Chunsong Zheng

CE-ESI-MS coupled with dynamic pH junction online concentration for analysis of peptides in human urine samples.

In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE‐ESI‐MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol–water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L‐Ala‐L‐Ala (pI=5.57), L‐Leu‐D‐Leu (pI=5.52), Gly‐D‐Phe (pI=5.52) and Gly‐Gly‐L‐Leu (pI=5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2–2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45–3.57 and 4.93–6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.

Affinity capillary electrophoresis coupling with partial filling technique and field‐amplified sample injection for enantioseparation and determination of DL‐tetrahydropalmatine

A novel, simple and sensitive method for the enantioseparation and determination of DL‐tetrahydropalmatine (DL‐THP) was developed using ACE in combination with partial filling technique and field‐amplified sample injection. A chiral selector, i.e. BSA, was used for the enantioseparation of DL‐THP in ACE. Effects of BSA concentration, pH and separation voltage on the effectiveness of the enantiomer separation were evaluated. In an optimal condition, D‐ and L‐THP were completely enantio‐separated in less than 9 min by partially filling an electrophoretic capillary with 50 μmol/L BSA (50 mbar, 100 s) and carrying out an electrophoresis with 20 mmol/L phosphate buffer (pH 7.4) at 15 kV. The sensitivity was further improved by making use of field‐amplified sample injection to lower the LOD (defined as S/N=3) down to 6 ng/mL. Real samples were also tested and promising results for the determination of DL‐THP enantiomers were obtained.

Achyranthes bidentata polysaccharides induce chondrocyte proliferation via the promotion of the G1/S cell cycle transition

Achyranthes bidentata polysaccharides (ABPS) are the major bioactive constituents of Radix Achyranthes bidentata (AB), which has been widely used in traditional Chinese medicine for the treatment of osteoarthritis. However, the molecular mechanisms behind the therapeutic effect of ABPS remain unclear. In the present study, chondrocytes were isolated from Sprague-Dawley rats. The effects of ABPS on the G1/S cell cycle transition in primary chondrocytes were investigated. The chondrocytes treated with and without ABPS were analyzed and it was observed that ABPS treatment was able to enhance chondrocyte proliferation in a dose- and time-dependent manner and promote the progression of chondrocyte cell cycle proliferation via the promotion of the G1 to S phase transition. Furthermore, using RT-PCR and western blot analysis, ABPS were observed as significantly upregulating the expression of cyclin D1 and the cyclin-dependent kinases (CDKs) CDK4 and CDK6. These results suggest that ABPS are able to promote chondrocyte proliferation via the promotion of the G1/S cell cycle transition.

Solid‐phase extraction‐field‐amplified sample injection coupled with CE‐ESI‐MS for online pre‐concentration and quantitative analysis of brain‐gut peptides

In order to improve the sensitivity of CE‐ESI‐MS for the analysis of brain‐gut peptides, a solid‐phase extraction combined with field‐amplified sample injection was used for the pre‐concentration of the brain‐gut peptides. Compared with the conventional pressure injection method, the sensitivity in the detection of brain‐gut peptides was improved more than 100‐fold. The possible factors affecting sample stacking, such as the sample matrix, the composition and the length of the water column, the types and the volumes of eluent, have been investigated in detail. Under the optimum conditions, the detectable concentration of brain‐gut peptides was found to be as low as 0.02 μM. A linear response concentration for the detection was developed in the range of 0.08–25 μmol/L. A real sample of human cerebrospinal fluids, which was spiked with brain‐gut peptides, was also examined in order to evaluate the reliability of the proposed approach. The recovery of the method was in a range from 69.2 to 85.4%. The method was found to be reliable, accurate and potentially applicable for clinical drug analysis.

Duhuo Jisheng Decoction promotes chondrocyte proliferation through accelerated G1/S transition in osteoarthritis

Duhuo Jisheng Decoction (DHJSD), a well known traditional Chinese folk medicine, is used for eliminating stagnation, removing blood stasis, promoting blood circulation and alleviating pain; it is commonly used for the treatment of various diseases, including osteoarthritis (OA). However, the molecular mechanisms behind the therapeutic effects of OA remain unclear. In the present study, the effects of DHJSD on the morphology of articular cartilage and the G1/S cell cycle progression in chondrocytes, as well as the underlying mechanisms, were investigated. A total of 27 two‑month‑old male Sprague Dawley rats were randomly divided into 3 groups: the control group (no papain-induced OA; received an equivalent amount of saline only), the model group (papain-induced OA; received an equivalent amount of saline only) and the DHJSD group [papain-induced OA; received a clinical oral dose of DHJSD (9.3 g/kg/day)]. After 8 consecutive weeks of treatment, the morphological changes in articular cartilage were observed under an optical microscope and by transmission electron microscopy (TEM) and the mRNA and protein expression levels of cyclin D1, CDK4, CDK6, retinoblastoma protein (Rb) and p16 were measured by RT‑PCR and immunohistochemistry, respectively. Treatment with DHJSD significantly improved the arrangement of collagen fibers in the articular cartilage, as well as its structure and reduced cell degeneration compared with the model group. The mRNA and protein expression levels of cyclin D1, CDK4, CDK6 and Rb in the DHJSD‑treated group were significantly increased compared with those in the model group, whereas p16 expression was significantly downregulated. Taken together, these results indicate that DHJSD treatment promotes chondrocyte proliferation by promoting the G1/S checkpoint transition in the cell cycle and by upregulating the expression of cyclin D1, CDK4, CDK6 and Rb and downregulating the expression of p16 and this may, in part, explain its clinical efficacy in the treatment of osteoarthritis.

Millimeter wave radiation induces apoptosis via affecting the ratio of Bax/Bcl-2 in SW1353 human chondrosarcoma cells.

The efficacy and safety of millimeter wave radiation has been proven for various types of malignant tumors. However, the mechanisms underlying effects of millimeter wave radiation on apoptosis are still unclear. The present study was undertaken to examine the effects of millimeter wave radiation on cell apoptosis and mitochondrial membrane potential, and to determine the molecular mechanism of millimeter wave radiation-induced apoptosis by investigating the expression of Bcl-2 family proteins (Bcl-2, Bax), caspase-9 and caspase-3 in SW1353 cells. We found that millimeter wave radiation suppressed the viability of SW1353 cells, demonstrating that millimeter wave radiation induced cell apoptosis and reduced cell viability in a time-dependent manner. Furthermore, we observed that treatment of cells with millimeter wave radiation significantly induced loss of mitochondrial membrane potential, upregulated proapoptotic Bax, caspase-9 and caspase-3, but did not significantly change levels of antiapoptotic Bcl-2. These data suggested that millimeter wave radiation may induce apoptosis via affecting the ratio of Bax/Bcl-2 in SW1353 cells.

Computational pharmacology study of Tougu Xiaotong Granule (透骨消痛颗粒) in preventing and treating knee osteoarthritis

ObjectiveTo study the pharmacological properties of Tougu Xiaotong Granule (透骨消痛颗粒,: TGXTG) in preventing and treating knee osteoarthritis (KOA) at the molecular level.MethodsThe computational methods, including principal component analysis, molecular docking, target-ligand space distribution, and the predictions of absorption, distribution, metabolism, excretion and toxicity (ADMET), were introduced to characterize the molecules in TGXTG.ResultsThe structural properties of molecules in TGXTG were more: diverse than those of the drug/drug-like molecules, and TGXTG could interact with significant target enzymes related to KOA. In addition, the cluster of effective components was preliminarily identified by the target-ligand space distributions. As to the results of ADMET properties, some of them were unsatisfactory, and were merely regarded as references here.ConclusionBased on this computational pharmacology study, TGXTG is a broad-broadspectrum recipe inhibiting many important target enzymes, which could effectively postpone the degeneration of spectrum cartilage by coordinately inhibiting the biological effects of cytokines, matrix metallopeptidase 3, and oxygen free radicals. radicals.

Millimeter wave treatment promotes chondrocyte proliferation via G1/S cell cycle transition

Millimeter waves, high-frequency electromagnetic waves, can effectively alleviate the clinical symptoms in osteoarthritis patients, as a non-pharmaceutical and non-invasive physical therapy regimen. However, the molecular mechanisms of the therapeutic effects of millimeter wave treatment are not well understood. In the present study, the effect of millimeter waves on the G1/S cell cycle progression in chondrocytes and the underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without millimeter waves for 48 h. Chondrocyte viability was analyzed using the MTT assay. The cell cycle distribution of chondrocytes was analyzed by flow cytometry. mRNA and protein expression levels of cyclin D1, cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and p21 were detected using real-time PCR and western blotting, respectively. Millimeter wave stimulation was found to significantly enhance chondrocyte viability. Moreover, the percentage of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the S phase was significantly increased. In addition, following millimeter wave treatment, cyclin D1, CDK4 and CDK6 expression was significantly upregulated, whereas p21 expression was significantly downregulated. The results indicate that millimeter wave treatment promotes chondrocyte proliferation via cell cycle progression.

Potential Synergistic and Multitarget Effect of Herbal Pair Chuanxiong Rhizome-Paeonia Albifora Pall on Osteoarthritis Disease: A Computational Pharmacology Approach

ObjectiveTo study the polypharmacological mechanism of herbal pair Chuanxiong Rhizome-Paeonia Albifora Pall (HP CXR-PAP) on the treatment for osteoarthritis (OA).MethodsChemical space was used to discuss the similarities and differences between the molecule sets of HP CXR-PAP and drugs. Docking protocol was used to study the interaction between HP CXR-PAP and OA target enzymes. The similarities and differences of HP CXR-PAP and drugs in target spaces were elucidated by network features.ResultsThe plots between the molecule sets of HP CXR-PAP and drugs in chemical space had the majority in the same region, and compounds from HP CXR-PAP covered a much larger additional region of space than drug molecules, which denoted the diverse structural properties in the molecule set of HP CXR-PAP. The molecules in HP CXR-PAP had the properties of promiscuous drugs and combination drug, and both HP CXR-PAP ligand-target interaction network and drug ligand-target interaction network were similar in the interaction profiles and network features, which revealed the effects of multicomponent and multitarget.ConclusionThe clue of potential synergism was obtained in curing OA disease by Chinese medicine, which revealed the advantages of Chinese medicine for targeting osteoarthritis disease.

Xiao Jin Wan, a traditional Chinese herbal formula, inhibits proliferation via arresting cell cycle progression at the G2/M phase and promoting apoptosis via activating the mitochondrial‑dependent pathway in U-2OS human osteosarcoma cells

Xiao Jin Wan (XJW) is a well-known traditional Chinese folk-medicine, which is commonly used for the treatment of various types of diseases including cancers. However, the mechanism of the anticancer activity of XJW against U-2OS human osteosarcoma cells, have not yet been reported. In the present study, we investigated the cellular effects of the XJW on the U-2OS human osteosarcoma cell line. Our results showed that XJW induced cell morphological changes, reduced cell viability in a dose- and time-dependent manner and arrested in the G2/M phase of the cell cycle suggesting that XJW inhibited the proliferation of U-2OS cells. Hoechst 33258 staining and Annexin V/propidium iodide double staining exhibited the typical nuclear features of apoptosis and increased the proportion of apoptotic Annexin V-positive cells in a dose-dependent manner, respectively. In addition, XJW treatment caused loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, the results indicate that the U-2OS cell growth inhibitory activity of XJW was due to cell cycle arrested and mitochondrial-mediated apoptosis, which may partly explain the anticancer activity of Xiao Jin Wan.