Baimai Ointment relieves chronic pain induced by chronic compression of dorsal root ganglion in rats by regulating neuroactive ligand-receptor interaction and HIF-1 signaling pathway / 中国中药杂志

The Baimai Ointment with the effect of relaxing sinew and activating collaterals demonstrates a definite effect on Baimai disease with pain, spasm, stiffness and other symptoms, while the pharmacodynamic characteristics and mechanism of this agent remain unclear. In this study, a rat model of chronic compression of L4 dorsal root ganglion(CCD) was established by lumbar disc herniation, and the efficacy and mechanism of Baimai Ointment in the treatment of CCD were preliminarily explored by behavioral tests, side effect evaluation, network analysis, antagonist and molecular biology verification. The pharmacodynamic experiment indicated that Baimai Ointment significantly improved the pain thresholds(mechanical pain, thermal pain, and cold pain) and gait behavior of CCD model rats without causing tolerance or obvious toxic and side effects. Baimai Ointment inhibited the second-phase nociceptive response of mice in the formalin test, increased the hot plate threshold of normal mice, and down-regulated the expression of inflammatory cytokines in the spinal cord. Network analysis showed that Baimai Ointment had synergistic effect in the treatment of CCD and was related to descending inhibition/facilitation system and neuroinflammation. Furthermore, behavioral tests, Western blot, and immunofluorescence assay revealed that the pain-relieving effect of Baimai Ointment on CCD may be related to the regulation of the interaction between neuroactive ligand and receptors(neuroligands) such as CHRNA7, ADRA2A, and ADRB2, and the down-regulation of the expression of NOS2/pERK/PI3K, the core regulatory element of HIF-1 signaling pathway in spinal microglia. The findings preliminarily reveal the mechanism of relaxing sinew and activating collaterals of Baimai Ointment in the treatment of Baimai disease, providing a reference for the rational drug use and further research of this agent.

Neurorotective Effect of Chaihu Jia Longgu Mulitang on Parkinson's Disease with Depression Model Rats and Its Mechanism Based on AMPK/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo observe the protective effect of Chaihu Jia Longgu Mulitang （CLMT） on dopaminergic neurons in Parkinson's disease with depression （PDD） model rats， and to explore the mechanism based on adenosine monophosphate-activated protein kinase/mammalian target of rapamycin （AMPK/mTOR） signaling pathway. MethodAmong the 80 male SD rats， 10 were randomly selected as normal group and the rest were treated with long-term low-dose subcutaneous injection of rotenone in the neck and back combined with chronic unpredictable mild stress （CUMS） to establish PDD rat model. The successfully modeled PDD rats were randomly divided into model group， western medicine group （madopar 0.032 g·kg-1+fluoxetine hydrochloride 0.002 g·kg-1）， CLMT low-dose， medium-dose and high-dose groups （5， 10 and 20 g·kg-1）， 10 rats in each group. Normal group and model group were administrated with the same amount of normal saline by gavage for 4 consecutive weeks. Behavioral changes of rats in each group were evaluated by open field test and pole climbing test. The content of dopamine （DA） and 5-hydroxytryptamine （5-HT） in cerebrospinal fluid was determined by high performance liquid chromatography （HPCL）. The pathological changes of dopaminergic neurons in substantia nigra of rats were observed by hematoxylin-eosin （HE） staining. The positive expression of tyrosine hydroxylase （TH） and expression of α-synuclein in substantia nigra were detected by immunohistochemistry （IHC） and immunofluorescence （IF）， repsectively. The protein expression of microtubule-associated protein 1 light chain 3 （LC3）， adenosine monophosphate-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， mammalian target of rapamycin （mTOR） and phosphorylated mTOR （p-mTOR） was detected by Western blot. ResultCompared with the conditions in normal group， the total horizontal distance and the activity time in the central region in open field test and the content of DA and 5-HT in cerebrospinal fluid were decreased （P<0.05， P<0.01）， and the time of pole climbing was shortened （P<0.01）， with increased score （P<0.01） in model group. Compared with model group， CLMT high-dose group and western medicine group increased the total horizontal distance and activity time in the central region and the content of DA and 5-HT （P<0.05， P<0.01）， and extended the time of climbing pole （P<0.05）， with decreased score （P<0.05， P<0.01）. Compared with those in normal group， the number of dopaminergic neurons in the substantia nigra was reduced， with narrowed and loosely arranged cell body. The fluorescence expression of α-synuclein was enhanced （P<0.01）， and the positive expression of TH was decreased （P<0.01） in model group. Compared with model group， CLMT high-dose group and western medicine group showed elevated number of dopaminergic neurons in the substantia nigra， with enlarged cell body， and decreased fluorescence expression of α-synuclein， and enhanced the positive expression of TH （P<0.05， P<0.01）. Compared with normal group， model group had lowered expression of LC3Ⅱ/Ⅰ， p-AMPK/AMPK in striatum （P<0.05， P<0.01） and increased expression of p-mTOR/mTOR （P<0.01）. Compared with those in model group， LC3Ⅱ/Ⅰ and p-AMPK/AMPK expression were increased （P<0.05， P<0.01） and p-mTOR /mTOR expression was decreased （P<0.01） in CLMT high-dose group and western medicine group. ConclusionCLMT exerts a neuroprotective effect by inhibiting rotenone neurotoxicity. It enhances the level of DA， and thus improves the depression condition in rats with Parkinson's disease. The underlying mechanism may be related to the regulation of AMPK/mTOR signaling pathway， activation of autophagy， and promotion of degrading α-synuclein.

Mechanism of Qingke Pingchuan Granule in Treatment of Acute and Chronic Bronchitis Complicated with Chronic Obstructive Pulmonary Disease： An Exploration Based on Network Pharmacology / 中国实验方剂学杂志

Objective:To explore the potential mechanism of Qingke Pingchuan granule in treating acute and chronic bronchitis complicated with chronic obstructive pulmonary disease （COPD） by network pharmacology. Method:The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） was retrieved to collect the active components of Qingke Pingchuan granule and predict the action targets， followed by the construction of component-target network using Cytoscape 3.8. GeneCards， Online Mendelian Inheritance in Man（OMIM）， and DrugBank were used to harvest disease targets， whose names were put into UniProt for standardization. The treatment targets of Qingke Pingchuan Granule against the two diseases were obtained based on Venn diagram， which were then imported into the STRING platform for constructing the protein-protein interaction （PPI） network. Following the gene ontology（GO） and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment analysis based on MetaScape， the active component-common target-signaling pathway network of Qingke Pingchuan granule against acute and chronic bronchitis complicated with COPD was finally constructed. The accuracy of the target was confirmed by literature. Result:A total of 165 active components， 374 related targets， 512 disease-related targets， and 130 common targets were obtained. Among them， the 14 core therapeutic targets were further subjected to GO enrichment analysis， which yielded 390 biological processes， nine cell components， and 23 molecular functions. The KEGG pathway analysis revealed 22 signaling pathways. Conclusion:Qingke Pingchuan granule alleviates the diseases possibly by regulating such targets as vascular endothelial growth factor receptor 2（KDR）， transforming growth factor beta-1 （TGF-<italic>β</italic>₁）， caveolin 1（CAV1）， hypoxia-inducible factor-1alpha（HIF-1<italic>α</italic>）， and interleukin-2（IL-2）， affecting the synthesis and transport of regulatory factors in cytoplasm， and controlling the cell proliferation and apoptosis.

Protective Effect and Mechanism of Velvet Antler Peptide on Rotenone-induced SH-SY5Y Cell Damage / 中国实验方剂学杂志

Objective:To investigate the intervening effect of velvet antler peptide （VAP） on rotenone-induced neuroblastoma （SH-SY5Y） cell damage and explore its related mechanism. Method:0.5 μmol·L-1 rotenone was used to SH-SY5Y cells to establish an in vitro model of Parkinson's disease （PD）. A blank control group， a model group， high， medium and low dose VAP groups （150，100，50 mg·L-1， respectively） and a rapamycin group were established. The number of lewy bodies， changes in mitochondrial membrane potential， content of reactive oxygen species （ROS） and α-synuclein （α-syn）， protein kinase B （Akt）， and mammalian target of rapamycin （mTOR） were observed by hematoxylin-eosin（HE） staining， rhodamine 123 staining， DCFH-DA staining and immunohistochemical staining expression respectively. Result:The results of HE staining showed that as compared with the blank group， the number of cells in model group was reduced， the tentacle structure became dull， the shape became round， and eosinophilic Lewy bodies were visible in cytoplasm. As compared with model group， there was no significant difference in cell morphology from rapamycin group and VAP high， medium and low dose groups， but there were fewer Lewy bodies in cytoplasm in these four groups. Rhodamine 123 staining showed that as compared with blank group， the mitochondrial membrane potential was increased significantly in model group （P<0.05）. As compared with the model group， the mitochondrial membrane potential was decreased in rapamycin group and VAP high， medium and low dose groups （P<0.05）. DCFH-DA staining results showed that as compared with blank group， the content of ROS was increased significantly in cells of model group （P<0.05）. As compared with model group， the content of ROS was decreased in rapamycin group and VAP high， medium and low dose groups （P<0.05）. Immunohistochemical staining showed that as compared with blank group， the protein expression levels of α-syn，Akt，and mTOR were increased significantly in model group （P<0.05）. As compared with model group， the protein expression levels of α-syn and mTOR were significantly reduced in rapamycin group and VAP high and medium dose groups （P<0.05）， and the expression levels of Akt were significantly reduced in rapamycin group and VAP high-dose group （P<0.05）. Conclusion:Velvet antler peptides may play a neuroprotective role by regulating the Akt/mTOR signaling pathway and promoting the degradation of α-syn in SH-SY5Y cells.

Analgesic effect and central mechanisms of CQ prescription on cancer invasion induced mirror image pain in model mice / 中国中药杂志

This study aimed to analyze the analgesic effect and related central mechanisms of CQ prescription on cancer invasion induced mirror image pain (CIIMIP)in model mice.In the study, male BALB/c mice were randomly divided into normal group, operation control group (injected with 0.2 mL inactivated S180 sarcoma cell sap), model group (injected with 0.2 mL S180 sarcoma cell sap on the right leg near the greater trochanter of femur) and CQ prescription low dose group (intraperitoneally injected with CQ prescription 100 mg•kg⁻¹ on the basis of model mice), CQ prescription middle dose group (intraperitoneally injected with CQ prescription 150 mg•kg⁻¹ on the basis of model mice), and CQ prescription high dose group (intraperitoneally injected with CQ prescription 200 mg•kg⁻¹ on the basis of model mice). Mechanical withdraw threshold (MWT) of the mirror image lateral hind paws were evaluated by Von Frey hairs before modeling and after surgery. The levels of glutamate (Glu), gamma aminobutyric acid (GABA), glycine (Gly), and taurine (Tau) in the L3-L5 spinal cord were measured by the high performance liquid chromatography-fluorescence detector (HPLC-FLD); AimPlex detection technology with multiple factors was used to detect the levels of regulated on activation in normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP-3) in the L3-L5 spinal cord. Then we observed the influence of GABAa receptor antagonist (Bicuculline) on analgesic effect of CQ prescription.The results indicated that CQ prescription could remarkably increase MWT of model mice(P<0.01, P<0.05), decrease the level of Glu(P<0.01, P<0.05), improve the levels of GABA, Gly, Tau(P<0.01, P<0.05), lower the ratio of Glu/GABA(P<0.01, P<0.05), and reduce the levels of RANTES, MCP-3(P<0.05) in the L3-L5 spinal cord, and GABAa receptor antagonist significantly blocked the analgesic effect of CQ prescription at two time points(P<0.05).This study showed that CQ prescription had significant analgesic effect on CIIMIP model mice, and its mechanism was associated with regulating the balance between excitability amino acid(EAA) and inhibitory amino acid (IAA) transmitters in central nervous system, partially activating GABAa receptor, and reducing the release of RANTES and MCP-3 in the spinal cord.