Neuroprotective effects of Shaoyao Gancao decoction against excitatory damage in PC12 cells based on the Src-NR2-nNOS pathway

Objective:To explore the neuroprotective effects of the Shaoyao Gancao decoction(SGD)against excitatory damage in PC12 cells and the role of the Src-NR2-nNOS pathway mediation by SGD in regulatingγ-aminobutyric acid(GABA)-glutamate(Glu)homeostasis.Methods: N-Methyl-d-aspartic acid(NMDA)was used to establish a PC12 cell excitability injury model.To investigate the neuroprotective effect of SGD,a cell counting kit-8(CCK-8)assay was used to determine PC12 cell viability,Annexin V/Propidium Iodide(Annexin V/PI)double staining was used to determine PC12 cell apoptosis,and Ca^(2+)concentration was observed using laser confocal microscopy.GABA receptor agonists and antagonists were used to analyze the neuroprotective interactions betweenγ-aminobutyric acid(GABA)and NMDA receptors.Additionally,molecular biology techniques were used to determine mRNA and protein expression in the Src-NR2-nNOS pathway.We analyzed the correlations between the regulatory sites of GABA and NMDA interactions,excitatory neurotoxicity,and brain damage at the molecular level.Results: NMDA excitotoxic injury manifested as a significant decrease in cell activity,increased apoptosis and caspase-3 protein expression,and a significant increase in intracellular Ca^(2+)concentration.Administration of SGD,a GABAA receptor agonist(muscimol),or a GABAB receptor agonist(baclofen)decreased intracellular Ca^(2+)concentrations,attenuated apoptosis,and reversed NMDA-induced upregulation of caspase-3,Src,NMDAR2A,NMDAR2B,and nNOS.Unexpectedly,a GABA_(A)receptor antagonist(bicuculline)and a GABA_(B)receptor antagonist(saclofen)failed to significantly increase excitatory neurotoxicity.Conclusions: Taken together,these results not only provide an experimental basis for SGD administration in the clinical treatment of central nervous system injury diseases,but also suggest that the Src-NR2A-nNOS pathway may be a valuable target in excitotoxicity treatment.

Therapeutic Effect of Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)with Acupoint Application on Chronic Lumbar Muscle Strain and the Recovery of Lumbar Strength

Objective:To study the therapeutic effect of Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)with acupoint application on chronic lumbar muscle strain and the recovery of lumbar strength.Methods:From August 2018 to August 2019,100 patients with chronic lumbar muscle strain admitted to our hospital were selected and randomly divided into an acupoint application group and a combined group of 50 cases each.The acupoint application group was treated by acupoint application,and the combined group was treated with Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)based on acupoint application.The indexes scores,VAS scores,ADL scores,ODI scores,lumbar endurance,TCM syndrome scores and the treatment effects of the two groups before and after treatment were statistically analyzed,and TNF-αand TXB2 levels were detected.Results:After treatment,the scores of indexes,VAS,ODI,TCM syndrome,TNF-αand TXB2 in the combined group were lower than those in the acupoint application group,and the ADL score,lumbar endurance and treatment efficiency were higher than those in the acupoint application group,with a statistical difference(P<0.05).Conclusion:Shaoyao Jiawei Gancao Decoction(芍药加味甘草汤)with acupoint application can effectively reduce pain,improve lumbar function,restore lumbar strength,effectively relieve inflammation,with significant effects.

Therapeutic targets and signal transduction mechanisms of medicinal plant formula Gancao Xiexin decoction against ulcerative colitis:A network pharmacological study

Background:Ulcerative colitis(UC)is a chronic disease that often presents with abdominal pain,diarrhea,hematochezia,and significant morbidity.Gancao Xiexin decoction(GXD),a traditional Chinese medicine,has been applied for the clinical treatment of UC,while its action mechanisms are unclear.Methods:The active ingredients and their targets of GXD,and UC-related targets,were derived from public databases.Protein-protein interaction,Gene Ontology(GO),and the Kyoto Encyclopedia of Genes and Genomes(KEGG)were used to analyze the important active compounds,key targets,and signaling pathways.Then,molecular docking and animal experiments were performed to verify the findings.A total of 213 active compounds and 89 common targets of GXD for UC were obtained.Results:The hub gene network showed ALB,AKT1,IL6,TNF,VEGFA,TP53,CXCL8,MAPK1,PTGS2,and IL1βmay be potential targets of GXD against UC.GO and KEGG pathway enrichment analyses suggested that the action of GXD against UC was mainly related to response to oxygen levels,lipopolysaccharide,and molecule of bacterial origin,etc.,and achieved by advanced glycation endproducts/receptors for advanced glycation endproducts signaling pathway in diabetic complications,hypoxia-inducible factor(HIF)-1 signaling pathway,interleukin-17/HIF-1 signaling pathway,TNF signaling pathway,etc.Molecular docking results showed that the GXD had good potency of action with the hub target.In vivo experiments showed that GXD significantly alleviated the symptoms of UC and down-regulated the expression of inflammatory factors,nuclear factor-κB and signal transducer and activator of transcription 3.Conclusions:The anti-UC action of GXD is mainly attributed to its anti-oxidative stress,antiinflammatory,and immunomodulatory functions.

Mechanism of cAMP-PKA Signaling Pathway Mediated by Shaoyao Gancao Decoction(芍药甘草汤)on Regulation of Aquaporin 5 and Muscarinic Receptor 3 Levels in Sjogren’s Syndrome

Objective:To investigate the mechanism of c AMP-PKA signaling pathway mediated by Chinese medicine formula Shaoyao Gancao Decoction(芍药甘草汤,SGD)on the regulation of aquaporin 5(AQP5)and muscarinic receptor 3(M3 R)levels in Sjogren’s syndrome(SS).Methods:Of the 30 mice,5 were randomly selected as control,and others were used for creating SS model.After successful modeling,mice were randomly divided into 5 groups(n=5 per group)and intragastrical y administered with saline(8 m L/kg),pilocarpine(1.4 mg/kg),or low,medium and high doses SGD(0.14,0.21,0.35 g/kg Radix paeoniae with 0.01 g/kg Radix glycyrrhizae,respectively)for 6 weeks.Human labial gland acinar cel s were treated with pilocarpine or varying doses of SGD with saline as the placebo.Hematoxylin and eosin staining was used to observe the histopathological changes of the submandibular glands of mice.The serum levels of anti-SS antigen A(SS-A),anti-SS antigen B(SS-B),M3 R,andα-fodrin in submandibular glands of mice were measured by enzyme-linked immunosorbent assay.Immunofluorescence staining was used to observe the spatial localization of AQP5 and M3 R in acinar cells.Reverse transcriptase polymerase chain reaction and Western blot were used to detect the expressions of PKA,c AMP,Epac1,AQP5,M3 R,nuclear factor kappa-B(NF-κB),and tumor necrosis factor(TNF)-αin submandibular gland tissues and cel s of each group.Results:Compared to normal mice,body weight,5-min salivary secretion,30-min secretion of tears and breakup time of tear film of model mice decreased at 1–6 weeks after immunization(al P<0.05),whereas water intake increased(al P<0.05).In the model group,glands of the submandibular glands showed atrophy,accompanied by acini of different sizes,decreased numbers and loose arrangement,with catheter dilatation and different degrees of lymphocyte infiltration.Conditions of mice in SGD groups were improved.The positive expression of AQP5 and M3 R were higher in the acinar cel s treated with al doses SGD compared to the normal group;serum levels of SS-A,SS-B,andα-fodrin were lower,and that of M3 R was higher in al doses SGD treated animals than the model or pilocarpine treated ones(al P<0.05).Compared to the model and pilocarpine groups,the m RNA and protein levels of NF-κB and TNF-αwere lower in mice or cel s treated with medium or high-dose SGD(al P<0.05),while those of PKA,Epac1,AQP5 and M3 R were higher(al P<0.05).Conclusion:SGD can improve symptoms of SS by regulating the c AMP-PKA signaling pathway and increasing AQP5 and M3 R levels.

Pretreatment of Shaoyao Gancao Decoction(芍药甘草汤)Alters Pharmacokinetics of Intravenous Paclitaxel in Rats

Objective：To investigate the effect of Shaoyao Gancao Decoction（芍药甘草汤,SGD）on the pharmacokinetics of intravenously administered paclitaxel in rats.Methods：Paclitaxel was intravenously administered to rats（3 mg/kg）with or without the concomitant administration of SGD（752 mg/kg,a single day or 14 consecutive days pretreatment）.The paclitaxel in the serum was quantified using a simple and rapid ultra performance liquid chromatography（UPLC）method for the pharmacokinetic study.The pharmacokinetic parameters were calculated via a non-compartment model using the computer program DAS 2.0.Results：The pharmacokinetic parameters of paclitaxel were significantly altered in response to 14 consecutive days of pretreatment with SGD.The area under the curve（AUC0-t,from 4 820±197 to 4 205±186 ng·m L-1·h-1）and AUC0-∞（from 5 237±280 to 4 514±210 ng·m L-1·h-1）significantly decreased in response to the 14-day pretreatment with SGD.The values of V dss（L/kg）were 10.74±1.08 and 9.35±0.49,those of CL（L/kg）were0.67±0.03 and 0.57±0.03 and the t1/2（h）values were 11.17±0.84 and 11.32±0.93,respectively,for the14-day SGD pretreatment and intravenous paclitaxel alone.The AUC0-t and AUC0-∞values decreased by13%and 14%（P〈0.01）,respectively.The area under the curve decreased significantly（P〈0.01）,and the total clearance increased by 1.2-fold（P〈0.01）,after 14 consecutive days of pretreatment with SGD.A single-day pretreatment with SGD did not significantly affect the pharmacokinetic parameters of paclitaxel.Conclusions：SGD administration for 14 consecutive days increased the metabolism of paclitaxel,while a 1-day pretreatment had little effect.The results would contribute important information to the study on interaction between Chinese medicines and chemotherapy and also help to utilize SGD better in the adjunctive therapy of cancer patients.

Exploration of the Potential Mechanism of Gancao Yangyin Decoction on Aging Based on Network Pharmacology

Objective:To explore the targels and molecular mechanism of Gancao Yangyin Decoction(甘草养阴汤,GCYYD)based on network pharmacology.Methods:The effective chemical components of 7 kinds of Chinese materia medica in GCYYD and their relevant targets were obtai ned through the traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)and the encyelopedia of traditional Chinese medicine(ETCM).The aging-related targets were obtained through GeneCards database.The targets related to the effective chemical components were mapped with the aging-related targets,and the gene targets of GCY YD for intervening in aging were obtained.The protein interaction network diagram of GCY YD interfering with aging was drawn through String database and Cytoscape 3.7.1 software,and the core target genes were screened.The potential targets obtained were analyzed by gene ontology(GO)biological function enrichment analysis and kyoto encyelopedia of genes and genomes(KEGG)pathway enrichment analysis.Results:Totally 130 effective chemical components of the 7 kinds of Chinese materia medica of GCYYD and 276 related targets were obtained from TCMSP and ETCM databases.Totally 216 aging-related targets were obtained through GeneCards database.There were 63 target genes intervening in aging in GCYYD,with core target genes ALB,AKTI,TNF,L 6,MMP-3,VEGFA,CASP5,etc.Through biological function and signaling pathway enrichment analyses for the target genes with R software,147 KEGG signaling pathways were found,mainly related to age-RAGE signaling pathway in diabetic complications,proteoglycans in cancer,fluid shear stress and atherosclero-sis,HIF-1 signaling pathway,human cytomegalovirus infection,celluar senescence,prostate cancer,bladder cancer,elte.Conclusion:GCYYD can intervene in aging through"multicomponents-mulitargets-multipath-ways",which lays foundation for further experimental research.