Discovery and confirmation of protein action site AK1 of ginsenosides in brain based on DARTS technology / 中国中药杂志

This study aims to explore the targets of ginsenosides in brain based on drug affinity responsive target stability(DARTS) technology. Specifically, DARTS technology was combined with label-free liquid chromatography tandem mass spectrometry(LC-MS) to screen out the proteins in the brain that might interact with ginsenosides. Based on the screening results, adenylate kinase 1(AK1) was selected for further confirmation. First, the His-AK1 fusion protein was yielded successively through the construction of recombinant prokaryotic expression vector, expression of target protein, and purification of the fusion protein. Biolayer interferometry(BLI) was employed to detect the direct interaction of Rg_1, Re, Rb_1, Rd, Rh_2, F1, Rh_1, compound K(CK), 25-OH-PPD, protopanaxa-diol(PPD), and protopanaxatriol(PPT) with AK1, thereby screening the ginsenoside monomer or sapogenin that had strong direct interaction with the suspected target protein AK1. Then, the BLI was used to further determine the kinetic parameters for the binding of PPD(strongest interaction with AK1) to His-AK1 fusion protein. Finally, molecular docking technology was applied to analyze the binding properties between the two. With DARTS and LC-MS, multiple differential proteins were screened out, and AK1 was selected based on previous research for target verification. Fusion protein His-AK1 was obtained by prokaryotic expression, and the response(nm) of Re, Rg_1, Rd, Rb_1, Rh_1, Rh_2, F1, PPT, PPD, 25-OH-PPD, and CK with His-AK1 was respectively 0.003 1, 0.001 9, 0.042 8, 0.022 2, 0.013 4, 0.037 3, 0.013 9, 0.030 7, 0.140 2, 0.016 0, and 0.040 8. The K_(on), K_(off), and K_D values of PPD and His-AK1 were determined by the BLI as 1.22×10~2 mol~(-1)·L·s~(-1), 1.04×10~(-2) s~(-1), 8.52×10~(-5) mol·L~(-1). According to the molecular docking result, PPD bound to AK1 with the absolute value of the docking score of 3.438, and hydrogen bonds mainly formed between the two. Thus, AK1 is one of the protein action sites of ginsenosides in the brain. The direct interaction between ginsenoside metabolite PPD and AK1 is the strongest.

Discussion on medicinal attribution of Descurainia sophia and its nature and flavor splitting components based on typical energy metabolism of heat syndrome animal models / 中草药

Objective: The relevant indicators of energy metabolism in rats with heat syndrome were tested to verify the medicinal properties of Descurainia sophia and its nature and flavor splitting components, in order to explain the cold and heat properties of D. sophia and its splitting components. Methods: Healthy male Sprague-Dawley rats were randomly divided into normal control group (NC), model group (M), water extract of gardenia (DS), flavonoid glycosides composition of D. sophia (FG), flavonoid aglycone composition of D. sophia (FA), oligosaccharide resolution component group (Oli), gardenia polysaccharide decomposition component group (Pol), and D. sophia fatty oil component separation group (FO). The model of heat syndrome was established by intragastric administration of Euthyrox (120 mg/kg). After 15 days of continuous administration, blood was taken from the abdominal aorta and the liver and heart were taken to detect the indicators related to the energy metabolism of the substance. The enzyme expression of glucokinase (GCK) and fructose phosphokinase (PFK-1), phosphoglycerate kinase (PGK), pyruvate kinase (PK), pyruvate dehydrogenase (PDH), acetyl-CoA, citrate synthase (CS), isocitrate dehydrogenase (ICD), alpha-ketoglutarate dehydrogenase (alpha-KGDHC), fumarate (FUM), glycogen phosphorylase (PYGL), glycogen synthase kinase-3 (GSK-3), adipose triglyceride lipase (ATGL), cytochrome C reductase (CCR), cytochrome C oxidase (COX), ATP synthase (ATPs), adenylate kinase (ADK), Na+ K,+-ATPase and the content of adenosine triphosphate (ATP), adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD+), reduced nicotinamide adenine dinucleotide (NADH) were determined. Results: Compared with NC group, the weight of M group rats was decreased significantly (P < 0.01). Compared with M group, the body weights of rats in DS, FG, FA, Pol and tFO groups were significantly or highly significantly increased (P < 0.05, 0.01). Compared with NC group, the number of spontaneous activities in M group was increased significantly (P < 0.01) within 5 min, and the number of locomotor activities of each group within 5 min after administration was significantly decreased (P < 0.01). Compared with NC group, the levels of GCK, PFK-1, PK, PGK, PDH, acetyl-CoA, CS, ICD, α-KGDHC, FUM, PYGL, GSK-3, ATGL, ATP, CCR, COX, ATPs, ADK, Na+ K,+-ATP, NADH expression or content in M group of rats were significantly increased (P < 0.05, 0.01), the content of ADP, NAD+ and NAD+/NADH was decreased (P < 0.05, 0.01). After administration, the expression level of material energy metabolism of the rats in each group was significantly or extremely significantly reduced compared with the M group (P < 0.05, 0.01). Conclusion: D. sophia can improve the state of the energy-metabolism related indicators of the rats in the heat syndrome model group. It is verified that D. sophia nature and flavor splitting components show the cold (cool) properties. From the side, it reflects the mechanism of "treating heat with cold drug" is related with the substance energy metabolism.

Role of adenosine monophosphate-activated protein kinase in acute pancreatitis / 临床肝胆病杂志

Adenosine monophosphate-activated protein kinase (AMPK) is a critical enzyme for energy and metabolic regulation and can effectively maintain the homeostasis of energy and metabolism in cells and the body, and thus it plays an important role in both health and diseases. Current studies have shown that AMPK can regulate inflammatory response in the body through various cytokines and signaling pathways such as nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) and has become a potential therapeutic target for a variety of inflammatory diseases. In acute pancreatitis, abnormal activation of trypsin can cause the injury and necrosis of tissue cells, release various inflammatory factors including NF-κB, TNF-α, and IL-6, and induce systemic inflammatory response, leading to organ injury or acute inflammatory disease. Recent studies indicate that the activation of AMPK can alleviate the inflammatory damage of acute pancreatitis. Therefore, AMPK and its signaling pathway may become potential therapeutic targets for acute pancreatitis.

Expression of adenylate kinase 4 in prostate cancer and its clinical significance / 国际生物医学工程杂志

Objective To investigate the expression of adenylate kinase 4 (AK4) in prostate cancer and its clinical significance.Methods The clinical data of 85 patients with prostate cancer who have underwent radical surgery were retrospectively analyzed.The expression of AK4 in prostate cancer tissues and corresponding paracancerous tissues was detected by immunohistochemistry to analyze the relationship with clinicopathological features.Results The positive rates of AK4 in prostate cancer and paracancerous tissues were 85.9％ and 38.8％ respectively,and the difference was statistically significant (P＜0.05).The abnormal expression of AK4 was correlated with recurrence and distant metastasis (all P＜0.05).The survival time of AK4-overexpressing patients with prostate cancer was significantly lower than that of AK4 low-expression patients,and the difference was statistically significant (P＜0.05).Conclusions The expression of AK4 in prostate cancer tissues is up-regulated,which is related to the distant metastasis of carcinoma and vascular tumor thrombus,which suggests that the expression of AK4 may indicate the poor prognosis of patients with prostate cancer.

Effects of 5-aminoimidazole-4-carboxamide ribonucleotide on the palmitic acid-induced expression of mitochondrial function-related proteins in C2 C12 skeletal muscle cells / 中华老年医学杂志

Objective To investigate the effects of activated protein kinase(AMPK)agonists on the expression of peroxisome proliferator-activated receptor γ coactivator 1α(PGC1a),mitofusin 2 (Mfn2) and nuclear respiratory factor1 (NRF1)in the process of lipid-induced mitochondrial dysfunction of skeletal muscles.Methods The expression of PGC1α,Mfn2 and NRF1 in C2C12 skeletal muscle cells after intervention with palmitic acid was detected using reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting.The effect of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on Mfn2 and NRF1 and,the expression of Mfn2 and NRF1 in C2C12 cells induced by a PGC1α activator and PGC1α-siRNA were assessed by Western blotting.Results Palmitic acid decreased the mRNA and protein expression of PGC1α,Mfn2 and NRF1 in C2C12 cells (P＜0.05).Additionally,AICAR,rosiglitazone and metformin up-regulated PGC1α expression,regardless of the presence of palmitic acid and,AICAR reversed lipid-induced PGC1α,Mfn2 and NRF1 attenuation in C2C12 cells.Furthermore,Mfn2 and NRF1 protein expression increased with PGC1α over-expression,and decreased with down-regulated PGC1α expression.Conclusions AICAR can relieve the adverse effects of palmitic acid on PGC1α,Mfn2 and NRF1 in skeletal muscle cells.Moreover,it appears that AICAR can up-regulate Mfn2 and NRF1 expression through activating PGC1α.

Astragalus polysaccharides inhibit the growth and proliferation of human hepatocellular carcinoma cell via AMPK-mTOR pathway / 中国医师杂志

Objective To investigate the effect of astragalus polysaccharides (APS) on the growth and proliferation of hepatocellular carcinoma cells and its effect on adenosine monophosphate activated pro-tein kinase (AMPK) activity.Methods Hepatocellular carcinoma HepG2 cells cultured for 12,24,and 48 hours were treated with 200,300,and 400 mg/L concentration of astragalus polysaccharides.The cell inhibition rate was detected with methyl thiazolyl tetrazolium (MTT),and apoptosis was observed under the fluorescence microscope.Western blot method was used to measure the expression of total AMPK,phosphorylated AMPK (p-AMPK),and phosphorylate mammalian target of rapamycin (p-mTOR) protein expressions.Results Astragalus polysaccharides of each concentration significantly inhibited the proliferation of human hepatoma HepG2 cells (P ＜ 0.01),and the effect of 300 mg/L concentration astragalus polysaccharides was more significant than that of the 200 mg/L concentration (P ＜0.01);while inhibitory effect of 400 and 300 mg/L Astragalus polysaccharides on the proliferation of human hepatoma cell line HepG2 was not significant difference.We found that Astragalus polysaccharides of each concentration could promote the apoptosis of HepG2 cells,and the effect of 300 mg/L Astragalus polysaccharides was more significant.However,astragalus polysaccharide of 400 mg/L concentration could promote the apoptosis no more than the 300 mg/L concentration,which was observed by fluorescent microscope.Western blot results showed that astragalus polysaccharides could increase the expression of p-AMPK (P ＜ 0.05),and inhibit its downstream protein expressions of p-mTOR (P ＜ 0.05).The proliferation effect of astragalus polysaccharides was weakened after accession of AMPK antagonist compound C on hepatocellular carcinoma cells.Conclusions APS can inhibit the growth and proliferation of hepatocarcinoma cells,and its mechanism is related to the AMPK-mTOR pathway.

Activation of AMP-activated protein kinase is involved in doxorubicin-induced anti-breast cancer cell proliferation / 肿瘤研究与临床

Objective To investigate the mechanism of anti-breast cancer cell proliferation induced by doxorubicin (DOX).Methods AMP-Activated Protein Kinase (AMPK) activator AICAR,AMPK siRNA,AMPK inhibitor compound C(AMPKi) and doxorubicin treated MCF-7 cells at different time points; AMPK,acetyl CoA carboxylase (ACC),p38 activation were detected by Western blot.MTT was used as cell viability assay. Results Doxorubicin-induced activation of AMPK, AMPK agonist (AICAR)or in combination with doxorubicin activated AMPK and increased MCF-7 cell proliferation rate [the difference of cell viability between group AICAR+DOX(17.7±1.6 ) ％ and group DOX(71.4±1.8 ) ％ was significant(P＜0.001)].After AMPKi or AMPK siRNA and doxorubicin combined administration, P-AMPK and P-ACC expression was significantly decreased,the level of p38 was not affected,and MCF-7 cell proliferation inhibition rate decreased [the cell viability of group AMPKi+DOX(72.7±1.8 ) ％ vs group DOX(96.3±1.7 ) ％,P＜0.001 ;group AMPK siRNA +DOX( 76.9±2.2 ) ％ vs group scramble siRNA+DOX(95.9±1.8) ％,P＜0.001].Conclusion AMPK is involved in doxorubicin-induced anti-breast cancer cell proliferation.

Secretion of adenylate kinase 1 is required for extracellular ATP synthesis in C2C12 myotubes

Extracellular ATP (exATP) has been known to be a critical ligand regulating skeletal muscle differentiation and contractibility. ExATP synthesis was greatly increased with the high level of adenylate kinase 1 (AK1) and ATP synthase beta during C2C12 myogenesis. The exATP synthesis was abolished by the knock-down of AK1 but not by that of ATP synthase beta in C2C12 myotubes, suggesting that AK1 is required for exATP synthesis in myotubes. However, membrane-bound AK1beta was not involved in exATP synthesis because its expression level was decreased during myogenesis in spite of its localization in the lipid rafts that contain various kinds of receptors and mediate cell signal transduction, cell migration, and differentiation. Interestingly, cytoplasmic AK1 was secreted from C2C12 myotubes but not from C2C12 myoblasts. Taken together all these data, we can conclude that AK1 secretion is required for the exATP generation in myotubes.

Investigation of the Growth Rate Change in Recombinant BCG which was cloned Mycobacterium tuberculosis Adenylate Kinase Mutation Gene or Human Muscle-type Adenylate Kinase Synthetic Gene / 결핵및호흡기질환

BACKGROUND: Normal cell proliferation and viability is strongly depends on the availability of metabolic energy and the maintenance of the appropriate adenylate-nucleotide pools. Hypothetically, changes in adenylate kinase (AK) expression could therefore be associated with adaptation to altered growth characteristics or inversely altered growth characteristics of proliferating cells could drive the changes in the metabolic profile. This study investigated whether the expression of either AK1 or a Mycobacterium tuberculosis adenylate kinase mutant which has the same catalytic activity of AK1 could affect the growth rate of slow-growing BCG. METHOD: Recombinant BCGs, which were cloned the human muscle-type adenylate kinase synthetic gene (AK1) and adenylate kinase mutation gene (AKmtDM) of Mycobacterium tuberculosis into the Mycobacterium/E.coli expression vectors, were constructed. Recombinant BCGs and wild-type BCG were cultured in 7H9 media and the optical density at 600nm was measured at intervals of 2-3 days. RESULT: There wasn't the growth rate change induced by AK1 or AKmtDM expression in recombinant BCGs. CONCLUSION: The expression of AK1 or Mycobacterium tuberculosis adenylate kinase mutant in BCG does not affect the growth rate of BCG.

Protective Efficacy of Recombinant Proteins Adenylate Kinase, Nucleoside Diphosphate Kinase, and Heat-Shock Protein 70 against Mycobacterium tuberculosis Infection in Mice / 결핵및호흡기질환

BACKGROUND: Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. METHOD: M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and IFN-gamma after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. RESULT: Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and IFN-gamma responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. CONCLUSION: Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.

Serological Kinetics of Adenylate Kinase 3 in Rat Acute Myocardial Infarction

BACKGROUND AND OBJECTIVES: Currently used serological markers for the diagnosis of acute myocardial infarction differ in appearance time and specificity for myocardial infarction, allowing no ideal single serological marker for myocardial infarction. Adenylate kinase (AK) is a ubiquitous enzyme which contributes to the homeostasis of the cellular adenine nucleotides pool. AK is abundant in the myocardium, and we postulated that AK3 could be used as a biochemical marker for the diagnosis of acute myocardial infarction (AMI). MATERALS AND METHODS: We constructed an AMI rat model with ligation of the anterior descending artery. We measured the concentration of serum AK3 in the AMI rat model by enhanced chemiluminescence (ECL) sandwich ELISA using monoclonal antibodies against recombinant AK3. RESULTS: The serum AK3 level started to increase in 3 hours and reached a peak at 6 hours after ligation of the rat coronary artery. The significant elevation of AK3 was retained for 2 days (p<0.05). CONCLUSION: AK3 is a useful serological marker for acute myocardial infarction in the rat.

Expression of Adenylate Kinase of Schistosoma japonicum and Evaluation on the Immunoreactivity of the Recombinant Protein / 中国寄生虫学与寄生虫病杂志

Objective To express and purify the Schistosoma japonicum adenylate kinase(AK) protein of which the cDNA sequence was subcloned into the pET32a(+) soluble expression plasmid, and to evaluate its immunoreactivity. MethodsThe recombinant plasmid was transformed into {E.coli} BL21, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS_PAGE after boiling and centrifugation. The target protein was purified with the Ni_NTA agarose. The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti_6_His antibody, sera from rabbits 6 weeks after infected with cercariae or UV_attenuated cercariae. The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum. Results The molecular weight of the target fusion protein was {Mr 40 000} after being induced with IPTG. The fused protein showed a single band when reacted with anti_6_His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum. Conclusion The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about {Mr 40 000}. This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV_attenuated cercariae.