程丑夫教授“四治法则”在治疗失眠中的临床应用

主要介绍程丑夫教授通过治痰、治瘀、治郁、治虚的"四治法则"治疗失眠的经验。从痰、瘀、郁、虚来分析失眠发生的病机,通过清热化痰、活血化瘀、疏肝解郁、补益心脾等方法来治疗失眠,同时注重对患者心理疏导及因时治宜、因地治宜、因人治宜,临床治疗效果显著。

咳嗽变异型哮喘的中西医诊治探析

咳嗽变异型哮喘（caugh variant asthma,CVA）又称咳嗽型哮喘,过去曾称为过敏性哮喘。CVA亦是一种以多种细胞,特别是肥大细胞、嗜酸粒细胞、T淋巴细胞参与的气道慢性炎症,引起气道高反应导致可逆性气流受限,除此之外还与变态反应、遗传以及神经调节等因素相互作用有关。

Ophthalmic Syndrome Differentiation System and Digital Chinese Medicine

In traditional Chinese medicine(TCM),ophthalmic syndrome differentiation is an ophthalmology-specific method for identifying syndromes based on the“Five Orbiculi”theory.It was devised by Professor Qing-Hua PENG through an unprecedented combination of syndrome element differentiation and ophthalmic clinical practices,based on the Clinical Terminology of Chinese Medical Diagnosis and Treatment-Syndromes of the National Standards of the People's Republic of China.This approach integrates an ophthalmic syndrome differentiation system with digital Chinese medicine(DCM),and proposes the extraction of syndrome elements of ophthalmic diseases from research on DCM.These elements are then quantified and organized to form a model of digital diagnosis and treatment specific to ophthalmology,which should help to achieve synergistic development of the ophthalmic syndrome differentiation system and DCM.

Effects of Buddlejae Flos Granules on Inflammatory Factors TGF-β1,NF-κB,IL-10 and IL-12 in Lacrimal Gland Cells of Castrated Male Rabbits

Objective To observe the effects of Buddlejae Flos (Mi Meng Hua,密蒙花) granules on the expression of TGF-β1, NF-κB, IL-10 and IL-12 in lacrimal gland cells in castrated male rabbits. Methods A total of 30 New Zealand male white rabbits were randomly divided into 5 groups. Group A: blank group;group B: model group;group C: Buddlejae Flos (Mi Meng Hua,密蒙花) granules group;group D: placebo group;group E: testosterone group. Except for the group A, all rabbits were treated with bilateral testicular and epididymis resection before intervention. Rabbits in group C were administered with Buddlejae Flos (Mi Meng Hua,密蒙花) granules (100 mg/kg), 3 times per day;Rabbits in group D were administered with normal saline, 3 times per day. Rabbits in group E were injected with testosterone propionate (0.5 mL/kg) in the thigh muscle, every 3 days. Schirmer I test (SIT) and break-up time (BUT) were measured on 1st day before modeling and 30th day after modeling. After 30 days of intervention, all rabbits were sacrificed. The removal lacrimal gland was stained in immunohistochemica staining. The expression of TGF-β1, NF-κB, IL-10 and IL-12 in lacrimal gland tissue of each group was detected, and the structure of lacrimal gland was observed. Results The results of SIT and BUT showed that there was significant difference between group B and D compared with group A (P < 0.05). There were no significant differences between group C and E (P > 0.05). There were no significant differences in the comparison of inflammatory factors TGF-β1, NF-κB, IL-10 and IL-12 between group B and D (P > 0.05). There were significant differences between group B and D compared with other groups (P < 0.01). There was no significant difference between group A, C and E (P > 0.05). Conclusions Buddlejae Flos (Mi Meng Hua,密蒙花) granules have an inhibitory effect on the expression of TGF-β1, NF-κB, IL-10 and IL-12 in castrated male rabbit lacrimal gland cells, and alleviate the inflammation of lacrimal gland tissue by inhibiting the expression of TGF-β1, NF-κB, IL-10 and IL-12 in lacrimal gland tissue, so as to treat dry eye.

Pretreatment with Lithospermic Acid Attenuates Oxidative Stress-induced Apoptosis in Bone Marrow-derived Mesenchymal Stem Cells via Anti-oxidation and Activation of PI3K/Akt Pathway

Objective Despite the potential therapeutic approaches of bone marrow-derived mesenchymal stem cells(BMSCs)in orthopaedic,their applications are hampered by harsh oxidative stress conditions after transplantation.In this study,the antiapoptotic and anti-oxidative properties of lithospermic acid(LSA)on BMSCs exposed to hydrogen peroxide(H2O2)were investigated.Methods In the present study,we used H2O2 to induce oxidative injury on BMSCs.Reactive oxygen species(ROS)staining and superoxide dismutase(SOD)assay were performed.The expression levels of phosphorylated(p)-Akt,Bcl-2-associated X protein(Bax)and B-cell lymphoma 2(Bcl-2)were measured by Western blotting.Results LSA can significantly reduce H2O2-induced chromatin condensation and intracellular ROS levels,enhance the activity of SOD.Moreover,it can alleviate H2O2-induced apoptosis by upregulating Bcl-2 and p-Akt,down-regulating Bax,which was blocked by the PI3K inhibitor,LY294002.Conclusions Our results demonstrated that pretreatment with LSA could attenuate oxidative stress-induced apoptosis in BMSCs,which may be related with anti-oxidant properties and partly via modulating PI3K/Akt pathway,suggesting that pharmacologically manipulating BMSCs with LSA could be a promising drug to increase cell survival for BMSCs transplantation in musculoskeletal disorders of orthopaedic.

STAT3 Inhibition by Centipede Scolopendra Extract in Liver Cancer HepG2 Cells and Orthotopic Mouse Models of Hepatocellular Carcinoma

Objective To observe the effects of Centipede Scolopendra extraction(CSE)on human liver cancer HepG2 cells and the nude mouse tumor model of liver orthotopic transplantation,and to explore the anti-liver cancer mechanism of the extract.Methods HepG2 cells were respectively treated with CSE250(250μg/mL),CSE500(500μg/mL)and 5-FU,and control group was established.An enzymatic hydrolysis and acetone precipitation method was used to separate and purify CSE,which was then used to treat HepG2 cells.The CCK8 assay was used to detect the inhibition of cell proliferation and the half maximal inhibitory concentration(IC50)was calculated.Flow cytometry was used to analyze the cell cycle,and western blot was used to detect the expression of signal transduction and activator of transcription 3(STAT3)pathway-related proteins in HepG2 cells treated with CSE.A nude mouse model with an orthotopic liver tumor was prepared.The mice were randomly divided into four groups,each containing 12 animals:the model group,the 5-FU group,the CSE10 group[10 mg/(kg·d)]and the CSE50 group[50 mg/(kg·d)].The volume and mass changes in the nude mice with orthotopic transplanted tumors were observed.Western blot method was used to test the protein expression levels of p-STAT3 and p38 mitogen-activated protein kinase(p38MAPK).Tissues from the liver of mice in the model group and the CSE50 group were analyzed by using a protein tyrosine kinase(PTK)chip,and the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)function enrichment analysis of the differentially expressed proteins was performed.Results This study showed that CSE significantly inhibited the proliferation of HepG2 cells(P<0.05).After 48 h of CSE treatment,the cell cycle of HepG2 cells manifested as S phase and G2/M phase;p-STAT3 protein levels in the CSE groups were significantly lower than that in the control group(P<0.05).Analysis of the tumor inhibition in the mice showed that the tumor masses and volume in CSE groups were lower(P<0.05).The protein levels of p-STAT3 and p38MAPK in CSE50 group and 5-FU group decreased significantly(P<0.05).PTK antibody chip screening results showed that CSE groups had a bidirectional regulation trend,and there were 23 up-regulated PTKs and six down-regulated PTKs.The GO and KEGG analyses showed that CSE exerted its anticancer effects through regulation of biological processes,including mitogen-activated protein kinase(MAPK)cascade,chemotaxis,cell invasion,cell adhesion,angiogenesis and other biological processes,and through signaling pathways,including the MAPK,phosphatidylinositol-3-kinase/serine threonine protein kinase(PI3K/AKT),and RAS signaling pathways.Conclusions CSE can effectively inhibite the proliferation of HepG2 cells and effectively inhibite the growth of liver cancer orthotopic transplantation tumor.Its mechanism may be closely related to the regulation of STAT3,MAPK and PI3K/AKT signaling pathways.

Study on differential gene expression profile of serum exosomes in patients with acute cerebral infarction

Objective To analyze the differential gene expression profile of serum exosomes in patients with acute cerebral infarction(ACI)and clarify the changes in gene expression related to cerebral infarction injury and the potential serum markers.Methods Four patients with ACI and five healthy people were enrolled in the PhaseⅠstudy.After serum isolation from peripheral blood,exosomes were extracted with exosomes kits,highthroughput detection of m RNA was performed with gene chips,and differentially expressed m RNAs were screened.Gene Ontology(GO)functional analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were performed simultaneously.Furthermore,real-time polymerase chain reaction(q RT-PCR)was used to verify the expression levels of the screened differential m RNAs in the serum exosomes collected in PhaseⅡfrom 32 patients each in the ACI case and normal control groups.Results In the PhaseⅠstudy,there were 248 differentially expressed m RNAs(fold change≥2.0,P<0.05)among five patients in the normal control group and four patients in the case group,of which the expression of 242 was upregulated and that of six was downregulated.The results of GO functional enrichment analysis mainly included behavior regulation,cell connection,and antioxidant activity.The results of KEGG pathway enrichment analysis mainly included ribosomes,proteasomes,oxytocin signaling pathways,and oxidative phosphorylation.After researching and screening based on relevant literature,it was found that among the genes with significant differential expression,H3 F3 B m RNA may be associated with and might play an important role in ACI.The q RT-PCR method was used to detect the H3 F3 B mRNA expression in serum exosomes of 32 patients each in the normal control and case groups in PhaseⅡ;the expression was significantly higher in serum exosomes of the case group than in those of the normal control group(P<0.001).H3 F3 B mRNA expression in serum exosomes of the case group positively correlated with age,the National Institutes of Health Stroke Scale(NIHSS)score,and the maximum infarct size(P<0.05).Conclusion ACI can lead to changes in the serum exosomes mRNA expression profile,which may be closely related to the occurrence,development,and prognosis of this condition.These findings will provide direction for research on the molecular mechanism,diagnostic markers,and therapeutic targets of ACI.

Compound Chai Jin Jie Yu Tablets,Acts as An Antidepressant by Promoting Synaptic Function in the Hippocampal Neurons

Objective To investigate the effectiveness of compound Chai Jin Jie Yu Tablets(CJJYT)in ameliorating cognitive impairment associated with depression and its potential mechanism of action.Methods In vitro experiments,the hippocampus was isolated from the whole brain of the fetal rat and cultured into hippocampal neuron cells.50μM corticosterone(CORT)was added to each group 18 h before the experiment for modeling depression,with the exception of the control group.After modeling,the blank serum group was added with 10%blank serum,the CJJYT group and the venlafaxine group were added with the corresponding 10%drug-containing serum,and the control group and the model group were added with equal volumes of culture medium.The intracellular Ca^2+mean fluorescence intensity,miniature excitatory postsynaptic current(mEPSC)current amplitude,and frequency of different hippocampal neurons were evaluated as indicators of synaptic function in the hippocampal neurons.In addition,the expression of synaptic plasticity related proteins,synaptophysin-α(SYN-α),N-methyl-D-aspartate receptor 2A(NR2A),N-methyl-Daspartate receptor 2B(NR2B),post synaptic density 95 protein(PSD-95),calcium/calmodulin dependent protein kinaseⅡ(CaMKⅡ)and synaptic associated protein(SynGAP)were detected in the hippocampal neurons by immunofluorescence staining and high content analysis(HCA)system.Then,reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the mRNA expression levels of SYN-α,NR2A,NR2B,PSD-95,CaMKⅡand SynGAP.For in vivo experiments,except for those in the blank control group,all rats were treated within a single cage for chronic unpredictable stress-induced depression modeling and subjected to corresponding drug interventions.Behavioral tests were used to detect depressive behavior and determine learning,memory and other cognitive abilities,whereas enzyme-linked immunosorbent assay(ELISA)was used to detect the CORT levels.Golgi-Cox staining was used to observe changes in the synaptic morphology of parahippocampal gyrus CA1 area(CA1)and dentategyrus(DG).Results In vitro,CJJYT treatment reduced the intracellular Ca^2+mean flurorescence intensity in the hippocampal neurons(P<0.05),causing a reduction in the frequency and current amplitude of mEPSC(P<0.05),and thus inhibited the excessive activation of post-synaptic receptors.CJJYT treatment reduced the protein and mRNA expression of SYN-α,NR2A,NR2B and PSD-95 in the hippocampal neurons(P<0.05),increased the mRNA and protein expression of CaMKⅡand SynGAP(P<0.05),and thereby improved the synaptic plasticity of the hippocampal neurons.In vivo,CJJYT intervention improved sucrose preference,voluntary activity,learning and memory ability of Morris water maze test,and suppressed appetite(P<0.05),and increased the despair feeling of forced swimming test(P<0.05).The CORT level was reduced(P<0.05),leading to the repair of synaptic damage in the hippocampal neurons.Conclusions CJJYT can improve the synaptic function of hippocampal neurons and has obvious protective effects on neurons.It can repair the structural damage in the hippocampal neurons,improving the cognitive ability of the depressed model rats.The mechanism of CJJYT improving cognition in depressed rats may be related to the transmission and function of SYN-α/NR and its downstream neurotransmitters.

Compound Chaijin Jieyu Tablets ameliorating insomnia complicated with depression by improving synaptic plasticity via regulating orexin A,melatonin,and acetylcholine contents

Objective To investigate the efficacy and mechanism of action of Compound Chaijin Jieyu Tablets(复方柴金解郁片,CCJJYT)in rats with insomnia complicated with depression.Methods Seventy-two Sprague-Dawley rats were randomly assigned into eight groups:the control,chronic unpredictable mild stress(CUMS),sleep deprivation(SD),CUMS+SD,positive drug(venlafaxine hydrochloride+diazepam),CCJJYT high-dose(CCJJYT˗2×),medium-dose(CCJJYT˗1×),and low-dose(CCJJYT˗0.5×)groups,with nine rats in each group.Depression-like behavior was evaluated by body weight,food intake,and behavioral tests such as the sucrose preference test(SPT),open field test(OFT),forced swimming test(FST),and pentobarbital-induced sleep test(PST).Hematoxylin-eosin(HE)staining and Golgi-Cox staining were used to observe changes in pathological tissue and synaptic morphology,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to detect the contents of orexin-A and acetylcholine.The expression levels of orexin receptor 1(OXR1),melatonin receptor 1(MT1A),melatonin receptor 2(MT1B),acetylcholinesterase(AChE),and choline acetyltransferase(ChAT)were detected by immunohistochemistry and Western blot.Results In the present study,rats in the model group showed significant behavioral changes as well as a reduction in hippocampal dendritic branch length and synaptic number,along with increasing the content of orexin A and acetylcholine(P<0.05),and altered expression levels of OX1R,MT1A,MT1B,ChAT,and AChE in the hippocampus and prefrontal cortex after modeling(P<0.05).CCJJYT can improve depressive insomnia behavior and synaptic plasticity of rats(P<0.05),which is similar to that of the positive drug group.It can also decrease the content of orexin A and acetylcholine,and reduce the expression levels of OXR1 and ChAT in hippocampus and prefrontal cortex(P<0.05),and increase the expression levels of MT1A,MT1B,and AChE proteins(P<0.05).Conclusion CCJJYT has good antidepressant and insomnia effects,probably through the regulation of orexin-A,melatonin,and acetylcholine content in hippocampus and prefrontal cortex of rats,improving synaptic plasticity and thus exerting antidepressant and insomnia effects.

A Network Pharmacology Approach to Uncover the Molecular Targets and Associated Potential Pathways of Lycii Fructus for the Treatment of Retinitis Pigmentosa

Objective To explore the molecular targets and associated potential pathways of Lycii Fructus(LF,Gou Qi Zi,枸杞子)in the treatment of retinitis pigmentosa(RP)by the approaches of network pharmacology and bioinformatics.Methods The potential blood-entry active ingredients and targets of LF were retrieved by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).RP-related gene targets were retrieved through disease comprehensive databases.Protein-protein interaction(PPI)network of LF component-targets and RP disease-targets was constructed by STRING,and the intersection of the 2 networks was extracted.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of theintersection network were conducted by Database for Annotation,Visualization and Integrated Discovery(DAVID).CytoHubba was used to screen the key targets.Results A total of 188 chemical constituents related to LF was retrieved from TCMSP database.45 active ingredients were screened according to pharmacokinetic parameters oral bioavailability(OB)and drug similarity(DL).36 active ingredients were further screened and 201 targets related to these constituents were obtained.206 target genes directly related to RP were obtained from the disease comprehensive databases,and 89 genes were obtained from the intersection of componenttarget and disease-target PPI network.These genes were mainly involved in intracellular signal transduction,GTPase activity regulation,cell morphology regulation,and other biological processes.Molecular functions were mainly related to Rho guanine nucleotide exchange factor activity,GTPase activator activity,receptor signal protein serine/threonine kinase activity and so on.They were enriched in the cytoplasm,cell membrane,Golgi apparatus,and other regions.The mechanism was related to cell cycle pathways,neurotrophin signaling pathways,Ras signaling pathways,and so on.10 key gene targets of LF in the treatment of RP were screened.Conclusions The material basis for LF to exert its pharmacodynamic effect is 36 active ingredients such as cycloartenol,mandenol,and so on.The key targets of LF in the treatment of RP include 10 genes,such as Rho,PAK,and so on.The main mechanism is related to the regulation of the Ras signaling pathway,neurotrophin signaling pathway,cell cycle related pathway,and other signaling networks.

Protective effects of Zuogui Jiangtang Jieyu Formula on hippocampal neurons in rats of diabetes complicated with depression via the TRP/KYN metabolic pathway

Objective To explore the protective effects and mechanism of Zuogui Jiangtang Jieyu Formula(左归降糖解郁方,ZGJTJYF)on hippocampal neurons in rats of diabetes complicated with depression(DD)via the TRP/KYN metabolic pathway.Methods(i)In vivo experiments:60 specified pathogen free(SPF)grade male Sprague-Dawley(SD)rats were randomly divided into six groups with 10 rats in each groups:control,DD model,positive(1.8 mg/kg fluoxetine+0.18 g/kg metformin),high-dose ZGJTJYF(ZGJTJYFH,40.500 g/kg ZGJTJYF),middle-dose ZGJTJYF(ZGJTJYF-M,20.250 g/kg ZGJTJYF),and lowdose ZGJTJYF(ZGJTJYF-L,10.125 g/kg ZGJTJYF)groups.Except for the control group,other groups were established DD model by high-fat emulsion intake with single tail vein streptozotocin(STZ)and four weeks of chronic unpredictable mild stress(CUMS).All drug administration groups were treated by gavage during CUMS modeling,and the control and model groups were given equal amount of distilled water.After four weeks,the serum levels of blood glucose and glycosylated hemoglobin were measured to determine the hypoglycemic effect of ZGJTJYF.Moreover,the open field test and Morris water maze test were performed to evaluate the antidepressant effect of ZGJTJYF.Changes in 5-hydroxytryptamine(5-HT)level were detected via high-performance liquid chromatography with electrochemical detection(HPLC-ECD);the levels of tryptophan(TRP),kynurenine(KYN),and indoleamine 2,3-dioxygenase(IDO)in the hippocampus were detected using enzyme-linked immunosorbent assay(ELISA);the protein expression levels of synaptophysin(SYN)and postsynaptic density material-95(PSD-95)were detected via immunohistochemistry(IHC);and the protein expression levels of N-methyl-D-aspartate receptor(NR)2 A and NR2 B were detected using Western blot.(ii)In vitro experiments:five SPF grade SD pregnant rats(E16–18)were used to obtain primary hippocampal neurons(Ne),six SD new-born rats were used to collected primary astrocytes(As)and microglia(MG),and to establish a Ne-As-MG co-culture system.All co-culture systems were divided into six groups:control(PBS),model[150 mmol/L glucose+200μmol/L corticosterone(G&P)+PBS],blank(G&P+blank serum),positive(G&P+positive drug-containing serum),ZGJTJYF(G&P+ZGJTJYF serum),and 1-methyl-D-tryptophan(1-MT,IDO inhibitor)(G&P+1-MT)groups.After 18 h of intervention by corresponding treatment,immunofluorescence was used to analyze the protein expression levels of SYN,PSD-95,NR2 A,and NR2 B;ELISA was performed to measure the levels of interleukin(IL)-1β,IL-6,tumor necrosis factor(TNF)-α,and TRP/KYN metabolic pathway-related factors[TRP,KYN,kynurenine acid(KYNA),quinolinic acid(QUIN)].Results(i)In vivo experimental results showed that ZGJTJYF-M and ZGJTJYF-L significantly improved the elevated blood glucose state of DD rats(P<0.01 and P<0.05,respectively);ZGJTJYF-H,ZGJTJYF-M,and ZGJTJYF-L increased their autonomous activity,learning,and memory ability(P<0.01,P<0.01,and P<0.05,respectively).Moreover,the levels of 5-HT and TRP were significantly increased(P<0.01),and the levels of KYN and IDO were significantly decreased in the hippocampus(P<0.01)of rats after ZGJTJYF-M treatment.The protein expression levels of SYN and PSD-95 were significantly upregulated in hippocampal neurons(P<0.01),while the abnormal activation of NR2A and NR2B was markedly inhibited in hippocampus(P<0.05)of rats after ZGJTJYF-M treatment.(ii)In vitro experimental results showed that ZGJTJYF-containing serum significantly increased the protein expression levels of SYN and PSD-95 in hippocampal neurons(P<0.01),decreased the levels of IL-1β(P<0.01),IL-6(P<0.05),TNF-α(P<0.01),IDO(P<0.05),KYN(P<0.05),and QUIN(P<0.01),and increased the levels of TRP and KYNA(P<0.01)in the simulated DD state.ZGJTJYF also had an significantly inhibitory effect on the abnormal activation of NR2A and NR2B in neurons(P<0.05)in a stimulated DD state.Conclusion ZGJTJYF can effectively improve 5-HT deficiency in the hippocampus of rats by inhibiting IDO expression and regulating the TRP/KYN metabolic pathway,and it has a favorable protective effect on hippocampal neuron injury caused by DD.Therefore,ZGJTJYF is an effective potential therapeutic drug for the prevention and treatment of DD.

Niuhuang(Bovis Calculus)-Shexiang(Moschus)combination induces apoptosis and inhibits proliferation in hepatocellular carcinoma via PI3K/AKT/mTOR pathway

Objective To investigate the effects of Niuhuang(Bovis Calculus,BC)and Shexiang(Moschus)(BC-Moschus)on human hepatocellular carcinoma(HCC)cells SMMC-7721 and a nude mouse model of subcutaneous xenografts,and to explore its anti-HCC mechanism.Methods The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro.SMMC-7721 was divided into the BC-Moschus group and the control group,and different doses(rude drug dosage 0.625,1.25,2.5,and 5 mg/m L)of BC-Moschus extract were used for the intervention.The proliferation ability of HCC cells was detected using the Cell Counting Kit-8(CCK-8)assay,and the migration ability was detected by a wound healing assay.A subcutaneous xenograft model was prepared using nude mice with human HCC.Specific pathogen-free-grade BALB/c nude mice(5-week-old)were randomly divided into the following groups(n=6 per group):control(0.9%physiological saline 0.2 m L/d),BC-Moschus[BC 45.5 mg/(kg·d)+Moschus 13 mg/(kg·d)],and cisplatin(DDP,intraperitoneal injection5 mg/kg per week)groups.All groups were administered for 14 d.The volume and mass of the subcutaneous xenografts in nude mice were observed.The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway,apoptosis-associated factor p70 S6 Kinase(S6K),Bax,Bcl-2,caspase-3,and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR(RT-qPCR)and Western blot.Terminal Deoxynucleotidy Transferase-Mediated d UTP NickEnd Labeling(TUNEL)was used for quantitative analysis of apoptotic cells.Results The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone,and the inhibition effect was dose-and time-dependent(P<0.01).The wound healing assay showed that the BC-Moschus combination inhibited HCC cell migration(P<0.01).In the subcutaneous xenograft model of nude mice with human HCC,we found that the tumor volume and weight of the BC-Moschus group were lower than those of the control group(P<0.01).The levels of the PI3K/AKT/m TOR signaling pathway and S6K protein in the BC-Moschus and DDP groups were significantly decreased(P<0.01).The expression level of the anti-apoptotic gene Bcl-2 was downregulated(P<0.05),and the expression of the pro-apoptotic gene Baxand apoptosis-related factors caspase-3 and caspase-9 were significantly upregulated(P<0.01).The TUNEL assays further confirmed that the combination of the BC-Moschuas could promote HCC(P<0.01).Conclusion The BC-Moschus combination inhibited the proliferation and migration ability of HCC cells SMMC-7721 and effectively inhibited the growth of subcutaneous xenografts in nude mice.The mechanism may be closely related to the downregulation of the PI3K/AKT/mTOR pathway,regulation of apoptosis-related protein caspase-3,caspase-9,Bcl-2,and Bax expression,and promotion of apoptosis.

Effects of Lycii Fructus and Salviae Miltiorrhizae on the Syndrome of Deficiency with Blood Stasis in RCS(rdy-/-, p-/-) Rats with Retinitis Pigmentosa: An Intervention Study

Objective To investigate the effects of Lycii Fructus(LF,Gou Qi Zi,枸杞子)and Salviae Miltiorrhizae Radix Ex Rhizoma(SM,Dan Shen,丹参)on the syndrome of deficiency with blood stasis in the RCS(rdy-/-,p-/-)rats with retinitis pigmentosa(RP).Methods A total of 32 RCS(rdy-/-,p-/-)rats were divided into 4 groups(equal amounts of female and male rats in each group):model group treated with 0.9%normal saline,LF group treated with LF formula granules,SM group treated with SM formula granules,and LF and SM(L·S)group treated with LF and SM formula granules.Eight RCS(rdy+/+,p+/+)rats(4 males and 4 females)were treated with 0.9%normal saline to serve as blank group.The contents of E2,PG,P-Selectin,plasma viscosity,whole blood relative index of the high shear rate and fibrinogen content in plasma,and the content of cAMP and cGMP in retinal homogenate were detected.The retina was evaluated by hematoxylin-eosin staining.Results The contents of E2,PG,P-Selectin,plasma viscosity,whole blood relative index of the high shear rate,and fibrinogen content in the plasma of L·S group significantly differed from those of model group(P<0.01),but were similar to those of blank group.The contents of cAMP and cGMP in the retinal homogenate of L·S group significantly differed from those in model group(P<0.01)but were similar to those in blank group(P>0.05).Conclusions LF and SM can effectively treat retinitis pigmentosa by ameliorating the syndrome of deficiency with blood stasis.

Effects of Qingguang’an Granules on mitochondrial autophagy of retinal ganglion cells in rats with chronic ocular hypertension

Objective To investigate the effect and underlying mechanism of Qingguang’an Granules(青光安颗粒剂,QGAG)on mitochondrial autophagy(mitophagy)of retinal ganglion cells(RGCs)in rats with chronic ocular hypertension(COH).Methods Sixty Sprague Dawley(SD)rats,half males and half females,were randomly assigned to three groups:the control,model,and QGAG(2.5 g/kg)groups,with 20 rats in each group.Rats’model of COH was established by cauterizing episcleral veins in the model group and QGAG group.Three weeks after successful modeling,rats in the QGAG group were intra-gastrically administered with QGAG,while rats in the control group and the model group received an equal dose of normal saline.After three months of intragastric administration,intraocular pressure(IOP)of all rats was measured.The mitophagy was monitored by the immunofluorescence method,the mitochondrial membrane potential was measured using the JC-1 method,and the morphological changes of mitophagy in RGCs were observed by transmission electron microscopy.Meanwhile,rat RGCs were labeled using the fluorescent gold method,and RGCs density in each group was calculated.Moreover,RGCs apoptosis was observed by TdT-mediated dUTP Nick-End Labeling(TUNEL)assay.Finally,the expression levels of Parkin,optineurin,microtubule-associated protein 1 light chain 3-Ⅱ/microtubule-associated protein 1 light chain 3-Ⅰ(LC3-Ⅱ/LC3-Ⅰ),recombinant lysosomal associated membrane protein 1(LAMP1),and B-cell lymphoma-2(Bcl-2)in RGCs were determined by Western blot assay.The corresponding mRNAs were detected through quantitative real-time polymerase chain reaction(qRT-PCR).Results The QGAG reduced IOP in COH rats,and inhibited mitophagy and apoptosis of RGCs(P<0.05).Besides,the QGAG significantly increased the expression levels of Parkin and Bcl-2(P<0.05),and inhibited the expression levels of optineurin,LAMP1,and LC3-Ⅱ/LC3-Ⅰ(P<0.05)in RGCs of COH rats.Conclusion The QGAG can inhibit mitophagy in RGCs of COH rats and show a protective effect against optic nerve damage caused by glaucoma,which may be mediated through the mitophagy ubiquitination via the Parkin/PINK1-related pathway.

Linarin ameliorates innate inflammatory response in an experimental dry eye model via modulation of the NLRP3 inflammasome

Objective To investigate the effect and mechanism of linarin(LA) in an experimental dry eye model.Methods LA or vehicle was applied in two dry eye models: an in vitro hyperosmotic stress model and an in vivo desiccating stress(DS) murine model. The viability of human corneal epithelial cells(HCECs) was measured using a cell counting kit(CCK-8).Tear secretion was assessed using the phenol red cotton test. The tear break-up time(TBUT) was recorded using 0.1% liquid fluorescein sodium. Corneal epithelial permeability was evaluated through Oregon green dextran(OGD) staining.Conjunctival goblet cells were counted using periodic acid-Schiff(PAS) staining. Terminal deoxynucleotidyl transfer d UTP nickend labeling(TUNEL) staining was used to quantify apoptotic cells in both models. The expression of Ki-67 was measured in HCECs in the cell model while that of matrix metalloproteinase(MMP)-3 and-9 was measured in the murine model through immunofluorescence staining. Real-time quantitative PCR(RTqPCR) was performed to assess the expression of MMP-3 and MMP-9 in the corneal epithelium and NLRP3, ASC, Caspase-1,interleukin(IL)-1β, IL-18, and tumor necrosis factor(TNF)-α in the conjunctiva. The protein expression levels of NLRP3, ASC,Caspase-1, IL-1β, and IL-18 in the conjunctiva were assessed via Western blot.Results In the in vitro model, treatment of HCECs with LA showed no toxicity, increased proliferation, and reduced apoptosis. In the murine model, compared to the control, LA significantly increased tear production and TBUT, improved OGD staining, and increased the number of goblet cells. Topical treatment of LA to mice provided decreased expression of MMP-3, MMP-9, TNF-α, and apoptotic corneal epithelium. Topical administration of LA also suppressed the NLRP3 inflammasome in the dry eye disease(DED) murine model by decreasing the expression of NLRP3, ASC, Caspase-1, IL-1β, and IL-18 in the conjunctiva.Conclusion Our findings support the safety and efficacy of LA in the treatment of DED. LA alleviated corneal epithelial damage and suppressed NLRP3 inflammasome-mediated immunity in the conjunctiva in a murine model of DED.

Inflammatory Mechanism of Total Flavonoids of Chrysanthemum and Medicated Serum on Castrated Dry Eye Animal and Cell Models

Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.

Analysis of the hotspots and trends in traditional Chinese medicine immunomodulation research based on bibliometrics

Objective To analyze the intellectual structure,hotspots and trends of traditional Chinese medicine(TCM)in immune regulation research.Methods The data were extracted from the Web of Science Core Collection(WoSCC)and the China National Knowledge Infrastructure(CNKI)and verified by two experienced TCM researchers.The time of literature retrieval is up to 2020.CiteSpace 5.7.R1 and Microsoft Excel 2016 were used for the statistical analysis and bibliometric diagrams,including the co-occurrence network of authors,institutions,countries,keywords,references,dual-map overlays of journals and citation bursts,etc.Results A total of 12270 publications related to TCM in immune regulation were included.The annual number of publications has increased in this field.There was close cooperation of countries and institutions,while the distribution of scholars was scattered.China was the core of the cooperation network.The dual-map overlays analysis of journals showed that core and marginal fields had increased.The keywords and references analysis showed that network pharmacology,metabolism and cancer were the most high-frequency keywords with high-intensity bursts.Conclusion TCM in immune regulation has attracted wider attention,with multi-country,multi-field,multi-disciplinary and multi-level research developing toward informatization.Network pharmacology,metabolism and cancer may be the focus of future research in this field.

Physical Therapy Modalities of Western Medicine and Traditional Chinese Medicine for Meibomian Gland Dysfunction

The meibomian gland is a unique sebaceous gland located in the eyelid.Its main function is to secrete lipids and form the lipid layer of the tear film to delay the evaporation of waterborne tears,increase the surface tension of the tear film,and to lubricate the contact area of the eyelid and eyeball.Abnormal secretion of the meibomian gland is known as meibomian gland dysfunction(MGD),which has become the most important cause of evaporative dry eye disease(DED).The clinical pathophysiological process and underlying molecular mechanisms of MGD are not clear.As serious side effects may occur with the long-term use of hormonotherapy and non-steroidal anti-inflammatory drugs(NSAIDs)for the treatment of MGD,meibomian gland physiotherapy is considered the most effective and safest therapy for MGD.This review summarizes the physical therapy modalities of western medicine(WM)and traditiond Chinese medicine(TCM)for the treatment of MGD to provide optimal treatments for these patients and to further lay a foundation for mechanistic studies of MGD.

Hub Genes of Astrocyte Involved in Glaucoma with Ocular Hypertension by Integrated Bioinformatics Analysis

This study was conducted to elucidate the potential key candidate genes and pathways in role of astrocyte involved in glaucoma with ocular hypertension.Methods Expression profiles GSE2378 and GSE758 including 27 reactive optic nerve head astrocytes(ONHAs)by hypertensions and 26 normal controls,were integrated and deeply analyzed.Differentially expressed genes(DEGs)were sorted and candidate genes and pathways enrichment were analyzed.DEGs-associated protein-protein interaction network(PPI)was performed.Results A total of 119 consistently expressed genes were identified from 281 commonly changed DEGs,including 68 up-regulated genes and 51 down-regulated genes.PPI network complex filtered 75 DEGs(43 up-regulated and 32 down-regulated genes)of the 119 consistently altered DEGs and developed 117 edges,and 10 hub genes were identified.The most significant 3 modules were filtered from PPI,pathway enrichment analysis showed that module 1 was associated with extracellular exosome.Module 2 was mainly associated with antibody-dependent cellular cytotoxicity(ADCC)and module 3 was mainly associated with Hippo signaling pathway.Conclusion Taken above,using integrated bioinformatical analysis,we have identified DEGs candidate genes and pathways in role of astrocyte involved in glaucoma with ocular hypertension,which could improve our understanding of the cause and underlying molecular events,and these candidate genes and pathways could be therapeutic targets for glaucoma.

Traditional Chinese medicine Master XIONG Jibo’s medication experience in treating arthralgia syndrome through data mining

Objective This study aimed to examine and propagate the medication experience and group formula of traditional Chinese medicine(TCM)Master XIONG Jibo in diagnosing and treat-ing arthralgia syndrome(AS)through data mining.Methods Data of outpatient cases of Professor XIONG Jibo were collected from January 1,2014 to December 31,2018,along with cases recorded in A Real Famous Traditional Chinese Medicine Doctor:XIONG Jibo's Clinical Medical Record 1,which was published in December 2019.The five variables collected from the patients’data were TCM diagnostic information,TCM and western medicine diagnoses,syndrome,treatment,and prescription.A database was established for the collected data with Excel.Using the Python environment,a custom-ized modified natural language processing(NLP)model for the diagnosis and treatment of AS by Professor XIONG Jibo was established to preprocess the data and to analyze the word cloud.Frequency analysis,association rule analysis,cluster analysis,and visual analysis of AS cases were performed based on the Traditional Chinese Medicine Inheritance Computing Platform(V3.0)and RStudio(V4.0.3).Results A total of 610 medical records of Professor XIONG Jibo were collected from the case database.A total of 103 medical records were included after data screening criteria,which comprised 187 times(45 kinds)of prescriptions and 1506 times(125 kinds)of Chinese herbs.The main related meridians were the liver,spleen,and kidney meridians.The properties of Chinese herbs used most were mainly warm,flat,and cold,while the flavors of herbs were mainly bitter,pungent,and sweet.The main patterns of AS included the damp heat,phlegm stasis,and neck arthralgia.The most commonly used herbs for AS were Chuanniuxi(Cyathu-lae Radix),Huangbo(Phellodendri Chinensis Cortex),Cangzhu(Atractylodis Rhizoma),Qinjiao(Gentianae Macrophyllae Radix),Gancao(Glycyrrhizae Radix et Rhizoma),Huangqi(Astragali Radix),and Chuanxiong(Chuanxiong Rhizoma).The most common effect of the herbs was“promoting blood circulation and removing blood stasis”,followed by“supple-menting deficiency(Qi supplementing,blood supplementing,and Yang supplementing)”,and“dispelling wind and dampness”.The data were analyzed with the support≥15%and con-fidence=100%,and after de-duplication,five second-order association rules,39 third-order association rules,39 fourth-order association rules,and two fifth-order association rules were identified.The top-ranking association rules of each were“Cangzhu(Atractylodis Rhizoma)→Huangbo(Phellodendri Chinensis Cortex)”“Cangzhu(Atractylodis Rhizoma)+Chuanniuxi(Cyathulae Radix)→Huangbo(Phellodendri Chinensis Cortex)”“Chuanniuxi(Cyathulae Radix)+Danggui(Angelicae Sinensis Radix)+Gancao(Glycyrrhizae Radix et Rhizoma)→Qinjiao(Gentianae Macrophyllae Radix)”and“Chuanniuxi(Cyathulae Radix)+Danggui(Angelicae Sinensis Radix)+Gancao(Glycyrrhizae Radix et Rhizoma)+Huangbo(Phello-dendri Chinensis Cortex)→Qinjiao(Gentianae Macrophyllae Radix)”,respectively.Five clusters were obtained using cluster analysis of the top 30 herbs.The herbs were mainly dry-ing dampness,supplementing Qi,and promoting blood circulation.The main prescriptions of AS were Ermiao San(二妙散),Gegen Jianghuang San(葛根姜黄散),and Huangqi Chongteng Yin(黄芪虫藤饮).The herbs of core prescription included Cangzhu(Atractylodis Rhizoma),Chuanniuxi(Cyathulae Radix),Gancao(Glycyrrhizae Radix et Rhizoma),Huangbo(Phellodendri Chinensis Cortex),Mugua(Chaenomelis Fructus),Qinjiao(Gentianae Macro-phyllae Radix),Danggui(Angelicae Sinensis Radix),and Yiyiren(Coicis Semen).Conclusion Clearing heat and dampness,relieving collaterals and pain,and invigorating Qi and blood are the most commonly used therapies for the treatment of AS by Professor XIONG Jibo.Additionally,customized NLP model could improve the efficiency of data mining in TCM.