Inhibition of N-methyl-N-nitrosourea-induced gastric tumorigenesis by Liuwei Dihuang Pill in db/db mice

AIM To investigate the inhibitory effect of Liuwei Dihuang Pill(LDP) on gastric tumorigenesis induced by N-methyl-N-nitrosourea(MNU) in diabetic mice.METHODS Four-week-old mice were divided into four groups: A, 12 db/m mice treated with MNU and saline, as the non-diabetic control; B, 12 db/db mice treated with MNU and saline, as the diabetic control; C, 12 db/db mice treated with MNU and metformin, as the positive control; and D, 12 db/db mice treated with MNU and LDP. MNU was administrated for 20 wk to induce gastric carcinogenesis. LDP was administrated for 10 wk for improvement of insulin resistance. Body weight and food intake were measured every week. Blood samples were collected for assays of fasting blood glucose, insulin, insulin-like growth factor(IGF)-1, adiponectin and leptin. Stomach tissues were collected for histopathological analysis, immunohistochemical staining of Ki67, quantitative reverse transcriptionpolymerase chain reaction and western blotting. RESULTS The incidence of MNU-induced gastric dysplasia was significantly elevated in diabetic(db/db) mice relative to the control(db/m) mice. The incidence of gastricdysplasia was significantly reduced by LDP with suppression of cell proliferation, as demonstrated by a decrease in Ki67 staining. Hyperglycemia, hyperinsulinemia and serum IGF-1 were inhibited by LDP. Expression of IGF-1 and insulin receptor m RNAs was decreased, phosphorylation of IGF-1 receptor and AKT protein was reduced in the stomach tissues by LDP. In addition, adiponectin was increased and leptin was decreased in the serum by LDP. CONCLUSION LDP decreased risk of gastric dysplasia in type 2 diabetic mice by down-regulation of IGF and insulin activity and correction of adipokines disorders.

The Nano Pulverization of Traditional Chinese Medicine Liuwei Dihuang

The crude drug of Liuwei Dihuang was pulverized to nano particles to improve its bioavailability. The appropriate technique parameters were studied. Paeonol, typical marker of Liuwei Dihuang , was extracted with organic solveat in ultrasonic and its content was determined by HPLC. The appropriate techrdques parameters are as follows ： rotating speed control 1200 r/ min , grinding time control 50min and mass percent concentration control 3.8% . The experimental results show that the average particle diameter is 161.9 nm and the great majority of the plant cell wall is broken into pieces after nano pulverization. The extraction efficiency of paeonol is increased by 23.5% .

Active fraction combination from Liuwei Dihuang decoction improves adult hippocampal neurogenesis and neurogenic microenvironment in cranially irradiated mice

OBJECTIVE Cranial radiotherapy is clinically used in the treatment of brain tumors;however,the consequent cognitive and emotional dysfunctions seriously impair the life quality of patients.LW-AFC,an active fraction combination extracted from classical traditional Chinese medicine prescription Liuwei Dihuang decoction,can improve cognitive and emotional dysfunctions in many animal models;however,the protective effect of LW-AFC on cranial irradiation-induced cognitive and emotional dysfunctions has not been reported.Recent studies indicate that impairment of adult hippocampal neurogenesis(AHN)and alterations of the neurogenic microenvironment in the hippocampus constitute critical factors in cognitive and emotional dysfunctions following cranial irradiation.Here,our research further investigated the potential protective effects and mechanisms of LW-AFC on cranial irradiation-induced cognitive and emotional dysfunctions in mice.METHODS LW-AFC(1.6 g·kg^(-1))was intragastrically administered to mice for 14 d before cranial irradiation(7 Gyγ-ray).AHN was examined by quantifying the number of proliferative neural stem cells and immature neurons in the dorsal and ventral hippocampus.The contextual fear conditioning test,open field test,and tail suspension test were used to assess cognitive and emotional functions in mice.To detect the change of the neurogenic microenvironment,colorimetry and multiplex bead analysis were performed to measure the level of oxidative stress,neurotrophic and growth factors,and inflammation in the hippocampus.RESULTS LW-AFC exerted beneficial effects on the contextual fear memory,anxiety behavior,and depression behavior in irradiated mice.Moreover,LW-AFC increased the number of proliferative neural stem cells and immature neurons in the dorsal hippocampus,displaying a regional specificity of neurogenic response.For the neurogenic microenvironment,LW-AFC significantly increased the contents of superoxide dismutase,glutathione peroxidase,glutathione,and catalase and decreased the content of malondialdehyde in the hippocampus of irradiated mice,accompanied by the increase in brain-derived neurotrophic factor,insulin-like growth factor-1,and interleukin-4 content.Together,LW-AFC improved cognitive and emotional dysfunctions,promoted AHN preferentially in the dorsal hippocampus,and ameliorated disturbance in the neurogenic microenvironment in irradiated mice.CONCLUSION LW-AFC ameliorates cranial irradiation-induced cognitive and emotional dysfunctions,and the underlying mechanisms are mediated by promoting AHN in the dorsal hippocampus and improving the neurogenic microenvironment.LW-AFC might be a promising therapeutic agent to treat cognitive and emotional dysfunctions in patients receiving cranial radiotherapy.

Effects of Liuwei Dihuang decoction and its compatible prescriptions on the function of hypothalamus-pituitary-adrenal axis in senescence-accelerated mouse

OBJECTIVE To investigate age-related functional change of hypothalamus-pituitary-adrenal(HPA)axis in senescence accelerated mouse(SAM)and the effects of Liuwei Dihuang decoction(LW)and its San-bu(three tonics)and San-xie(three eliminators)components on the function of HPA axis.METHODS Male SAM-resistance/1(SAMR1)and SAM-prone/8(SAMP8)at the ages of 6and 12 months old were used.SAMP8 were orally administered with LW,three tonics and three eliminators at the doses of 10,6.4and 3.6g·kg-1·d-1,respectively,for consecutive 60 d.Serum level of CORT was assayed with ELISA method.The levels of hypothalamic CRH and pituitary ACTH was determined with radioimmunoassay.RESULTS The levels of hypothalamic CRH,pituitary ACTH and serous CORT were much higher in 6 and 12 months old SAMP8 than those in age-matched SAMR1,which suggested the abnormal function of HPA axis in SAMP8.Oral administration of LW and three tonics significantly decreased the level of hypothalamic CRH of 6 and 12 months old SAMP8,and reduced the levels of pituitary ACTH and serous CORT of 6 month old SAMP8.Three eliminators significantly decreased the level of hypothalamic CRH of 6 months old SAMP8.The results indicated that oral administration of LW,three tonics and three eliminators improved the function of HPA axis of SAMP8.CONCLUSION The results showed the hyperactivity of HPA axis of SAMP8,and LW improved the hyperactivity of 6 month old SAMP8.Three tonics and three eliminators had similar effects as LW,which had better effect after compatibility.

Liuwei Dihuang soft capsules inhibits phenotypic conversion of VSMCs via up-regulating expression of myocardin by promoting interaction between ERα and SRC3

OBJECTIVE To reveal the under mechanism of how Liuwei Dihuang(LWDH)inhibits the phenotypic con⁃version of VSMCs.METHODS 24 ApoE-/-mice were divided into 4 groups:sham group,model group,E2 group,and LWDH group.Six C57BN/L6 mice were used as control group.The primary VSMCs were divided into control group,model group,E2 group,LWDH group,LWDH+MPP group,and LWDH+PHTPP group with or without control siRNA,ERαsiRNA,ERβsiRNA,and myocardin siRNA.Oil red staining was used to evaluate the lipid deposition in the cardiac aorta.Serum chemistry analysis to test serum TG,TC,LDL,and HDL.Immunofluorescence staining was used to testα-SMA,osteopontin and F-actin.Immunohistochemical staining was performed to check out the myocardin in the cardiac aorta.The mRNA levels ofα-SMA,osteopontin,ERα,ERβ,SRC3 and myocardin were detected by real time-PCR,and the protein expression levels of them were detected by Western blotting.Co-immunoprecipitation was proceeded to test the interaction between ERαand SRC3 and SRC3 and myocardin.Flow cytometry was used to check out the cell cycle.Wound healing assay and Transwell were managed to evaluate the migration capacity of VSMCs.RESULTS In vivo administration of LWDH suppressed atherosclerotic symptoms,decreases phenotypic marker of vascular endothelial cell,and increases phenotypic marker of VSMC in ovariectomized ApoE-/-female mice.Moreover,LWDH significantly increased the mRNA and protein expression levels of ERα,ERβ,SRC3 and myocardin in the cardiac aorta of ovariecto⁃mized ApoE-/-female mice.In vitro,LWDH altered cell cycle and reduced the elevated cyclinD protein expression migra⁃tion capacity and in the model VSMCs.In addition,LWDH inhibited phenotypic conversion and promoted the expression of ER,SRC3,and myocardin of the primary VSMC phenotypic conversion model.Inhibition of ERαalmost completely eliminated the impacts of LWDH onα-SMA and osteopontin.Furthermore,LWDH promoted the interaction between ERαand SRC3 and up-regulated the co-activation of SRC3 and myocardin.CONCLUSION LWDH could inhibit the pheno⁃typic conversion of VSMCs in vitro and in vivo by increasing the activity of myocardin through up-regulating the expres⁃sion of ERαand promoting the interaction between ERαand SRC3.Our research reveals the under mechanism of how LWDH inhibits the phenotypic conversion of VSMCs.

Effects of LW-AFC,a new formula derived from Liuwei Dihuang decoction,on intestinal microbiome in senescence-accelerated mouse prone 8 strain,a mouse model of Alzheimer disease

OBJECTIVE To investigate the effects of LW-AFC,a new formula derived fromLiuwei Dihuang decoction,on gut microbiota and the behavior of learning and memory of SAMP8 mice,a mouse model of Alzheimer Disease(AD),and identify the specific intestinal microbiota correlating with cognitive ability.METHODS Morris-water maze test,novel object recognition test and shuttle-box test were conducted to observe the ability of learning and memory.16S rRNA amplicon sequencing(Illumina,San Diego,CA,USA)was employed to investigate gut microbiota.RESULTS The treatment of LW-AFC improved cognitive impairments of SAMP8 mice,including spatial learning and memory ability,active avoidance response,and object recognition memory capability.Our data indicated that there were significantly 8 increased and 12 decreased operational taxonomic units(OTUs)in the gut microbiota of SAMP8 mice compared with senescence accelerated mouse resistant 1(SAMR1) strains,the control of SAMP8 mice.The treatment of LW-AFC altered 22(16 increased and 6 decreased)OTUs in SAMP8 mice and among them,15 OTUs could be reversed by LW-AFC treatment resulting in a microbial composition similar to that of SAMR1 mice.We further showed that there were7(3 negative and 4 positive correlation)OTUs significantly correlated with all the three types of cognitive abilities,at the order level,including Bacteroidales,Clostridiales,Desulfovibrionales,CW040,and two unclassified orders.LW-AFC had influences on bacterial taxa correlated with the abilities of learning and memory in SAMP8 mice and restored them to SAMR1 mice.CONCLUSION The effects of LW-AFC on improving cognitive impairments of SAMP8 mice might be via modulating intestinal microbiome and LW-AFC could be used as a potential anti-AD agent.

Construction of NIM sub-network regulated by Liuwei Dihuang decoction and its characteristics

OBJECTIVE To construct the neuroendocrine immunomodulation(NIM) sub-network regulated by Liuwei Dihuang decoction(LW) and analyze its characteristics.METHODS We took the GSE57273 in GEO database and screened the differentially expressed genes(DEGs)(P<0.01) by the GEO2 R tool as gene expression signature of LW.The global PPI network was constructed in the context of whole PPI network through direct interaction algorithm and forest algorithm respectively.Then the enrichment and the topological characteristics of NIM signaling molecules were evaluated by permutation test.Finally,we abstracted the NIM sub-network by NIMNT,which combined the NIM molecular network and forest algorithm,and analyzed the topological characteristics of it by the Network Analyzer(release 2.7) plugin in Cytoscape v3.5.1.RESULTS We got 2468 DEGs in the gene expression signature of LW.After analyzing the global PPI network of LW got by two kinds of algorithms,we found that the NIM signaling molecules significantly enriched and located in important positions in the global PPI network.The NIM sub-network regulated by LW contained 1099 nodes and 1056 edges.We screened out 22 hub nodes(Degree>10).Among them,there were 19 NIM signaling molecules in which only ESR1 changed significantly and 3 non-DEGs(NFATC2,RARA,TP53).However,the down.stream of the hub nodes were significantly changes.CONCLUSION The results suggested that LW might mainly regulate the non-hub nodes to recovery of the network imbalance of the body in the state of disease.

六味地黄丸治疗早发性卵巢功能不全模型小鼠的分子机制

背景:在临床上,大多数治疗早发性卵巢功能不全的方剂均以六味地黄丸为基础方演变而来,并且取得了较好的疗效。目前对六味地黄丸的实验研究大多为体内动物模型的形态学观察及生理生化指标的检测,而分子机制方面的研究报道较少。目的:探讨六味地黄丸治疗早发性卵巢功能不全的分子作用机制。方法:环磷酰胺120 mg/kg联合白消安12 mg/kg腹腔注射制备早发性卵巢功能不全小鼠模型,然后用六味地黄丸混悬液对早发性卵巢功能不全小鼠进行干预,干预12周采用ELISA法检测小鼠血清中促卵泡刺激素、促黄体生成素、雌激素、抗苗勒氏管激素、8-羟脱氧鸟苷、总抗氧化能力、活性氧水平;苏木精-伊红染色观察小鼠卵巢形态学改变;透射电镜观察小鼠卵泡颗粒细胞超微结构及颗粒细胞线粒体的凋亡情况;免疫组化法检测六味地黄丸对小鼠卵巢颗粒细胞中核受体转录辅激活因子(receptor gamma coactivator-1 alpha,PGC-1α)、线粒体转录因子A(mitochondrialtranscriptionfactorA,TFAM)的表达水平。结果与结论:①与模型组比较,实验组小鼠血清促卵泡刺激素、促黄体生成素、活性氧、8-羟脱氧鸟苷水平降低(P<0.05),雌激素、抗苗勒氏管激素、总抗氧化能力水平升高(P<0.05);②苏木精-伊红染色发现模型组小鼠卵巢组织中闭锁卵泡和黄体较多,个别可见次级卵泡,间质纤维化增生;实验组小鼠卵巢组织可见大量闭锁卵泡,黄体较少,边缘可见原始卵泡,次级卵泡较少,未见明显成熟卵泡;③透射电镜发现实验组小鼠卵巢颗粒细胞内细胞器较完整;④免疫组化结果显示,实验组小鼠卵巢组织PGC-1α的表达水平在第4周稍有升高至第8,12周无明显变化,与模型组有明显差异;实验组小鼠卵巢组织TFAM的表达水平在第4周时短暂升高,然后稍有所下降,但结果均与模型组有明显差异;⑤结果表明,六味地黄丸通过PGC-1α-TFAM-ROS信号通路在一定程度上抑制早发性卵巢功能不全小鼠卵巢颗粒细胞凋亡,从而改善卵巢的内分泌功能,提高机体抗氧化能力,减轻氧化应激损伤程度。

Liuwei Dihuang Decoction improves cognitive impairments via regulating immune system in APP/PS1 transgenic mouse, a mouse model of Alzheimer disease

OBJECTIVE To investigate the effect and mechanisms of Liuwei Dihuang Decoction(LW) on cognition in PrP-hAβPPswe/PS1ΔE9(APP/PS1) transgenic mice.METHODS LW was adminis.trated with oral for 3 months.The locomotor activity test was performed to investigate the spontaneous motor activity of mice.The Morris water maze test and shuttle box test were performed to investigate the spatial learning and memory and active avoidance response respectively.The Αβ deposits and neuron loss in the hippocampus was detected by immunofluorescence staining and nissl staining respectively.The flow cytometry was employed to investigate the lymphocyte subsets of the mice.The 3 H-thymidine incorporation was performed to investigate the splenocytes proliferation.RESULTS The treatment of LW ameliorated the impairments of spatial learning and memory and active and passive avoidance in APP/PS1 mice.The administration of LW alleviated neuron loss in the brain,suppressed amyloid-β(Αβ) deposits in the hippocampus of APP/PS1 mice.The treatment of LW significantly increased ConAand LPS-induced proliferation of splenocytes,increased CD3+ T cells and CD19+ B cells in the spleen lymphocytes and reduced Gr1+ cells in APP/PS1 mice.CONCLUSION This data indicated the adminis.tration of LW ameliorated behavioral and pathological deterioration via regulating immune function.

Pharmacological mechanism of Liuwei Dihuang Decoction in treating Osteoporosis and Alzheimer’s disease based on Network Pharmacology

Objective:To predict the active components,targets of Liuwei Dihuang Decoction for the treatment of osteoporosis and Alzheimer’s disease with network pharmacology analysis.Methods:The components and targets of all the herbs of Liuwei Dihuang Decoction were obtained from ETCM database,Osteoporosis and Alzheimer’s disease targets were extracted from the GeneCards and DrugBank databases,selected based on STRING database for PPI network construction and build the network through the software of Cytoscape,Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses were carried out by R software.Results:181 active compounds and 762 disease-related from Liuwei Dihuang Decoction,there were 144 disease-related common targets obtained from Venn Diagram.IL6,TNF,PPARA,PPARG,SRC and other 11 targets are the node protein of the whole network.In the multisystem biological reaction process such as neurotransmitter,glucose and lipid metabolism,calcium and phosphorus metabolism.Conclusion:Liuwei Dihuang pill may play a co-therapeutic role in Osteoporosis and Alzheimer’s disease by participating in the multi-system biological reaction process such as neurotransmitter,glucose and lipid metabolism,calcium and phosphorus metabolism.

针灸联合六味地黄丸序贯治疗肾精亏虚型早发性卵巢功能不全临床观察

目的探究针灸联合六味地黄丸序贯治疗对肾精亏虚型早发性卵巢功能不全(POI)的临床效果。方法将63例POI患者纳入试验,于月经期、经后期口服六味地黄丸,排卵期、经前期行灸法序贯治疗,针刺治疗贯穿后三期,隔日1次,每周治疗3次;连续治疗3个月经周期。观察序贯治疗对患者促卵泡生成激素(FSH)、促黄体生成素(LH)、雌二醇(E2)水平及中医证候积分的影响。结果治疗3个月后,患者E2增高,FSH、LH均降低,中医证候得到有效改善(P<0.05)。结论采用针灸联合六味地黄丸序贯治疗肾精亏虚型POI,能较好地改善性激素水平及临床症状,且具有良好安全性。

六味地黄汤对老化模型大鼠视网膜组织铁死亡通路的影响

目的:观察六味地黄汤对老化模型大鼠视网膜组织中铁蛋白、溶质载体家族7成员11(SLC7A11)、谷胱甘肽(GSH)、谷胱甘肽过氧化物酶4(GPX4)表达水平的影响。方法:将45只SD大鼠随机分成空白组、模型组、中药组,各15只。空白组腹腔注射生理盐水,模型组、中药组腹腔注射D-半乳糖[500 mg/(kg·d)],同时,中药组灌服六味地黄汤[6.75 g/(kg·d)],空白组、模型组灌服等体积生理盐水,连续8 wk。检测各组大鼠视网膜组织中铁蛋白、SLC7A11、GSH、GPX4的表达水平。结果:模型组较空白组大鼠视网膜组织中铁蛋白表达增高(P<0.05),GSH、SLC7A11、GPX4表达降低(P<0.05)。中药组较模型组大鼠视网膜组织中GSH、SLC7A11、GPX4表达增高(P<0.05)。结论:六味地黄汤可能通过调控铁死亡途径发挥延缓视网膜老化的作用。

六味地黄汤对阿霉素肾病小鼠尿蛋白及肾小球p-p38 MAPK、α-actinin-4、synaptopodin的影响

目的探讨六味地黄汤对阿霉素肾病(adriamycin nephropaghy,ADRN)小鼠尿蛋白的干预作用及可能机制。方法30只SPF级雄性C57BL/6小鼠随机选取5只为空白组,余下小鼠以单次尾静脉注射阿霉素(0.01 g·kg^(-1))复制ADRN模型,2周后将造模成功的小鼠随机分为模型组、贝那普利组及低、中、高剂量六味地黄汤组,每组5只。空白组、模型组予等体积注射用水灌胃,贝那普利组予0.0013 g·kg^(-1)贝那普利灌胃,低、中、高剂量六味地黄汤组分别予9.75、19.5、39 g·kg^(-1)六味地黄汤灌胃,每日1次,连续8周。自动生化仪检测小鼠24 h尿蛋白定量、血清白蛋白、血清肌酐、血清钾;HE染色法、电镜观察小鼠肾脏病理改变及足突形态;免疫组织化学法、Western bolt检测小鼠肾脏磷酸化p38 MAPK(phosphorylated p38 MAPK,p-p38 MAPK)、α-辅肌动蛋白-4(α-actinin-4)、突触孔蛋白(synaptopodin)的表达;RT-PCR检测小鼠肾脏α-actinin-4、synaptopodin mRNA表达。结果与空白组比较,模型组小鼠体质量、24 h尿量、血清白蛋白降低,24 h尿蛋白定量升高(P<0.05);肾小球系膜细胞增生,部分系膜区扩大、肾小管蛋白管型,间质可见炎性细胞浸润,足突广泛融合;α-actinin-4蛋白及mRNA、p-p38 MAPK蛋白表达升高(P<0.05),synaptopodin蛋白及mRNA表达降低(P<0.05)。与模型组比较,各干预组病理改变及足突融合均得到不同程度改善,贝那普利组及中、高剂量六味地黄汤组24 h尿蛋白定量减少,高剂量六味地黄汤组p-p38 MAPK蛋白表达降低(P<0.05),低、中、高剂量六味地黄汤组synaptopodin蛋白及mRNA表达均升高(P<0.05)。贝那普利组及低、中、高剂量六味地黄汤组组间比较,各指标差异无统计学意义(P>0.05)。结论六味地黄汤可能通过抑制p38 MAPK通路活化和上调α-actinin-4、synaptopodin蛋白及mRNA表达,改善肾脏病理及足突融合,减轻足细胞损伤,减少ADRN小鼠尿蛋白。

六味地黄丸联合大黄液治疗慢性牙周炎的效果分析

目的:分析六味地黄丸联合大黄液治疗慢性牙周炎的效果及对牙龈指数(GI)、探针深度(PD)的影响。方法:选取2021年1月-2022年1月广州市胸科医院诊治的慢性牙周炎患者86例作为研究对象,按照随机数字表法分为对照组和联合组,各43例。两组患者均采用常规治疗,对照组在此基础上局部应用大黄液治疗,联合组在对照组基础上给予六味地黄丸治疗。比较两组临床疗效、中医证候积分、牙周健康指标和炎性因子水平。结果:联合组临床疗效总有效率高于对照组,差异有统计学意义(P=0.044)。治疗后,两组牙齿松动、肢体沉重、牙龈红肿、胸闷口苦、反复咽痛评分均下降,且联合组低于对照组,差异有统计学意义(P<0.05);治疗后,两组GI和PD均下降,联合组低于对照组,差异有统计学意义(P<0.05);治疗后,两组C反应蛋白、肿瘤坏死因子-α和白细胞介素-1β水平均下降,且联合组低于对照组,差异有统计学意义(P<0.05)。结论:六味地黄丸联合大黄液治疗慢性牙周炎的临床效果显著,可有效改善患者中医证候积分和牙周健康指标,降低炎性因子水平。

调补肺肾治疗慢性阻塞性肺疾病缓解期临床研究

目的探讨调补肺肾治疗慢性阻塞性肺疾病(COPD)缓解期的临床效果。方法选取2021年5月至2023年5月江西省胸科医院收治的94例COPD缓解期患者作为研究对象,按照随机数字表法分为对照组(47例)和观察组(47例)。对照组患者采用常规西医治疗,观察组患者加用调补肺肾治疗(六味地黄汤加减和揿针特定穴位治疗),两组均连续治疗12周。比较两组患者的临床疗效、肺功能、血气指标、呼吸困难程度、生活质量、中医证候积分及不良反应。结果观察组治疗总有效率高于对照组,差异有统计学意义(P<0.05);两组治疗前第1秒用力呼气容积(FEV_(1))、用力肺活量(FVC)、FEV_(1)/FVC、动脉二氧化碳分压(PaCO_(2))、动脉氧分压(PaO_(2))、中医证候积分、改良版英国医学研究委员会呼吸问卷(mMRC)、COPD评估测试问卷(CAT)评分比较,差异无统计学意义(P>0.05);两组治疗后FEV_(1)、FVC、FEV_(1)/FVC、PaO_(2)高于本组治疗前,PaCO_(2)、中医证候积分及mMRC、CAT评分低于本组治疗前,差异有统计学意义(P<0.05);观察组治疗后FEV_(1)、FVC、FEV_(1)/FVC、PaO_(2)高于对照组,PaCO_(2)、中医证候积分及mMRC、CAT评分低于对照组,差异有统计学意义(P<0.05);两组不良反应总发生率比较,差异无统计学意义(P>0.05)。结论缓解期COPD患者应用调补肺肾治疗具有较高的价值,可改善通气及肺功能,缓解呼吸困难,提高生活质量,安全可靠,效果显著。

蒙药安神补心六味丸联合针刺治疗睡眠障碍的临床研究

目的 评价安神补心六味丸联合针刺治疗睡眠障碍的有效性和安全性。方法 选取2020年8月至2022年10月收治的120例睡眠障碍患者为研究对象,按照随机数字表法将其分为治疗组(57例,安神补心六味丸+针刺治疗)和对照组(56例,针刺治疗)。比较两组的治疗效果。结果 治疗组的治疗总有效率高于对照组(P<0.05);治疗后,两组的匹兹堡睡眠质量指数(PSQI)评分、中医证候积分低于治疗前,生活质量评分高于治疗前,且治疗组优于对照组(P<0.05)。结论 安神补心六味丸联合针刺治疗睡眠障碍效果显著,且安全性较高。

六味地黄丸介导RAGE抑制MMP-2/MMP-9对Aβ_(1-40)损伤bEnd.3细胞紧密连接蛋白的影响

目的探讨六味地黄丸对β淀粉样蛋白1-40(Aβ_(1-40))损伤的小鼠脑微血管内皮细胞(bEnd.3)的保护作用及其机制。方法采用CCK8法检测Aβ_(1-40)和六味地黄丸含药血清(MSLDP)对细胞活性的影响,筛选合适的作用浓度。将bEnd.3细胞分为对照组、Aβ_(1-40)组、MSLDP+Aβ_(1-40)组和MSLDP组,采用Western blot检测低密度脂蛋白相关蛋白1(LRP1)、晚期糖基化终末产物受体(RAGE)、基质金属蛋白酶2(MMP-2)、MMP-9、闭锁小带蛋白-1(ZO-1)、脑源性神经营养因子(BDNF)蛋白表达,免疫荧光检测LRP1、RAGE、ZO-1表达;再将bEnd.3细胞分为对照组、Aβ_(1-40)组、FPS-ZM1(RAGE抑制剂)+Aβ_(1-40)组和FPS-ZM1+Aβ_(1-40)+MSLDP组,Western blot检测RAGE、MMP-9、MMP-2、ZO-1蛋白表达。结果Aβ_(1-40)呈剂量依赖性降低bEnd.3细胞活性(P<0.01),MSLDP对Aβ_(1-40)损伤的细胞活性具有保护作用(P<0.05,P<0.01),因此选择10μmol/L Aβ_(1-40)和10%MSLDP进行后续实验。与对照组比较,Aβ_(1-40)组RAGE、MMP-2、MMP-9蛋白表达升高(P<0.01),LRP1、ZO-1、BDNF蛋白表达降低(P<0.05,P<0.01),并且LRP1、ZO-1荧光强度降低(P<0.01),RAGE荧光增强(P<0.01);与Aβ_(1-40)组比较,MSLDP组RAGE、MMP-2、MMP-9蛋白表达和RAGE荧光强度降低(P<0.05,P<0.01),而LRP1、ZO-1、BDNF蛋白表达和LRP1、ZO-1荧光强度升高(P<0.05,P<0.01)。与Aβ_(1-40)组比较,Aβ_(1-40)+FPS-ZM1组MMP-2、MMP9、RAGE蛋白表达降低(P<0.05,P<0.01),ZO-1蛋白表达升高(P<0.05);Aβ_(1-40)+FPS-ZM1+MSLDP组MMP-2、MMP9、RAGE蛋白表达降低(P<0.01),ZO-1蛋白表达升高(P<0.01),FPS-ZM1和MSLDP联合使用的效果更佳。结论六味地黄丸能够保护Aβ_(1-40)损伤的脑微血管内皮的细胞紧密连接,减轻血脑屏障障碍,保护神经血管单元防治阿尔茨海默病,可能通过调节RAGE途径抑制MMP-2/MMP-9途径实现。

基于网络药理学和分子对接探讨六味地黄丸异病同治老年性聋和阿尔茨海默病的作用机制

目的借助网络药理学和分子对接的方法探讨六味地黄丸异病同治老年性聋和阿尔茨海默病(AD)的共同分子机制,并采用细胞实验进行验证。方法利用中药系统药理学数据库与分析平台(TCMSP)、成分靶点数据库(SwissADME)获取六味地黄丸有效成分,并结合基因和化学物质相互作用预测数据库(STITCH)获取作用靶点。利用人类基因综合数据库(Genecard)、人类疾病相关基因与突变位点信息数据库(DisGeNET)获取老年性聋和AD靶点。使用Cytoscape 3.7.1软件构建“中药-成分-靶点-疾病”网络,借助蛋白互作网络分析数据库(STRING)构建蛋白-蛋白相互作用(PPI)网络,获取核心靶点。利用富集分析数据平台(Metascape)对共有靶点进行基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析。采用AutoDock Vina 1.1.2软件对核心靶点与相关成分进行分子对接。采用过氧化氢(H2O2)致PC12细胞氧化损伤模型,观察六味地黄丸的作用。采用细胞计数(CCK-8)法观察细胞存活率,活性氧(ROS)检测试剂盒检测ROS水平,酶联免疫吸附测定(ELISA)法检测肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)水平,Western blot法检测基质金属蛋白酶-9(MMP-9)、蛋白激酶B(AKT)、磷酸化-蛋白激酶B(p-AKT)蛋白表达。结果共获得六味地黄丸有效成分86种,主要来自薯蓣皂素、视黄醇、丹皮酚、软脂酸等化合物,六味地黄丸防治老年性聋和AD共有靶点49个,核心靶点为AKT1、TNF、IL-6、MMP-9等。GO分析表明这些共有靶点主要参与对氧化应激的反应、对脂多糖的反应等生物进程。KEGG通路分析显示,共有靶点主要涉及缺氧诱导因子-1(HIF-1)、核因子-κB(NF-κB)等信号通路。分子对接结果显示,TNF-α、IL-6、AKT1、MMP-9与相关成分均存在结合可能。细胞实验结果发现,六味地黄丸含药血清可以增强细胞存活率,降低细胞ROS、TNF-α、IL-6水平,增加MMP-9蛋白表达水平,降低p-AKT蛋白表达水平。结论六味地黄丸异病同治老年性聋和AD具有物质基础,其共同的分子机制,可能是通过薯蓣皂素、视黄醇、丹皮酚等多种成分,作用于AKT、TNF-α、IL-6、MMP-9等核心靶点,抑制炎症反应和氧化应激,减少神经元死亡。

六味地黄丸通过干预缝隙连接通讯功能调控树突状细胞抗原交叉提呈能力研究

【目的】探讨六味地黄丸是否通过提高缝隙连接通讯功能(GJIC)增强树突状细胞(DC)抗原交叉提呈能力,并对其机制进行相关探索。【方法】采用Western Blot实验、免疫荧光法观察六味地黄丸含药血清对黑色素瘤细胞(B16)缝隙连接蛋白43(Cx43)表达及膜定位的影响;采用钙黄绿素(Ca-AM)/DiI荧光示踪实验观察六味地黄丸含药血清对B16细胞GJIC功能的影响;采用流式细胞术观察GJIC在六味地黄丸含药血清增强DC抗原提呈能力中的作用;采用碘化丙啶(PI)/Hoechst染色实验观察CD8+T淋巴细胞的免疫杀伤效果。【结果】Western Blot及免疫荧光实验结果显示,六味地黄丸含药血清上调Cx43表达;荧光示踪实验证明,六味地黄丸含药血清显著增强B16细胞GJIC功能;流式细胞术分析表明,六味地黄丸含药血清增强DC抗原提呈能力;PI/Hoechst染色结果显示,六味地黄丸含药血清干预B16-OVA后CD8+T细胞的免疫杀伤效果更加显著。【结论】六味地黄丸通过上调黑色素瘤细胞Cx43表达,改善GJIC功能,进而增强DC的交叉提呈能力,从而激活更强的CD8+T细胞免疫杀伤反应。

基于BDNF/TrkB/CREB通路研究六味地黄丸对丙戊酸钠诱导的孤独症谱系障碍模型仔鼠的作用机制

目的基于脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)/酪氨酸激酶受体B(tyrosine kinase receptor B,TrkB)/cAMP反应元件结合蛋白(cAMP response element binding protein,CREB)通路,探讨六味地黄丸对丙戊酸钠(sodium valproate,VPA)诱导的孤独症谱系障碍(autism spectrum disorder,ASD)仔鼠的作用机制。方法将13只SD孕鼠随机分为两组,其中10只孕鼠在第12.5天时腹腔注射VPA溶液(600 mg·kg^(-1))为VPA组,另外3只孕鼠注射等体积生理盐水为对照组。第21天对两组雄性仔鼠开展行为学检测,筛选出符合ASD疾病模型的仔鼠30只,随机分为模型组(等体积生理盐水),维生素D组(1480 IU·kg^(-1)),六味地黄丸高(3 g·kg^(-1))、中(1.5 g·kg^(-1))、低(0.75 g·kg^(-1))剂量组,每组6只。正常雄性仔鼠6只,设为空白组(等体积生理盐水)。各组仔鼠连续灌胃14 d,1次/d,给药后再次开展行为学检测。尼氏染色观察各组仔鼠海马组织神经元形态学变化,比色法检测各组仔鼠海马组织中谷氨酸(glutamic acid,GLU)、γ-氨基丁酸(gamma-aminobutyric acid,GABA)含量;qRT-PCR检测各组仔鼠海马组织中BDNF、TrkB、CREB mRNA相对表达。结果与对照组比较,VPA组仔鼠体质量、身长、尾长更小(P<0.05)。与空白组比较,模型组社交障碍症状明显(P<0.01),焦虑障碍症状明显(P<0.01),重复刻板行为增多(P<0.05或P<0.01),海马神经元结构损伤,GLU升高(P<0.01)、GABA下降(P<0.01),BDNF、TrkB、CREB mRNA表达降低(P<0.05或P<0.01);与模型组比较,维生素D组及六味地黄丸中、低剂量组仔鼠社交能力增强(P<0.05或P<0.01),焦虑障碍减轻(P<0.05或P<0.01),重复刻板行为减少(P<0.01或P<0.05),海马神经元结构明显复原,GLU下降(P<0.01),BDNF、TrkB、CREB mRNA表达增加(P<0.05或P<0.01),六味地黄丸中、低剂量组GABA上升(P<0.05或P<0.01)。结论六味地黄丸能显著改善VPA诱导的ASD仔鼠行为表现,增强海马组织神经元的再生与修复,其机制可能与平衡GLU、GABA水平,上调仔鼠海马组织中BDNF/TrkB/CREB的表达有关。