Inhibiting Matrix Metalloproteinase-2 Activation by Perturbing Protein-Protein Interactions Using a Cyclic Peptide.

We report on a cyclic peptide that inhibits matrix metalloproteinase-2 (MMP2) activation with a low-nM-level potency. This inhibitor specifically binds to the D570-A583 epitope on proMMP2 and interferes with the protein-protein interaction (PPI) between proMMP2 and tissue inhibitor of metalloproteinases-2 (TIMP2), thereby preventing the TIMP2-assisted proMMP2 activation process. We developed this cyclic peptide inhibitor through an epitope-targeted library screening process and validated its binding to proMMP2. Using a human melanoma cell line, we demonstrated the cyclic peptide's ability to modulate cellular MMP2 activities and inhibit cell migration. These results provide the first successful example of targeting the PPI between proMMP2 and TIMP2, confirming the feasibility of an MMP2 inhibition strategy that has been sought after for 2 decades.

Alteration of Amino Acid Profiling Influenced by the Active Ingredients of DanHong Injection After Prescription Optimization.

INTRODUCTION: The aim of this work was to optimize the formulation composition of DanHong injection and to study the disturbance of microscopic components of cerebral ischemia in amino acid metabolites and metabolic pathways. The subtle relationship among these three substances and the influence of metabolic pathways were also studied. METHODS: In this study, the central composite design (CCD) matrix and response surface methodology (RSM) were used to design the experiments and to evaluate the interactive effects of three substances. Targeted metabolomics was used to detect the amino acid variation in CCD sets. RESULTS: Response surfaces were generated, and the formulation was optimized by superimposing the contour plots. It was found that the optimum values of the responses could be obtained at an SAB concentration (x1) of 8-9 mg/kg, a TSN concentration (x2) of 14-16 mg/kg, and an HSYA yellow A concentration (x3) of 6 mg/kg. Statistical analysis showed that the three independent variables had significant effects (p < 0.05) on the responses. A total of 22 experimental runs were performed, and the kinetic data were analyzed using a second-order polynomial. Model algorithm calculation indicated that glutamic acid, serine, leucine, glycine, and valine had a very close correlation with the active ingredients. Methionine, aspartic acid, asparagine, glutamic acid, and valine were important for distinguishing different groups, and they were identified as potential biomarkers. Cluster analysis and pathway analysis indicated that the valine, leucine, and isoleucine degradation (VLI degradation) pathway was the major metabolic pathway. Arginine and proline metabolites were most frequently detected, and they were closely associated with other networks according to the network analysis results. VLI degradation pathway and arginine and proline metabolism pathway had a significant influence on cerebral ischemia. DISCUSSION: The integration of CCD and metabolomics may be an effective strategy for optimizing the formulation composition and identifying the mechanism of action of traditional chinese medicine.

Curcumin Alleviates the Functional Gastrointestinal Disorders of Mice In Vivo.

Curcumin is a natural polyphenol extracted from the turmeric rhizome, which has a wide range of biological activities, but until now the effects of curcumin on the gastrointestinal peristalsis have not been fully understood. In vivo study, we observed the effects of curcumin on gastric emptying and intestinal propulsion rates of mice in normal state and in delayed state by atropine (ATR) or nitric oxide precursor L-arginine (L-Arg). An in vitro study explored the direct effects of curcumin on the intestinal contractility, but were studied through measuring spontaneous contraction of isolated jejunum of mice. Our results showed that intragastric administration of curcumin (200 mg/kg/day) for 10-20 days significantly improved gastric emptying and intestinal propulsion rates of mice delayed by ATR. Moreover, intragastric administration of curcumin (200 mg/kg/day) for 15 days also significantly improved mice gastric emptying and intestinal propulsion rates delayed by L-Arg. There was no significant effect on normal gastrointestinal propulsion of mice after intragastric administration of curcumin (200 mg/kg/day) for 1-20 days. When normal isolated jejunum of mice were incubated with curcumin in vitro, the amplitude of the spontaneous contractile waves of jejunum was reduced in a concentration-dependent manner. Moreover, curcumin reduced the amplitude of the contractile waves of jejunum in both contracted and relaxed state induced by acetylcholine or ATR individually. Taken together, our results suggest that curcumin has quite different effects on gastrointestinal peristalsis in vivo and in vitro. Moderate dose of curcumin by intragastric administration for more than 10 days can alleviate the functional gastrointestinal disorders of mice, but cannot affect normal gastrointestinal propulsion.

DanHong injection dose-dependently varies amino acid metabolites and metabolic pathways in the treatment of rats with cerebral ischemia.

AIM: To determine how the relative amino acid contents and metabolic pathways regulate the pharmacological phenotypes in rats with cerebral ischemia after treatment with varying doses of DanHong injection (DHI). METHODS: Adult male rats underwent middle cerebral artery occlusion (MCAO), and were injected with DHI (DH-1: 1 mL/kg; DH-2: 2.5 mL/kg; DH-3: 5 mL/kg, and DH-4: 10 mL/kg, iv) daily for 3 d. The neurological deficit score, body weights and infarct volume were assessed. Serum levels of 20 free amino acids were determined using HPLC, and the values were transformed through the quantitative analysis of the amino acids in the serum metabolic spectrum. Multivariate statistical analysis methods (PCA and PLS-DA) and web-based metabolomics tools (MetPa and MetaboAnalyst) were used to analyze the biological data sets for the amino acids. RESULTS: Administration of DHI dose-dependently decreased cerebral infarct volume, and ameliorated neurological deficits. A total of 5, 6, 7 and 7 non-overlapping metabolites were identified in the DH-1, DH-2, DH-3, and DH-4 groups, respectively. Eight metabolites were shared between the DHI groups and the vehicle group. In addition, the serum levels of glutamic acid, aspartic acid and serine increased with increasing DHI dose. A total of 3, 2, 2 and 5 non-overlapping metabolic pathways were identified in the DH-1, DH-2, DH-3 and DH-4 groups, respectively, and glycine, serine, threonine and histidine metabolism were identified as overlapping pathways among the 4 dose groups. CONCLUSION: Overlapping and non-overlapping amino acid metabolites and metabolic pathways are associated with the dose-dependent neuroprotective effect of DHI.

Phenotype-dependent alteration of pathways and networks reveals a pure synergistic mechanism for compounds treating mouse cerebral ischemia.

AIM: Our previous studies have showed that ursodeoxycholic acid (UA) and jasminoidin (JA) effectively reduce cerebral infarct volume in mice. In this study we explored the pure synergistic mechanism of these compounds in treatment of mouse cerebral ischemia, which was defined as synergistic actions specific for phenotype variations after excluding interference from ineffective compounds. METHODS: Mice with focal cerebral ischemia were treated with UA, JA or a combination JA and UA (JU). Concha margaritifera (CM) was taken as ineffective compound. Cerebral infarct volume of the mice was determined, and the hippocampi were taken for microarray analysis. Particular signaling pathways and biological functions were enriched based on differentially expressed genes, and corresponding networks were constructed through Ingenuity Pathway Analysis. RESULTS: In phenotype analysis, UA, JA, and JU significantly reduced the ischemic infarct volume with JU being superior to UA or JA alone, while CM was ineffective. As a result, 4 pathways enriched in CM were excluded. Core pathways in the phenotype-positive groups (UA or JA) were involved in neuronal homeostasis and neuropathology. JU-contributing pathways included all UA-contributing and the majority (71.7%) of JA-contributing pathways, and 10 new core pathways whose effects included inflammatory immunity, apoptosis and nervous system development. The functions of JU group included all functions of JA group, the majority (93.1%) of UA-contributing functions, and 3 new core functions, which focused on physiological system development and function. CONCLUSION: The pure synergism between UA and JA underlies 10 new core pathways and 3 new core functions, which are involved in inflammation, immune responses, apoptosis and nervous system development.

Forty-six cases of acute cerebral infarction treated with the combined use of acupuncture and drugs.

OBJECTIVE: To observe the clinical therapeutic effects of treatment for acute cerebral infarction with the combined use of acupuncture and drugs, and study the therapeutic mechanism. METHODS: 88 cases of acute cerebral infarction were divided randomly into two groups, a treatment group of 46 cases treated with acupuncture, Danshen Zhushe Ye (Saliva Injection) and routine western drugs and a control group of 42 cases treated simply with drugs. The therapeutic effects were evaluated based on the evaluation criteria for damage of the nerve function. Observation was also made on changes in the skull CT images, and in the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px), and nitric oxide synthase (NOS) in serums, and levels of malondialdehyde (MDA) and nitric oxide (NO) in serums. RESULTS: After treatment for 15 days, the total effective rate of the treatment group reached 80.43%, while that of the control group was 61.90%, showing significant differences. The effective rate shown by the skull CT images of the treatment group was 51.61%, while that of the control group was 45.16%, showing no significant differences. But as compared with the control group, the activities of SOD and GSH-px in serums were significantly enhanced, and the levels of MDA and NO, and activity of NOS in serums were significantly decreased in the treatment group. CONCLUSION: The prompt acupuncture treatment of acute cerebral infarction can markedly raise the clinical therapeutic effects, improve the activity of SOD and GSH-px in serums, lower down the level of MDA in serum, and markedly decrease NO level and NOS activity in serums.

[Effect of mailuoning on C-Fos protein expression in rats with cerebral infarction].

OBJECTIVE: To investigate the protective effect of mailuoning (MLN) on nerve cells after cerebral infarction induced by photochemistry. METHODS: Eighty SD rats were divided into three groups, the control group (n = 20), the model group (n = 30) and the MLN group (n = 30). Focal cerebro-ischemia induced by photothrombosis in adult rat was used as a model. Changes of C-Fos protein expression before and after MLN treatment were observed using immunohistochemistry, computerized imaging technique and transmission electron microscopy (TEM). RESULTS: C-Fos positive cells located in the transitional zone between the necrotic core and normal cortex. C-Fos protein expression began to show 3 hrs after cerebral infarction, peaked at 6 hrs. As compared with the model group, C-Fos expression was significantly reduced in the MLN group (P<0.01). CONCLUSION: MLN could markedly reduce the injury of nerve cell in the transitional zone, the protection may be related with its inhibition on C-Fos protein expression.