve crush+normal saline group(vehicle group),optic nerve crush+EGCG group.Optic nerve crush models were established by clamping optical nerve for 60 seconds by minitype optic nerve clipper with the force of 40 gram.Only ocular tissue was cut in the rats in sham group.Normal saline solution or EGCG(25 mg/kg)was intraperitoneally injected daily for 5 days consecutively and orally administered(2 mg/kg)daily afterwards.The expression of GFAP in optic nerve was detected by immunohistochemistry and quantified by Western blotting analysis on day 7,14 and 28 after modeling. Results lmmunochemistry showed that GFAP were weakly expressed in the rats of both normal group and sham+EGCG group with the sliSht brown staining in optic nerve tissue.The deeply brown staining for GFAP was seen in vehicle group,and the staining intensity weakened in optic nerve crush+EGCG group compared to vehicle group on days 7,14 and 28 after modeling.Western blotting analysis revealed that the expression level of GFAP in rat optic nerve tissue of vehicle group was significantly enhanced in comparison with normal control group(P＜0.01).On day 7 and 14 after optic nerve modeling,the expression levels of GFAP were evidently decreased in optic nerve crush+EGCG group in comparison with vehicle group(P＜0.05).However,on day 28 after modeling,no significant difference wag found in the expression levels of GFAP between vehicle group and optic nerve erush+EGCG group(P＞0.05). Conclusion EGCG down-regulates optic nerve crush-induced of GFAP in the optic nerve and therefore attenuates the activity of astrocytes,suggesting that EGCG might reduce the formation of glial scar.

Background Researches demonstrated that epigallocatechin-3-gallate(EGCG) can protect retinal ganglion cells(RGCs) against damage induced by retinal ischemia-reperfusion and optic nerve crush(ONC),but the effect of EGCG on lateral geniculate nucleus(LGN) was under study.Objective This study was designed to detect neuroprotective effect of EGCG on LGN in the rat model with ONC.Methods Forty-eight 7-week-old female clean Wistar rats were randomly divided into normal control group,sham operation+EGCG group,ONC+normal saline(NS) group and ONC+EGCG group.ONC models were created by clamping the optical nerve for 60 seconds with the clipper with the force of 40 grams in the right eyes of 24 rats.The EGCG solution(25mg/kg) was intraperitoneally injected from 2 days before operation daily for 5 consecutively days and orally administered(2mg/kg) after that,and NS was used in the same way for ONC+NS group.Four weeks after ONC,the brain tissue of the rats was obtained,and the neurons of dorsal LGN(dLGN) were counted by Nissl staining under the light microscopy.The expression of neurofilament triplet L(NF-L) was detected by immunohistochemistry and Western blot analysis.Meanwhile,the neuronal nitric oxide synthase(nNOS) positive cells were counted.Results Compared with normal control group,no significant differences were found in neuron number both between sham operation+EGCG group or ipsilateral LGN of operative eyes in ONC+normal saline group and ONC+EGCG group(P=0.906,0.561,0.794,0.646 respectively) in 4 weeks after ONC,but loss of neurons in contralateral LGN in both ONC+normal saline group and ONC+EGCG group were observed(P=0.000,0.015 respectively).However,compared with ONC+normal saline group,the density of neurons in ONC+EGCG group was higher(P=0.007).Moreover,a higher expression level of NF-L protein was seen in ONC+EGCG group compared with ONC+normal saline group at contralateral LGN of operative eyes(P=0.002).Concerning the number of nNOS positive cells in LGN,there was no significant difference among normal control group,sham operation+EGCG group and ONC+EGCG group(P＞0.05).The number of nNOS positive cells in the contralateral LGN of operative eyes of ONC+normal saline group was higher than that of ONC+EGCG group(P=0.000).Conclusion EGCG plays the protective effect on LGN after ONC in rats through mediating the expression of nNOS.

This study examined the ability of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (β-PGG) to induce the expression of heme oxygenase-1 (HO-1) in the PC12 cells and its regulation in the PC12 cells. One week before treatment with the drug, nerve growth factor (NGF) was added to the cultures at a final concentration of 50 ng/mL to induce neuronal differentiation. After drug treatment, HO-1 gene transcription was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Expression of HO-1 and NF-E2-related factor2 (Nrf2) and activation of extracellular signal-regulated kinase (ERK) and Akt were detected by Western blotting. The viability of the PC12 cells treated with different medicines was examined by MTT assay. The oxidative stress in the PC12 cells was evaluated qualitatively and quantitatively by DCFH-DA. The results showed that β-PGG up-regulated HO-1 expression and this increased expression provided neuroprotection against MPP(+)-induced oxidative injury. Moreover, β-PGG induced Nrf2 nuclear translocation, which was found to be upstream of β-PGG-induced HO-1 expression, and the activation of ERK and Akt, a pathway that is involved in β-PGG-induced Nrf2 nuclear translocation, HO-1 expression and neuroprotection. In conclusion, β-PGG up-regulates HO-1 expression by stimulating Nrf2 nuclear translocation in an ERK- and Akt-dependent manner, and HO-1 expression by β-PGG may provide the PC12 cells with an acquired antioxidant defense capacity to survive the oxidative stress.

[ ABSTRACT] AIM:To investigate the role of ATP-sensitive potassium ( KATP ) channels in the inhibitory effect of hydrogen sulfide ( H2 S) on high glucose ( HG)-induced inflammation mediated by necroptosis in H 9c2 cardiac cells. METHODS:The expression levels of receptor-interacting protein 3 ( RIP3; an indicator of necroptosis ) and cyclooxyge-nase-2 (COX-2) were determined by Western blot.The levels of interleukin-1β(IL-1β) and tumor necrosis factor-α( TNF-α) were detected by ELISA .RESULTS:After H9c2 cardiac cells were treated with 35 mmol/L glucose ( HG) for 24 h, the expression of RIP3 was significantly increased .Pre-treatment of the cells with 100 μmol/L diazoxide ( DZ; a KATP channel opener) or 400 μmol/L NaHS (a donor of H2S) for 30 min considerably blocked the up-regulation of RIP3 induced by HG.Moreover, pre-treatment of the cells with 100 μmol/L 5-hydroxydecanoic acid (5-HD; a KATP channel
blocker) attenuated the inhibitory effect of NaHS on HG-induced up-regulation of RIP3.On the other hand, co-treatment of the cells with 100μmol/L necrostatin-1 ( a specific inhibitor of necroptosis ) or pre-treatment of the cells with 100 μmol/L DZ or 400 μmol/L NaHS attenuated HG-induced inflammatory responses , evidenced by decreases in the expression of COX-2 and secretion levels of IL-1βand TNF-α.However , pre-treatment of the cells with 100 μmol/L 5-HD significantly attenuated the above anti-inflammatory effects of NaHS.CONCLUSION:KATP channels play an important role in the inhib-itory effect of H2 S on HG-induced inflammation mediated by necroptosis in H 9c2 cardiac cells.