Effect of Danhong Injection on platelet metabolites based on metabonomics technology / 中国中药杂志

The effect of Danhong Injection on the endogenous metabolites of rabbit platelets was analyzed by the liquid chromatography-mass spectrometry( LC-MS) based metabonomic approach. Anti-platelet aggregation was detected after Danhong Injection treatment and the changes of platelet metabolites were analyzed by metabonomics. Principal component analysis( PCA) and partial least squares discriminant analysis( PLS-DA) were performed to investigate the effect of Danhong Injection on endogenous metabolites of platelets,characterize the biomarkers,and explore the relevant pathways and the underlying mechanism. As demonstrated by the pharmacodynamic results,Danhong Injection of different doses and concentrations antagonized platelet aggregation in a dose-and concentration-dependent manner. In contrast to the control group,25 differential metabolites such as nicotinic acid,nicotinic acid riboside,and hypoxanthine were screened out after platelets were treated by Danhong Injection. These metabolites,serving as important biomarkers,were mainly enriched in the nicotinic acid-niacinamide metabolic pathway and purine metabolic pathway. This study explored the therapeutic mechanism of Danhong Injection from a microscopic perspective by metabonomics,which is expected to provide a new idea for the investigation of platelet-related mechanisms.

Analysis and comparison of metabolic processes of salvianolic acid A and salvianolic acid B in rats based on UHPLC-LTQ-Orbitrap MS technology / 中国中药杂志

The metabolites of salvianolic acid A and salvianolic acid B in rats were analyzed and compared by ultra-high-perfor-mance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS). After the rats were administrated by gavage, plasma at different time points and urine within 24 hours were collected to be treated by solid phase extraction(SPE), then they were gradient eluted by Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) and 0.1% formic acid solution(A)-acetonitrile(B) mobile phase system, and finally all biological samples of rats were analyzed under negative ion scanning mode. By obtaining the accurate relative molecular mass and multi-level mass spectrometry information of metabolites, combined with the characteristic cleavage law of the reference standard and literature reports, a total of 30 metabolites, including salvianolic acid A and B, were identified. Among them, there were 24 metabolites derived from salvianolic acid A, with the main metabolic pathways including ester bond cleavage, dehydroxylation, decarboxylation, hydrogenation, methylation, hydroxylation, sulfonation, glucuronidation, and their multiple reactions. There were 15 metabolites of salvianolic acid B, and the main biotransformation pathways were five-membered ring cracking, ester bond cleavage, decarboxylation, dehydroxylation, hydrogenation, methylation, sulfonation, glucuronidation, and their compound reactions. In this study, the cross-metabolic profile of salvianolic acid A and B was elucidated completely, which would provide reference for further studies on the basis of pharmacodynamic substances and the exploration of pharmacological mechanism.

Analysis of carnosic acid metabolites in rats by UHPLC-Q-Exactive MS / 中国中药杂志

A method of ultra-high performance liquid chromatography coupled with quadrupole/electrostatic field Obitrap high-resolution mass spectrometry(UHPLC-Q-Exactive MS) was established to comprehensively identify the metabolites of carnosic acid in rats. After oral gavage of carnosic acid CMC-Na suspension in rats, urine, plasma and feces samples were collected and pretreated by solid phase extraction(SPE). Acquity UPLC BEH C_(18 )column(2.1 mm×100 mm, 1.7 μm) was used with 0.1% formic acid solution(A)-acetonitrile(B) as the mobile phase for the gradient elution. Biological samples were analyzed by quadrupole/electrostatic field Obitrap high-resolution mass spectrometry in positive and negative ion mode. Based on the accurate molecular mass, fragment ion information, and related literature reports, a total of 28 compounds(including carnosic acid) were finally identified in rat samples. As a result, the main metabolic pathways of carnosic acid in rats are oxidation, hydroxylation, methylation, glucuronide conjugation, sulfate conjugation, S-cysteine conjugation, glutathione conjugation, demethylation, decarbonylation and their composite reactions. The study showed that the metabolism of carnosic acid in rats could be efficiently and comprehensively clarified by using UHPLC-Q-Exactive MS, providing a reference for clarifying the material basis and metabolic mechanism of carnosic acid.

Bioassay method for Compound Danshen Tablets based on a platelet aggregation inhibition test / 药学学报

A bioassay method for inhibiting platelet aggregation in vitro was established to quantify the pharmacological effects of Compound Danshen Tablets and support its quality control. The inhibition of platelet aggregation in rabbit plasma in vitro by Compound Danshen Tablets was used as the experimental system. The titer was calculated by using the method of dose-response parallel lines. As a result, a bioassay for the inhibition of platelet aggregation in vitro by Compound Danshen Tablets was established. Linearity was good in the concentration range of 0.128 g·mL-1 to 0.205 g·mL-1, and the titer of standard Compound Danshen tablets was 7 659 U·g-1 according to the titer definition. This in vitro assay was simple, reliable, reproducible and convenient. The activity of Compound Danshen Tablets in inhibiting platelet aggregation was quantified by potency assay and the quality of different batches was evaluated. The method can be applied for the quality control of Compound Danshen Tablets.

Research progress in small molecule drugs based on high-density lipoprotein for anti-atherosclerosis / 药学学报

A large number of epidemiological data have shown that the high-density lipoprotein cholesterol level is negatively related to atherosclerotic cardiovascular disease, suggesting that high-density lipoprotein may have the effect of anti-atherosclerosis. It may play the role of anti-atherosclerosis, through the promotion of cholesterol reverse transport, anti-inflammatory, antioxidant, and against thrombosis and fibrinolysis and so on. Among them, reverse cholesterol transport which is mainly regulated by apolipoprotein A-I, ATP-binding cassette transporter 1, liver X receptor and cholesteryl ester transfer protein, may play a major role in the maintenance of cholesterol homeostasis and reversing the course of atherosclerosis. These regulatory factors may be potential targets in high density lipoprotein-based drug discovery. In this review, these key proteins are discussed for the current status of small molecule drugs against atherosclerosis.

High glucose suppresses the corneal limbal stem cell migration and changes the cell phenotype / 眼科新进展

Objeetive To explore the influence of extracellular high glucose on the proliferation,migration and biomarkers of corneal limbal stem cells.Methods Establishment of a model of high glucose in cultured human limbal stem cells to observe and investigate the effects of extracellular high glucose on the proliferation and migration of corneal limbal stem cells by immunoflurescence,CCK-8 and Transwell assay,respectively.Totally 16 SPF rats were collected and induced diabetic model by streptozotocin as the high-glucose group,and the normal rats of the same age served as the control group.Corneal epidermises of rats in both groups were scraped to observe the repair of corneal epithelium.And the corneas were treated with HE staining and immunohistochemical staining to detect the expression of biomarkers of corneal limbal stem cells and the modality changes of cells.Results The proliferation rate of human limbal epithelial cells was significantly decreased when exposed to high glucose,and the rate at 24 h,48 h and 72 h was 0.728,0.345 and 0.395,respectively,which was markedly lower than that in the control group,with a significant difference (P ＜ 0.05);meanwhile the cell migration rate of the high-glucose group was 17.6％ at 48 h,which was significantly slower than that of the control group (100％).And the inhibition was accompanied by the decreased expression of β-catenin and vimentin.Furthermore,the expression levels of β-catenin and vimentin mRNA and protein were down-regulated,with abnormal location,in the high-glucose group.And diabetic rats had poor corneal epithelial healing.The epithelial layer became thinner and the structures were disorganized in diabetic rats through HE staining.The immunohistochemical assay revealed the expression of β-catenin and vimentin of cornea limbal stem cells was down-regulated in high-glocose group when compared with the control group.Conclusion High glucose can significantly inhibit the proliferation and migration of cornea limbal stem cells,and its main damage mechanism is correlated with the abnormalities of β-catenin and vimentin.

P2X7 receptor and inflammatory bowel disease / 生理学报

P2X7 receptors are closely associated with inflammation, and they have been found to be expressed on colonic cells broadly. In animal model of colonic inflammation, ATP/P2X7 signaling mainly promotes inflammation, and a variety of cells, including macrophages, dendritic cells, T cells, mast cells and enteric neurons are involved in this process. However, in the toxoplasmic ileitis, P2X7 signaling plays a role in inhibiting the inflammation. But, the underlying mechanisms are still not clear. This review outlined the research progresses of P2X7 receptors in inflammatory bowel disease (IBD) to provide some clues for the further studies on the relationship between P2X7 receptors and IBD.

Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice / 生理学报

The study was aimed to investigate the changes in mechanical pain threshold in the condition of chronic inflammatory pain after transient receptor potential vanilloid 1 (TRPV1) gene was knockout. Hind-paw intraplantar injection of complete freund's adjuvant (CFA, 20 μL) produced peripheral inflammation in wild-type and TRPV1 knockout female mice. The mechanical pain thresholds were measured during the 8 days after injection and pre-injection by using Von-Frey hair. Nine days after injection, mice were killed and the differences of expression of c-Fos and P2X3 receptor in the dorsal root ganglia (DRG) and spinal cord dorsal horn were examined by Western blotting between the two groups. Compared with that in wild-type mice, the mechanical pain threshold was increased significantly in TRPV1 knockout mice (P < 0.05); 3 days after CFA injection, the baseline mechanical pain threshold in the TRPV1 knockout mice group was significantly higher than that in the wild-type mice group (P < 0.05); The result of Western blotting showed that the expression of c-Fos protein both in DRG and spinal cord dorsal horn of TRPV1 knockout mice group was decreased significantly compared with that in wild-type mice group (P < 0.01, P < 0.05), while the expression of P2X3 receptor in DRG of TRPV1 knockout mice group was increased significantly compared with that in wild-type mice group (P < 0.05). Our findings indicate that TRPV1 may influence the peripheral mechanical pain threshold by mediating the expression of c-Fos protein both in DRG and spinal cord dorsal horn and changing the expression of P2X3 receptor in DRG.

BK(Ca) channel agonist NS1619 and Kv channel antagonist 4-AP on the facial mechanical pain threshold in a rat model of chronic constriction injury of the infraorbital nerve / 生理学报

Trigeminal neuralgia is a paroxysmal disorder with severely disabling facial pain and thus continues to be a real therapeutic challenge. At present there are few effective drugs for treatment of this pain. The present study was aimed to explore the involvement of BK(Ca) channels and Kv channels in the mechanical allodynia in a rat model of trigeminal neuropathic pain. Here the effectiveness of drug target injection at the trigeminal ganglion through the infraorbital foramen was first evaluated by immunofluorescence and animal behavior test. Trigeminal neuropathic pain model was established by chronic constriction injury of the infraorbital nerve (ION-CCI) in rats. BK(Ca) channel agonist and Kv channel antagonist were administered into the trigeminal ganglion in ION-CCI rats and sham rats by the above target injection method, and the facial mechanical pain threshold was measured. The results showed that the drug could accurately reach the trigeminal ganglion by target injection which was more effective than that by the normal injection around infraorbital foramen. Rats suffered significant mechanical allodynia in the whisker pad of the operated side from 6 d to 42 d after ION-CCI. BK(Ca) channel agonist NS1619 significantly and dose-dependently attenuated the facial mechanical allodynia and increased the facial mechanical pain threshold in ION-CCI rats 15 d after operation. Kv antagonist 4-AP was able to reduce the threshold in ION-CCI rats when facial mechanical threshold was partly recovered and relatively stable on the 35th day after operation. These results suggest that BK(Ca) channel agonist NS1619 and Kv channel antagonist 4-AP can significantly affect the rats' facial mechanical pain threshold after ION-CCI. Activation of BK(Ca) channels may be related to the depression of the primary afferent neurons in trigeminal neuropathic pain pathways. Activation of Kv channels may exert a tonic inhibition on the trigeminal neuropathic pain.

Study an E protein epitopes and primary identification of main yellow virus / 中华流行病学杂志

Objective To analysis the E protein epitopes of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus and to distinguish the shared or specific epitopes among them. Methods Bioinformatic software DNAStar was used to analyze the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E prtein amino acid sequences. The influence of secondary structure was also considered. Based on the bio-informatic analysis of E protein epitopes, 6 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-c2x. The vectors was then transferred into E.coli BL21 (DE3) and Rosetta (DE3). Isopropyl-β-D-thiogalactoside (IPTG) was used to induce the expression of gene segments and SDS-PAGE were used identify the expression proteins. The antigenieity was tested, using Western blot. Results 15 shared epitopes and 47 specific epitopes were forecasted by bioinformatic analysis, and 6 specific epitopes from dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E protein were expressed in E.coli successfully. Two specific antigenic determinant from dengue virus type 1 and dengue virus type 2 were confirmed using Western blot, while the others epitopes shown no antigenic reaction property. Conclusion Two specific antigenic determinant were confirmed, under Western blot.

Changes in voltage-gated potassium currents in the trigeminal neurons after a chronic constriction of infraorbital nerve / 生理学报

The purpose of this study was to establish a model of trigeminal neuralgia (TN) through an approach from lower edge of cheekbone and to observe the functional changes in the voltage-gated potassium currents in the cultured trigeminal ganglion (TG) neurons. Thirty Sprague-Dawley male rats were divided into two groups, the sham-operated (sham) group and the operated group. The TN model was carried out by using a chronic constriction injury of the infraorbital nerve (ION-CCI) from lower edge of cheekbone. Peripheral pain threshold test and whole-cell patch clamp recording were used to determine the difference between sham and ION-CCI rats. The withdrawal threshold of whisker pad in operated side of ION-CCI rat was decreased significantly from 6 d after operation and then maintained until 21 d, with the lowest on the 15th day. The threshold of whisker pad in non-operated side of operated rats was also decreased significantly compared with that in the sham group. Delayed rectifier potassium current (I(K)) in cultured ION-CCI TG neurons was decreased significantly compared with that in the sham group. Transient outward potassium currents (I(A)) in both operated and non-operated sides of TG neurons from ION-CCI rats were also reduced significantly compared with that in the sham group. The present study provided a new method of ION-CCI. In this model, the decrease of I(A) and I(K) might contribute, at least in part, to the decrease in mechanical pain threshold of whisker pad and the subsequent hyperalgia.

Role of glutamate receptors in the spiral ganglion neuron damage induced by acoustic noise / 生理学报

The aim of the present study was to investigate the role of glutamate receptors in the damage of spiral ganglion neurons (SGNs) induced by acute acoustic noise. This investigation included in vivo and in vitro studies. In vivo, kynurenic acid (KYNA), a broad-spectrum antagonist of glutamate receptors, was applied to the round window of guinea pigs, and its protective effect was observed. The animals were divided into three groups: control (saline, 0.9%, 10 microL), saline (0.9%, 10 microL) + noise and KYNA (5 mmol/L, 10 microL) + noise. Saline and KYNA were applied to the round window membrane with a microsyringe. The animals were exposed to 110 dB SPL of white noise for 1 h. Hearing thresholds for auditory brainstem responses (ABRs) and compound action potentials (CAPs) in all animals were measured before and after treatment. The amplitudes of III waveform of ABR and N1 waveform of CAP and the latency of N1 waveform at different stimulation levels (intensity-amplitude and intensity-latency functions) were also measured. The cochleas were then dissected for transmission electron microscopy (TEM) after final electrophysiological measurement. In vitro, the SGNs of the normal guinea pigs were isolated and glutamate (100 micromol/L or 1 000 micromol/L) was added into the medium. The morphology of the SGNs was examined by light microscopy. In vivo results showed that the hearing function and morphology of the inner ear including hair cells and SGNs in the control group were normal. Compared with that in the control group the thresholds for ABR and CAP (click and tone burst) in saline + noise group were elevated significantly. The input-output functions showed that the amplitudes of III waveform of ABR and N1 waveform of CAP decreased and the latency of N1 waveform increased obviously. There was significant difference in the amplitude and latency between saline + noise group and KYNA + noise group (P<0.05). TEM indicated obvious swelling and vacuoles at the terminate of dendrites of SGNs in NS + noise group. On the contrary, the afferent dendrites in KYNA + noise group showed normal appearance without swelling and vacuoles. In vitro experiment showed that the isolated SGNs of guinea pigs obviously swelled and even died after application of 100 micromol/L or 1 000 micromol/L glutamate. These results suggest that noise exposure causes hearing impairment, damage of hair cells and hair cell/afferent synapse and death of SGNs. The antagonist of glutamate receptors provides protective effects against hearing loss and SGN damage. It is inferred that excessive release of glutamate from the inner hair cells induced by noise may be responsible for these damages. Glutamate receptors are involved in the degeneration and death of SGNs.

Effect of isoflavones in regulating the transcription of target genes through estrogen receptors / 中西医结合学报

To observe the transcriptional regulation of the two isoflavones genistein and daidzein on target genes.

Regulatory effect of Zn2+ on P2X receptor-mediated, ATP-induced currents in different autonomic ganglion neurons in rats / 第二军医大学学报

Objective: To compare the effects of Zn2+ on the P2X receptor-mediated, ATP-induced currents in neurons separated from rat superior cervical ganglion (SCG), nodose ganglion (NG), and otic ganglion (OTG). Methods: Whole-cell patch clamp recording technique was used to study the regulatory effects of Zn2+ on ATP/αβ-me ATP-induced currents in the above 3 ganlglion neurons. Results: All SCG neurons responded to ATP with a sustained current, while no neurons responded to αβ-me ATP; Zn2+ potentiated ATP-induced sustained currents to (1442±34)% of the original value. All NG neurons responded to ATP and αβ-me ATP with a similar sustained current; coapplication of Zn2+ (10 μmol/L) potentiated their responses to (180±12)% and (262±28)%, respectively. All OTG neurons responded to both ATP and αβ-me ATP with a sustained current. Coapplication of Zn2+ (10 μ mol/L) did not significantly potentiate the sustained currents induced by 10 μmol/L ATP, but when ATP was at 30 μmol/L, Zn2+ (10-100 μmol/L) inhibited ATP-induced sustained currents in a dose dependent manner. If TNP-ATP (100 nmol/L) was first used to inhibit ATP-induced current to (26±2)% of the original value, Zn2+ at 10 μmol/L potentiated the inhibited current to (127±9)% of its original value. Coapplication of Zn2+ (10 μmol/L) potentiated αβ-me ATP-induced currents to (146±5)% of the control. Zn2+ (300 μmol/L) had no effect on τon and τoff of ATP- and αβ-me ATP-induced (30 μmol/L) currents in OTG neurons. Conclusion: (1) Zn2+ is an allosteric modulator of P2X2 and P2X2/3 receptors in SCG and NG neurons and can potentiate the currents they induced. (2) The predominant receptor subtypes in OTG appear to be homomeric P2X2/3 and a little P2X2. Zn2+ has an inhibitory effect on the ATP-induced currents in OTG neurons, suggesting some novel members of the P2X purinoceptor exist in these neurons.

Enhancement effect of propofol on binding function of GABA_A receptor / 第二军医大学学报

Objective:To investigate the influence of propofol on the binding function of GABA A receptor. Methods: By using radioligand receptor binding assay, effects of propofol on the specific binding of 3H GABA and saturation curves of GABA A receptor were observed in cortical membrane preparations from mouse cerebral cortex. Results: Specific binding experiments showed that propofol at the concentrations of 10 300 ?mol/L markedly enhanced the specific binding of 3H GABA( P 0.05). Conclusion: Clinical concentrations of propofol can enhance the binding function of GABA A receptor through increasing the affinity of the low affinity binding site of GABA.

Regulatory effect of Zn~(2+) on P2X receptor-mediated,ATP-induced currents in different autonomic ganglion neurons in rats / 第二军医大学学报

Objective:To compare the effects of Zn~(2+)onthe P2X receptor-mediated,ATP-induced currents in neurons separated from rat superior cervical ganglion(SCG),nodose ganglion(NG),and otic ganglion(OTG).Methods: Whole-cell patch clamp recording technique was used to study the regulatory effects of Zn~(2+) on ATP/??-me ATP-induced currents in the above 3 ganlglion neurons.Results: All SCG neurons responded to ATP with a sustained current,while no neurons responded to ??-me ATP;Zn~(2+) potentiated ATP-induced sustained currents to(1 442?34)% of the original value.All NG neurons responded to ATP and ??-me ATP with a similar sustained current;coapplication of Zn~(2+)(10 ?mol/L) potentiated their responses to(180?12)% and(262?28)%,respectively.All OTG neurons responded to both ATP and ??-me ATP with a sustained current.Coapplication of Zn~(2+)(10 ? mol/L) did not significantly potentiate the sustained currents induced by 10 ?mol/L ATP,but when ATP was at 30 ?mol/L,Zn~(2+)(10-100 ?mol/L) inhibited ATP-induced sustained currents in a dose dependent manner.If TNP-ATP(100 nmol/L) was first used to inhibit ATP-induced current to(26?2)% of the original value,Zn~(2+) at 10 ?mol/L potentiated the inhibited current to(127?9)% of its original value.Coapplication of Zn~(2+)(10 ?mol/L) potentiated ??-me ATP-induced currents to(146?5)% of the control.Zn~(2+)(300 ?mol/L) had no effect on ?_(on) and ?_(off) of ATP-and ??-me ATP-induced(30 ?mol/L) currents in OTG neurons.Conclusion:(1) Zn~(2+) is an allosteric modulator of P2X_(2) and P2X_(2/3) receptors in SCG and NG neurons and can potentiate the currents they induced.(2)The predominant receptor subtypes in OTG appear to be homomeric P2X_(2/3) and a little P2X_(2).Zn~(2+) has an inhibitory effect on the ATP-induced currents in OTG neurons,suggesting some novel members of the P2X purinoceptor exist in these neurons.