Electroacupuncture pretreatment inhibits ferroptosis and inflammation after middle cerebral artery occlusion in rats by activating Nrf2.

OBJECTIVE: Electroacupuncture (EA) pretreatment can effectively increase the tolerance of the brain to ischemic stroke. The mechanism of ischemic tolerance induced by EA is related to Nrf2, but its specific mechanism has not been elucidated. This paper was designed to explore the effect of EA pretreatment on brain injury and the related mechanisms. METHODS: Rats were pretreated with EA before middle cerebral artery occlusion (MCAO) modeling. The symptoms of neurological deficit and the volume of cerebral infarction were measured. The levels of inflammatory factors, oxidative stress-related factors, LPO, ROS, and Fe2+ were evaluated by the corresponding kits. Cell apoptosis was determined through TUNEL staining. The mRNA expression of inflammatory factors was examined by RT-qPCR, and the protein expression of ferroptosis-related factors, pyroptosis-related proteins, Keap1, Nrf2, HO-1, and NQO1 by western blotting. RESULTS: EA pretreatment improved the symptoms of neurological deficit and reduced the volume of cerebral infarction. EA pretreatment significantly inhibited oxidative stress, inflammatory response, ferroptosis, pyroptosis, and apoptosis in brain tissues of MCAO rats. Mechanistically, EA pretreatment could activate Nrf2 expression and reduce Keap1 expression. CONCLUSION: EA pretreatment reduced inflammation and oxidative stress and inhibited ferroptosis by activating Nrf2 expression, ultimately delaying the development of ischemic stroke.

Attractor - a new turning point in drug discovery.

Drug discovery for complex diseases can be viewed as a challenging problem in which the influence of compounds on dynamic features of disease system should be considered, especially the strategies escaping from the disease attractors. Moreover, escaping from the disease-related attractors has been proved to be a cue for the treatment of the complex diseases. The drug discovery methodology based on the attractor theory indicates new solutions for target identification, drug discovery and drug combination design. The methodology is based on the holism level of the organism and the features of system dynamics, so it has advantages for the classification of complex diseases and drug discovery. Currently, research results of this method have increased, which expand the insight scope for drug discovery. This article introduces the major drug discovery methods in the history of pharmacy development and their characteristics, so as to illustrate the reasons and inevitability of the appearance of attractor method, its position in the history of pharmacy development, and its advantages for drug discovery and design, thereby to prove that the attractor method can indeed become the next major drug development method. In addition, it provides a comprehensive description about the concept of attractor, the pipeline of attractor analysis, the common methods of each process and its research progress, so as to provide a macroscopic framework and optional methods and tools for the follow-up researchers.

[Construction of database of growth environment of Dao-di herbs].

The Dao-di herbs are recognized as high-quality Chinese medicines with good medicinal properties. The factors of their growth environment affects the quality of Dao-di herbs. Choosing the right place to produce,rationally planting appropriate local medicinal materials,and improving the quality and yield of authentic medicinal materials are prerequisites for ensuring the steady development of Chinese medicine industry. In this paper,a knowledge model of the relationship between organic medicinal materials and growth environment factors was constructed by integrating the dataset of environmental factors of medicinal materials,and a knowledge base was constructed to provide a decision for the study of suitable medicinal materials under different environmental conditions. Based on this,it is expected to provide a platform for further research on the relationship between the medicinal properties of medicinal herbs and environmental factors.

[Effect of acupuncture treatment on gene expression of neurotransmitters of brain tissue in rats with post-stroke depression].

OBJECTIVE: To observe the effects of acupuncture on mRNA of the neurotransmitter serotonin transporter (5-HTT), 5-HT1Areceptor (5-HT1AR), norepinephrineα2 receptor (NEα2R) in brain tissue in rats with post-stroke depression. METHODS: Forty male SD rats were randomly divided into a normal group, a model group, a medication group and an acupuncture group, 10 rats in each one. The model of post-stroke depression was established by occlusion of middle cerebral artery and chronic unpredictable mild stress method. After model was successfully established, the rats in the normal group and model group received no treatment; the rats in medication group were treated with intragastric administration of fluoxetine (2 mg/kg); the rats in acupuncture group were treated with acupuncture at "Baihui" (GV 20), "Fengfu" (GV 16), "Shenmen" (HT 7) and "Taichong" (LR 3) for 20 min, during which manual stimulation was given once, once a day, 7 days as one course, and totally 3 courses were given with an interval of one day between courses. The changes of rat behavior and 5-HT, NE were observed after intervention; the RT-PCR method was applied to observe the mRNA of 5-HTT, 5-HT1AR, NEα2R in hippocampus, raphe nucleus and locus coeruleus. RESULTS: After treatment, compared with the normal group, the Zea Longa score in the model group was increased, while sugar water consumption, the number of horizontal and vertical movement of open-field test was reduced (all P<0.01); compared with the model group, the Zea Longa score in the medication group and acupuncture group was reduced, while sugar water consumption, the number of horizontal and vertical movement of open-field test were increased (P<0.01, P<0.05); compared between the medication group and acupuncture group, the behavior changes were not significantly different (all P>0.05). After treatment, compared with the normal group, the content of 5-HT and NE in brain tissue and mRNA expression of 5-HTT, 5-HT1AR in hippocampus, raphe nucleus and locus coeruleus in the model group were reduced (all P<0.01), but the mRNA expression of NEα2R was increased (P<0.01);compared with the model group, the content of 5-HT and NE in brain tissue and mRNA expression of 5-HTT, 5-HT1AR in hippocampus, raphe nucleus and locus coeruleus in the medication group and acupuncture group were increased (P<0.01, P<0.05), while the mRNA expression of NEα2R was reduced (all P<0.01). The differences between medication group and acupuncture group were not significantly different (all P>0.05). CONCLUSIONS: Acupuncture could significantly improve behavioral change in rats with post-stroke depression, which may be related to the regulation of 5-HT, NE in cerebral cortex as well as mRNA expressions of 5-HTT, 5-HT1AR, NEα2R in hippocampus, raphe nucleus and locus coeruleus.

Identification of potential ACAT-2 selective inhibitors using pharmacophore, SVM and SVR from Chinese herbs.

Acyl-coenzyme A cholesterol acyltransferase (ACAT) plays an important role in maintaining cellular and organismal cholesterol homeostasis. Two types of ACAT isozymes with different functions exist in mammals, named ACAT-1 and ACAT-2. Numerous studies showed that ACAT-2 selective inhibitors are effective for the treatment of hypercholesterolemia and atherosclerosis. However, as a typical endoplasmic reticulum protein, ACAT-2 protein has not been purified and revealed, so combinatorial ligand-based methods might be the optimal strategy for discovering the ACAT-2 selective inhibitors. In this study, selective pharmacophore models of ACAT-1 inhibitors and ACAT-2 inhibitors were built, respectively. The optimal pharmacophore model for each subtype was identified and utilized as queries for the Traditional Chinese Medicine Database screening. A total of 180 potential ACAT-2 selective inhibitors were obtained, which were identified using an ACAT-2 pharmacophore and not by our ACAT-1 model. Selective SVM model and bioactive SVR model were generated for further identification of the obtained ACAT-2 inhibitors. Ten compounds were finally obtained with predicted inhibitory activities toward ACAT-2. Hydrogen bond acceptor, 2D autocorrelations, GETAWAY descriptors, and BCUT descriptors were identified as key structural features for selectivity and activity of ACAT-2 inhibitors. This study provides a reasonable ligand-based approach to discover potential ACAT-2 selective inhibitors from Chinese herbs, which could help in further screening and development of ACAT-2 selective inhibitors.

[Effect of Manual Acupuncture Intervention on Levels of 5-HTT, 5-HT1AR, NEα2R of Brain Tissues in Rats with Post-stroke Depression].

OBJECTIVE: To observe the effect of manual acupuncture intervention on serotonin transporter (5-HTT), 5-HT 1 A receptor (5-HT1AR), norepinephrine (NE) alpha receptor 2 (NEα2R) protein of brain tissues in rats with post-stroke depression (PSD). METHODS: Forty SD rats were randomly divided into normal group, model group, medication group and acupuncture group, with 10 rats in each. The PSD model was established by the combination of middle cerebral artery occlusion and chronic non- predictable mild stress. Rats of the medication group were treated by gavage of fluoxetine (2 mg/kg, once daily for 21 days), and those of the acupuncture group treated by manual acupuncture stimulation of "Baihui"(GV 20), "Fengfu" (GV 16), bilateral"Shenmen" (HT 7) and "Taichong" (LR 3) for 20 min, once a day for 21 days, with one day's interval between each every 7 days. The animals' neurological deficit was detected by Zea Longa's scaling, and the depression status assessed by sugar solution consumption, and the locomotor activity (crossing and rearing times in 5 min) detected by open-field tests. The contents of 5-HT, NE, acetylcholine (ACh), gamma aminobutyric acid (GABA), and glutamate (Glu) of the cerebral cortex were detected using high performance liquid chromatography (HPLC) and expression levels of 5-HTT, 5-HT1AR, NEα2R proteins of the hippocampus, raphe nuclei and locus coeruleus(LC) tissues detected using western blotting (EB), respectively. RESULTS: After modeling, the neurological deficit score was significantly increased, while the sugar solution consumption and the locomotor activities (crossi-ng and rearing times in 5 min) were notably decreased (P<0.05, P<0.001). Findings of HPLC and WB indicated that after modeling, the cerebrocortical 5-HT and NE contents and 5-HTT protein expression levels in the hippocampus, raphe nucleus and LC were obviously decreased and the cortical ACh, GABA and Glu contents as well as the expression of 5-HT1AR and NEα2R proteins in the hippocampus, raphe nucleus and LC considerably increased in the model group (P<0.01). Following acupuncture intervention and medication, the increased neurological deficit score, cortical ACh, GABA and Glu contents, and the expression le-vels of 5-HT1AR and NEα2R proteins in the hippocampus, rahpe nucleus and LC, as well as the decreased sugar solution consumption, crossing and rearing times of open field tests, cortical 5-HT and NE contents, as well as cortical 5-HT protein expression were all reversed in both acupuncture and medication groups (P<0.05, P<0.01). No significant differences were found between the acupuncture and medication groups in all the above-mentioned indexes (P>0.05). CONCLUSIONS: Acupuncture can improve the locomotor function changes in stroke rats with depression after stroke, which may be associated with its effects in suppressing the up-regulated cortical ACh, GABA, Glu contents, the expression levels of 5-HT1AR and NEα2R proteins in the hippocampus, raphe nucleus and LC, and the down-regulated 5-HT and NE contents in the cerebral cortex and 5-HTT protein expression levels in the hippocampus, raphe nucleus and LC.

Virtual Screening and Molecular Dynamics Study of Potential Negative Allosteric Modulators of mGluR1 from Chinese Herbs.

The metabotropic glutamate subtype 1 (mGluR1), a member of the metabotropic glutamate receptors, is a therapeutic target for neurological disorders. However, due to the lower subtype selectivity of mGluR1 orthosteric compounds, a new targeted strategy, known as allosteric modulators research, is needed for the treatment of mGluR1-related diseases. Recently, the structure of the seven-transmembrane domain (7TMD) of mGluR1 has been solved, which reveals the binding site of allosteric modulators and provides an opportunity for future subtype-selectivity drug design. In this study, a series of computer-aided drug design methods were utilized to discover potential mGluR1 negative allosteric modulators (NAMs). Pharmacophore models were constructed based on three different structure types of mGluR1 NAMs. After validation using the built-in parameters and test set, the optimal pharmacophore model of each structure type was selected and utilized as a query to screen the Traditional Chinese Medicine Database (TCMD). Then, three different hit lists of compounds were obtained. Molecular docking was used based on the latest crystal structure of mGluR1-7TMD to further filter these hits. As a compound with high QFIT and LibDock Score was preferred, a total of 30 compounds were retained. MD simulation was utilized to confirm the stability of potential compounds binding. From the computational results, thesinine-4'-O-ß-d-glucoside, nigrolineaxanthone-P and nodakenin might exhibit negative allosteric moderating effects on mGluR1. This paper indicates the applicability of molecular simulation technologies for discovering potential natural mGluR1 NAMs from Chinese herbs.

[Acupuncture and drug for subcortical ischemic vascular disease complicated with depression: a randomized controlled trial].

OBJECTIVE: To explore the difference in the efficacy and effect mechanism of subcortical ischemic vascular disease (SIVD) complicated with depression between acupuncture and medication. METHODS: Sixty patients were randomized-into an acupuncture group and a medication group, 30 cases in each one. In the acupuncture group, acupuncture was applied to Baihui (GV 20), Shuigou (GV 26), Fengfu (GV 16), Fengchi (GB 20) and the others, once a day, 6 times a week. The treatment of 4 weeks made one session and totally 2 sessions were required. In the medication group, nimodipine 30 mg, three times a day and fluoxetine 20 mg, once a day were prescribed for oral administration, for 8 weeks totally. Before treatment, at the end of the 4th week and at the end of the 8th week of treatment, cerebral blood flow velocity (CBFV) and solubility CD40 ligand (sCD40L) were determined respectively. The scores in Montreal cognitive assessment (MoCA) and Hamilton' s depression scale (HAMD) were evaluated in the two groups. The efficacies on cognitive function and depression symptoms were compared in the patients between the two groups. Results Compared with the outcome before treatment, mean blood flow velocity (Vm) of middle cerebral artery (MCA), anterior cerebral artery (ACA) and posterior cerebral artery (PCA) was increased significantly at the end of the 4th week of treatment in the two groups (all P<0.05). At the end of the 8th week, Vm was increased much significantly (all P<0.01). The differences were not significant in comparison between the two groups (all P>0.05). Compared with the expression before treatment, sCD40L was reduced significantly after treatment in the patients of the two groups (all P<0.01), but the differ- ence was not significant between the two groups (all P>0.05). Compared with that before treatment, MoCA score was increased significantly after treatment in the two groups (all P<0.01), HAMD score was reduced sig- nificantly (all P<0.01), the differences were not significant in comparison between the two groups (all P>0.05). The total effective rate of cognitive improvement was 86.7% (26/30) in the acupuncture group and was 80.0% (24/30) in the medication group, the differences were not significant in comparison of the two groups (P>0.05). The total effective rate of the improvement in depression was 93.3% (21/30) in the acupuncture group and was 86.7% (26/30) in the medication group, the differences were not significant in comparison of the two groups (P>0.05). CONCLUSION: Acupuncture could significantly increases CBFV and reduces serum sCD40L expressions in the patients of SIVD complicated with depression, and significantly improves cognitive function and relieves depression symptoms. The efficacy of it is similar to that of western medication. The increase of serum sCD40L expression is possibly involved in the occurrence and development of SIVD. Reducing sCD40L expression contributes to the alleviation of damage induced by cerebral ischemia and reperfusion.

[Effect of nape cluster-acupuncture treatment on brain microcirculation in ischemic stroke rats].

OBJECTIVE: To observe the effect of nape cluster-acupuncture treatment on blood microcirculation of the cerebral piamater and the whole blood viscosity in cerebral ischemia (CI) rats so as to study its mechanism underlying improvement of CI. METHODS: Eighty male Wistar rats were randomly allocated to false operation group, CI model group, medication group and acupuncture group (n = 20). CI model was established by occlusion of the middle cerebral artery with intraluminal suture-ball blocking method. For rats of the nape cluster-acupuncture group, acupuncture needles were inserted into "Fengfu" (GV 16), "Yamen" (GV 15), "Xia-naohu" (GV 17), "Fengchi" (GB 20), and the points of evenly divided into three parts between GV 16 and GB 20 respectively, and rotated for 1 min, then retained for 15 min, once a day, for 14 sessions. Rats of the medication group were given with intragatric perfusion of nimodipine (12 mL/kg), once daily for 14 days. The blood microcirculation of the brain was detected by using a laser Doppler flowmeter with the probe put near the surface of cerebral piamater. The whole blood viscosity was determined using a blood rheological automatic detector. RESULTS: In comparison with the false operation group, the blood flow volume of the cerebral piamater was remarkably decreased and the whole blood viscosity level significantly increased in the model group (P < 0.01). In comparison with the model group, the blood flow volume of the cerebral piamater was obviously increased and the whole blood viscosity apparently decreased in both the acupuncture and medication groups (P < 0.01). The effects of acupuncture were obviously better than those of the medication in upregulating blood flow volume and in lowering whole blood viscosity (P < 0.01). CONCLUSION: The nape cluster acupuncture treatment has a favorable role in improving brain microcirculation by raising blood flow volume and reducing blood viscosity in ischemic stroke rats.