Integration of network pharmacology and bone marrow mesenchymal stem cells experimental research to reveal the molecular mechanisms for Hai Honghua medicinal liquor against osteoporosis

Background:Hai Honghua medicinal liquor(HHML)formula has been used in clinical practice for a long time,mainly for the treatment of freshly closed fractures,to promote osteogenesis,to increase bone mass,and thus to promote fracture healing.However,the underlying mechanisms of HHML in the treatment of osteoporosis(OP)are still unclear.Methods:Firstly,Traditional Chinese Medicines Systems Pharmacology Database and Analysis Platform and The Encyclopedia of Traditional Chinese Medicine were used to screen the targets of the active compounds of HHML.At the same time,OP targets were identified through GeneCards,Online Mendelian Inheritance in Man,DisGeNET,Therapeutic Target Database,Comparative Toxicogenomics Database and Human Phenotype Ontology databases.Next,protein-protein interaction and pathway networks were constructed for compound-disease common targets,and core targets and compounds were screened for molecular docking.Furthermore,rat bone marrow mesenchymal stem cells were extracted as model cells,and the osteogenic effects of HHML were verified via in vitro experiments.Results:Total of 343 common targets of HHML-OP and the top 10 target proteins in the protein-protein interaction network are TP53,AKT1,STAT3,HSP90AA1,ESR1,TNF,IL6,MAPK1,MAPK3 and MAPK8.Enrichment analysis yielded that the key molecular pathway was the PI3K/Akt signaling pathway.Molecular docking analysis showed that baicalein,erysodienone and naringenin docked with the target proteins AKT1,STAT3 and TP53,respectively,with low binding energy and high affinity.In addition,In vitro experiments demonstrated that HHML promoted the proliferation of bone marrow mesenchymal stem cells;compared with the control group,HHML-treated cells showed enhanced alkaline phosphatase staining,promoted the expression of OCN,RUNX2,BSP,and COL1 mRNAs as well as the expression of PI3K and AKT phosphorylated proteins.Secondly,the expression of target proteins revealed that HHML promoted the phosphorylation of STAT3 protein and inhibited the expression of P53.Conclusions:Our study investigated that HHML treatment with OP promotes bone formation possibly through activation of the PI3K/Akt signaling pathway and may involve STAT3 and TP53 target interactions.

Clinical safety and efficacy of allogenic human adipose mesenchymal stromal cells-derived exosomes in patients with mild to moderate Alzheimer’s disease:a phaseⅠ/Ⅱclinical trial

Background There have been no effective treatments for slowing or reversing Alzheimer’s disease(AD)until now.Growing preclinical evidence,including this study,suggests that mesenchymal stem cells-secreted exosomes(MSCs-Exos)have the potential to cure AD.Aims The first three-arm,drug-intervention,phase I/II clinical trial was conducted to explore the safety and efficacy of allogenic human adipose MSCs-Exos(ahaMSCs-Exos)in patients with mild to moderate AD.Methods The eligible subjects were assigned to one of three dosage groups,intranasally administrated with ahaMSCs-Exos two times per week for 12 weeks,and underwent follow-up visits at weeks 16,24,36 and 48.Results No adverse events were reported.In the medium-dose arm,Alzheimer’s Disease Assessment Scale–Cognitive section(ADAS-cog)scores decreased by 2.33(1.19)and the basic version of Montreal Cognitive Assessment scores increased by 2.38(0.58)at week 12 compared with baseline levels,indicating improved cognitive function.Moreover,the ADAS-cog scores in the medium-dose arm decreased continuously by 3.98 points until week 36.There were no significant differences in altered amyloid or tau deposition among the three arms,but hippocampal volume shrank less in the medium-dose arm to some extent.Conclusions Intranasal administration of ahaMSCs-Exos was safe and well tolerated,and a dose of at least 4×10^(8)particles could be selected for further clinical trials.

Research of the molecular mechanism of Hai Honghua medicinal liquor in the treatment of fracture based on network pharmacology combined with molecular docking

Background:Hai Honghua medicinal liquor(HHML),a famous hospital formula composed of 19 traditional Chinese medicines,has been successfully applied in treating soft tissue injury,fresh closed fracture,limb dysfunction after fracture healing,shoulder,neck and leg pain,knee joint pain and other clinical multiple diseases for 30 years in clinical.However,research on the material basis of HHML for the treatment of fracture healing-related disorders is still in a gap.Therefore,it is particularly important to explore the active ingredients,core targets and potential pharmacological mechanisms of HHML to promote fracture healing.Methods:We screened the core active components of each traditional Chinese medicine in formula and its action targets through the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Encyclopedia of Traditional Chinese Medicine database.The fracture related targets were retrieved from several different public databases,including GeneCards,Online Mendelian Inheritance in Man,DisGeNET and Therapeutic Target Database.Bioinformatics analysis to obtain key bioactive components,underlying targets and signaling pathways,containing the Venn diagram of the intersection with components and diseases gene targets,protein–protein interaction,as well as the Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes analysis,and finally molecular docking.Results:A total of 249 bioactive ingredients of HHML and 325 HHML-fracture-related targets were screened.The network analysis revealed that quercetin,luteolin,kaempferol,Licochalcone A,naringenin and 8-Isopentenyl-kaempferol may be potential candidate agents.Multiple targets are involved including TP53,MAPK3,STAT3,AKT1,MAPK1,HSP90AA1,ESR1 and PIK3CA may be closely linked targets.PI3K-AKT signaling pathway may play a significant role of HHML in treatment of fracture.What’s more,molecular docking suggested that 8-isopentenyl kaempferol,glycyrrhiza chalcone A,and naringenin bound to AKT1,PIK3CA,and ESR1,respectively,exhibiting lower energy and more stable characteristics.Conclusions:The findings indicate the potential active ingredients,target proteins and molecular mechanisms of HHML for the treatment of fractures to provide the exact idea for the next research on the mechanism of action of HHML formula for fracture treatment.

A biomimetic nanoplatform for customized photothermal therapy of HNSCC evaluated on patient-derived xenograft models

Cancer cell membrane(CCM)derived nanotechnology functionalizes nanoparticles(NPs)to recognize homologous cells,exhibiting translational potential in accurate tumor therapy.However,these nanoplatforms are majorly generated from fixed cell lines and are typically evaluated in cell line-derived subcutaneous-xenografts(CDX),ignoring the tumor heterogeneity and differentiation from inter-and intra-individuals and microenvironments between heterotopic-and orthotopic-tumors,limiting the therapeutic efficiency of such nanoplatforms.Herein,various biomimetic nanoplatforms(CCM-modified gold@Carbon,i.e.,Au@C-CCM)were fabricated by coating CCMs of head and neck squamous cell carcinoma(HNSCC)cell lines and patient-derived cells on the surface of Au@C NP.The generated Au@C-CCMs were evaluated on corresponding CDX,tongue orthotopic xenograft(TOX),immunecompetent primary and distant tumor models,and patient-derived xenograft(PDX)models.The Au@C-CCM generates a photothermal conversion efficiency up to 44.2% for primary HNSCC therapy and induced immunotherapy to inhibit metastasis via photothermal therapy-induced immunogenic cell death.The homologous CCM endowed the nanoplatforms with optimal targeting properties for the highest therapeutic efficiency,far above those with mismatched CCMs,resulting in distinct tumor ablation and tumor growth inhibition in all four models.This work reinforces the feasibility of biomimetic NPs combining modular designed CMs and functional cores for customized treatment of HNSCC,can be further extended to other malignant tumors therapy.

Rapid bioluminescence assay for monitoring rat CES1 activity and its alteration by traditional Chinese medicines

In traditional Chinese medicine herbs(TCM),including Radix Salviae Miltiorrhizae(Danshen),Radix Puerariae Lobatae(Gegen),Radix Angelicae Sinensis(Danggui),and Rhizoma Chuanxiong(Chuanxiong)are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with Western drugs as a part of integrative medicine practice.Carboxylesterase 1(CES1)plays a pivotal role in the metabolisms of pro-drugs,Since(S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydrothiazole-4-carboxylate(NLMe)has recently been identified by us as a selective CES1 bioluminescent sensor,we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats.This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above.The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in the blood,liver,kidney and heart than that in the small intestine,spleen,lung,pancreas,brain and stomach.In addition,the four herbs showed tissue-specific effects on rat CES1 expression.Based on the CES1 biodistribution and its changes after treatment in rats,the possibility that Danshen,Gegen and Danggui might alter CES1 activities in human blood and kidney should be considered.In summary,a selective and sensitive bioluminescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats.

Discovery of human pancreatic lipase inhibitors from root of Rhodiola crenulata via integrating bioactivity-guided fractionation,chemical profiling and biochemical assay

Although herbal medicines(HMs)are widely used in the prevention and treatment of obesity and obesity-associated disorders,the key constituents exhibiting anti-obesity activity and their molecular mechanisms are poorly understood.Recently,we assessed the inhibitory potentials of several HMs against human pancreatic lipase(hPL,a key therapeutic target for human obesity),among which the root-extract of Rhodiola crenulata(ERC)showed the most potent anti-hPL activity.In this study,we adopted an integrated strategy,involving bioactivity-guided fractionation techniques,chemical profiling,and biochemical assays,to identify the key anti-hPL constituents in ERC.Nine ERC fractions(retention time=12.5e35 min),obtained using reverse-phase liquid chromatography,showed strong anti-hPL activity,while the major constituents in these bioactive fractions were subsequently identified using liquid chromatography-quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS/MS).Among the identified ERC constituents,1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose(PGG)and catechin gallate(CG)showed the most potent anti-hPL activity,with pIC50 values of 7.59±0.03 and 7.68±0.23,respectively.Further investigations revealed that PGG and CG potently inhibited hPL in a non-competitive manner,with inhibition constant(Ki)values of 0.012 and 0.082 mM,respectively.Collectively,our integrative analyses enabled us to efficiently identify and characterize the key anti-obesity constituents in ERC,as well as to elucidate their anti-hPL mechanisms.These findings provide convincing evidence in support of the anti-obesity and lipid-lowering properties of ERC.

High performance thin layer chromatography as an effective tool for rapid screening of 5α-reductase inhibitors

Benign prostatic hyperplasia(BPH)is a common disease in men,and is known to be related to 5α-reductase,which can affect steroid metabolism.Under the promotion of 5α-reductase,testosterone can be converted into dihydrotestosterone(DHT),and excessive DHT will cause related diseases.Since BPH seriously affects the quality of life of patients,it is essential to discover effective 5α-reductase inhibitors.In this study,the simple analytical method of high performance thin layer chromatography(HPTLC)was used to screen active compounds,and seven compounds with strong activity were screened out.This research will provide a reference for studying the material basis of drugs for the treatment of BPH.

Huangqi Decoction,a compound Chinese herbal medicine,inhibits the proliferation and activation of hepatic stellate cells by regulating the long noncoding RNA-C18orf26-1/microRNA-663a/transforming growth factor-βaxis

Objective Huangqi Decoction(HQD),a classical traditional Chinese medicine formula,has been used as a valid treatment for alleviating liver fibrosis;however,the underlying molecular mechanism is still unknown.Although our previous studies showed that microRNA-663a(miR-663a)suppresses the proliferation and activation of hepatic stellate cells(HSCs)and the transforming growth factor-β/small mothers against decapentaplegic(TGF-β/Smad)pathway,whether long noncoding RNAs(lncRNAs)are involved in HSC activation via the miR-663a/TGF-β/Smad signaling pathway has not yet reported.The present study aimed to investigate the roles of lncRNA lnc-C18orf26-1 in the activation of HSCs and the mechanism by which HQD inhibits hepatic fibrosis.Methods The expression levels of lnc-C18orf26-1,miR-663a and related genes were measured by quantitative reverse transcription-polymerase chain reaction.HSCs were transfected with the miR-663a mimic or inhibitor and lnc-C18orf26-1 small interfering RNAs.The water-soluble tetrazolium salt-1 assay was used to assess the proliferation rate of HSCs.Changes in lncRNA expression were evaluated in miR-663a-overexpressing HSCs by using microarray to identify miR-663a-regulated lncRNAs.RNA hybrid was used to predict the potential miR-663a binding sites on lncRNAs.Luciferase reporter assays further confirmed the interaction between miR-663a and the lncRNA.The expression levels of collagen α-2(I)chain(COL1A2),α-smooth muscle actin(α-SMA)and TGF-β/Smad signaling pathway-related proteins were determined using Western blotting.Results Lnc-C18orf26-1 was upregulated in TGF-β1-activated HSCs and competitively bound to miR-663a.Knockdown of lnc-C18orf26-1 inhibited HSC proliferation and activation,downregulated TGF-β1-stimulatedα-SMA and COL1A2 expression,and inhibited the TGF-β1/Smad signaling pathway.HQD suppressed the proliferation and activation of HSCs.HQD increased miR-663a expression and decreased lnc-C18orf26-1 expression in HSCs.Further studies showed that HQD inhibited the expression of COL1A2,α-SMA,TGF-β1,TGF-βtype I receptor(TGF-βRI)and phosphorylated Smad2(p-Smad2)in HSCs,and these effects were reversed by miR-663a inhibitor treatment.Conclusion Our study identified lnc-C18orf26-1 and miR-663a as promising therapeutic targets for hepatic fibrosis.HQD inhibits HSC proliferation and activation at least partially by regulating the lnc-C18orf26-1/miR-663a/TGF-β1/TGF-βRI/p-Smad2 axis.

Cold exposure promotes coronavirus infection by altering the gut microbiota and lipid metabolism to reduce host immunity

Objective:Cold exposure has been suggested to be advantageous for the spread and infection of the coronavirus,and the gut microbiota influences the severity of the infection by modulating host inflammatory and immune responses.However,it remains unclear whether the promotion of viral infection through cold exposure is linked to the gut microbiota.Methods:In this study,we performed an unbiased analysis of gut microbiota,serum,and lung tissue metabolome changes in cold-exposed and virus-infected mice,alongside the assessment of immune-inflammatory indicators in serum and lung tissue.Results:The results revealed that both cold exposure and viral infection significantly decreased the percentage of peripheral blood lymphocytes(CD4^(+)T cells,CD8^(+)T cells,and B cell)and increased the expression of inflammatory factors(IL-6,IL-1β,TNF-α,and IFN-γ).Meanwhile,cold exposure disrupted the homeostasis of gut microbiota,elevating the abundance of pathogenic bacteria(Staphylococcus)and diminishing the abundance of beneficial bacteria(Alistipes).Notably,in virus-infected mice exposed to a cold environment,the reduction in the abundance of beneficial bacteria Alistipes was more pronounced than in cases of single virus infection and cold exposure.Analysis of altered serum and lung tissue metabolites highlighted glycerophospholipids,fatty acids,and eicosanoids as the most affected metabolites by cold exposure.These metabolites,closely associated with virus infection,exhibited a significant correlation with immune-inflammatory indicators.Conclusion:These findings establish a mechanistic connection between cold exposure and virus infection,suggesting that cold exposure-induced dysregulation of gut microbiota and lipid metabolism diminishes host immunity,promoting virus infection.

Metabolomics and its application in the treatment of coronary heart disease with traditional Chinese medicine

Traditional Chinese Medicine(TCM) is the treasure of Chinese Nation and gained the gradual acceptance of the international community. However, the methods and theories of TCM understanding of diseases are lack of appropriate modern scientific characterization systems. Moreover, traditional risk factors cannot promote to detection and prevent those patients with coronary artery disease(CAD) who have not developed acute myocardial infarction(MI) in time. To sum up, there is still no objective systematic evaluation system for the therapeutic mechanism of TCM in the prevention and cure of cardiovascular disease.Thus, new ideas and technologies are needed. The development of omics technology, especially metabolomics, can be used to predict the level of metabolites in vivo and diagnose the physiological state of the body in time to guide the corresponding intervention.In particular, metabolomics is also a very powerful tool to promote the modernization of TCM and the development of TCM in personalized medicine. This article summarized the application of metabolomics in the early diagnosis, the discovery of biomarkers and the treatment of TCM in CAD.

Desulfovibrio vulgaris,a potent acetic acid-producing bacterium,increased by Astragalus polysaccharides to attenuate nonalcoholic fatty liver disease in mice

OBJECTIVE Although the underlying mechanism is largely unknown,gut dysbiosis has emerged as a central initiator of obesity-related diseases including nonalcoholic fatty liver disease(NAFLD),type 2 diabetes and metabolic syndrome.The emerging evidence support the use of prebiotics like herb-derived polysaccharides for treating NAFLD by modulating gut microbiome.So,our study focused on the microbiota-dependent anti-NAFLD effect and the exact mechanisms of Astragalus polysaccharides(APS)extracted from Astragalus mongholicus Bunge in high-fat diet(HFD)fed mice.METHODS Co-housing experiment was used to assess the microbiota dependent anti-NAFLD effect of APS.Then,targeted metabolomics and metagenomics were adopted for determining short-chain fatty acids(SCFAs)and bacteria that were specifically enriched by APS.Further in vitro experiment was carried out to test the capacity of SCFAs-producing of identified bacterium.Finally,the anti-NAFLD efficacy of identified bacterium was tested in HFD fed mice.RESULTS Our results first demonstrated the anti-NAFLD effect of APS in HFD fed mice and the contribution of gut microbiota.Moreover,our results indicated that SCFAs,predominantly acetic acid were elevated in APS-supplemented mice and ex vivo experiment.Metagenomics revealed that D.vulgaris from Desulfovibrio genus was not only enriched by APS,but also a potent generator of acetic acid,which showed significant anti-NAFLD effects in HFD fed mice.In addition,D.vulgaris modulated the hepatic gene expression pattern of lipids metabolism,particularly suppressed hepatic fatty acid synthase(FASN)and CD36 protein expression.CONCLUSION APS enriched D.vulgaris is effective on attenuating hepatic steatosis possibly through producing acetic acid,and modulation on hepatic lipids metabolism in mice.Further studies are warranted to explore the long-term impacts of D.vulgaris on host metabolism and the underlying mechanism.

Functional Metabolomics Reveals that Astragalus Polysaccharides Improve Lipids Metabolism through Microbial Metabolite 2-Hydroxybutyric Acid in Obese Mice

Polysaccharides are widely present in herbs with multiple activities,especially immunity regulation and metabolic benefits for metabolic disorders.However,the underlying mechanisms are not well under-stood.Functional metabolomics is increasingly used to investigate systemic effects on the host by iden-tifying metabolites with particular functions.This study explores the mechanisms underlying the metabolic benefits of Astragalus polysaccharides(APS)by adopting a functional metabolomics strategy.The effects of APS were determined in eight-week high-fat diet(HFD)-fed obese mice.Then,gas chromatography–time-of-flight mass spectrometry(GC–TOFMS)-based untargeted metabolomics was performed for an analysis of serum and liver tissues,and liquid chromatography–tandem mass spectrom-etry(LC–MS/MS)-based targeted metabolomics was performed.The potential functions of the metabo-lites were tested with in vitro and in vivo models of metabolic disorders.Our results first confirmed the metabolic benefits of APS in obese mice.Then,metabolomics analysis revealed that APS supplemen-tation reversed the HFD-induced metabolic changes,and identified 2-hydroxybutyric acid(2-HB)as a potential functional metabolite for APS activity that was significantly decreased by a HFD and reversed by APS.Further study indicated that 2-HB inhibited oleic acid(OA)-induced triglyceride(TG)accumula-tion.It was also found to stimulate the expression of proteins in lipid degradation in hepatocytes and TG lipolysis in 3T3-L1 cells.Moreover,it was found to reduce serum TG and regulate the proteins involved in lipid degradation in high-fat and high-sucrose(HFHS)-fed mice.In conclusion,our study demonstrates that the metabolic benefits of APS are at least partially due to 2-HB generation,which modulated lipid metabolism both in vitro and in vivo.Our results also highlight that functional metabolomics is practical for investigating the mechanism underlying the systemic benefits of plant polysaccharides.

Strategy of Systems Biology for Visualizing the “Black Box” of Traditional Chinese Medicine

Traditional Chinese medicine(TCM)has been practiced for thousands of years in China.TCM formula,usually composed of several or even dozens of herbal medicines,is the main form of TCM practicing,which is extremely complex due to multiple components and therapeutic targets,especially the characteristics of formula compatibility.Thus,it is an enormous challenge for the modernization of TCM.Systems biology is a strategy for investigating the complex interactions between genes,mRNA,proteins,and metabolites by using integrated omics approaches.In recent years,systems biology has been increasingly adopted in TCM study.This review comprehensively summarized status of syndrome and application of TCM formulae in clinical and preclinical studies and discussed the advances of systems biology in TCM research.Then,a"Disease-Syndrome-Formulae-Effect"strategy was proposed for TCM research.Combination of systems biology and"Disease-Syndrome-Formulae-Effect"strategy provided a novel approach to understand the complex interactions among biological systems,drugs,and complex diseases from a network perspective,thus facilitating the modernization of TCM.The objective of this manuscript is to provide comprehensive and up-to-date review on the application of systems biology in TCM research,as well as the perspective of TCM modernization with systems biology.

An ultra-sensitive and easy-to-use assay for sensing human UGT1A1 activities in biological systems

The human UDP-glucuronosyltransferase 1A1(UGT1A1),one of the most essential conjugative enzymes,is responsible for the metabolism and detoxification of bilirubin and other endogenous substances,as well as many different xenobiotic compounds.Deciphering UGT1A1 relevance to human diseases and characterizing the effects of small molecules on the activities of UGT1A1 requires reliable tools for probing the function of this key enzyme in complex biological matrices.Herein,an easy-to-use assay for highly-selective and sensitive monitoring of UGT1A1 activities in various biological matrices,using liquid chromatography with fluorescence detection(LC-FD),has been developed and validated.The newly developed LC-FD based assay has been confirmed in terms of sensitivity,specificity,precision,quantitative linear range and stability.One of its main advantages is lowering the limits of detection and quantification by about 100-fold in comparison to the previous assay that used the same probe substrate,enabling reliable quantification of lower amounts of active enzyme than any other method.The precision test demonstrated that both intra-and inter-day variations for this assay were less than 5.5%.Furthermore,the newly developed assay has also been successfully used to screen and characterize the regulatory effects of small molecules on the expression level of UGT1A1 in living cells.Ove rall,an easy-to-use LC-FD based assay has been developed for ultra-sensitive UGT1A1 activities measurements in various biological systems,providing an inexpensive and practical approach for exploring the role of UGT1A1 in human diseases,interactions with xenobiotics,and characterization modulatory effects of small molecules on this conjugative enzyme.

Vancomycin pretreatment attenuates acetaminophen-induced liver injury through 2-hydroxybutyric acid

Liver injury caused by acetaminophen(AP)overdose is a leading public health problem.Although APinduced liver injury is well recognized as the formation of N-acetyl-p-benzoquinone(NAPQI),a toxic metabolite of AP,resulting in cell damage,emerging evidence indicates that AP-induced liver injury is also associated with gut microbiota.However,the gut microbiota-involved mechanism remains largely unknown.In our study,we found that vancomycin(Vac)pretreatment(100 mg/kg,twice a day for 4 days)attenuated AP-induced liver injury,altered the composition of gut microbiota,and changed serum metabolic profile.Moreover,we identified Vac pretreatment elevated cecum and serum 2-hydroxybutyric acid(2-HB),which ameliorated AP-induced cell damage and liver injury in mice by reducing AP bioavailability and elevating GSH levels.Our current results revealed the novel role of 2-HB in protecting AP-induced liver injury and add new evidence for gut microbiota in affecting AP toxicity.

DNA Methylation and Transcription of HLA-F and Serum Cytokines Relate to Chinese Medicine Syndrome Classification in Patients with Chronic Hepatitis B

Objective:To explore the molecular bases of Chinese medicine(CM) syndrome classification in chronic hepatitis B(CHB) patients in terms of DNA methylation,transcription and cytokines.Methods:Genome-wide DNA methylation and 48 serum cytokines were detected in CHB patients(DNA methylation:15 cases;serum cytokines:62 cases) with different CM syndromes,including dampness and heat of Gan(Liver) and gallbladder(CHB1,DNA methylation:5 cases,serum cytokines:15 cases),Gan stagnation and Pi(Spleen) deficiency(CHB2,DNA methylation:5 cases,serum cytokines:15 cases),Gan and Shen(Kidney) yin deficiency(CHB3,DNA methylation:5 cases,serum cytokines:16 cases),CHB with hidden symptoms(HS,serum cytokines:16 cases) and healthy controls(DNA methylation:6 cases).DNA methylation of a critical gene was further validated and its mRNA expression was detected on enlarged samples.Genome-wide DNA methylation was detected using Human Methylation 450 K Assay and further verified using pyrosequencing.Cytokines and mRNA expression of gene were evaluated using multiplex biometric enzyme-linked immunosorbent assay(ELISA)-based immunoassay and reverse transcription-quantitative polymerase chain reaction(RT-qPCR),respectively.Results:Totally 28,667 loci,covering 18,403 genes were differently methylated among CHB1,CHB2 and CHB3(P<0.05 and|△β value|> 0.17).Further validation showed that compared with HS,the hg19 CHR6:29691140 and its closely surrounded 2 CpG loci were demethylated and its mRNA expressions were significantly up-regulated in CHB1(P<0.05).However,they remained unaltered in CHB2(P>0.05).Levels of Interleukin(IL)-12 were higher in CHB3 and HS than that in CHB1 and CHB2 groups(P<0.05).Levels of macrophage inflammatory protein(MIP)-1αand MIP-1β were higher in CHB3 than other groups and leukemia inhibrtory factor level was higher in CHB1 and HS than CHB2 and CHB3 groups(P<0.05).IL-12,MIP-1α and MIP-1β concentrations were positively correlated with human leukocyte antigen F(HLA-F)mRNA expression(R;=0.238,P<0.05;R;=0.224,P<0.05;R;=0.447,P<0.01;respectively).Furthermore,combination of HLA-F mRNA and differential cytokines greatly improved the differentiating accuracy among CHB1,CHB2 and HS.Conclusions:Demethylation of CpG loci in 5’ UTR of HLA-F may up-regulate its mRNA expression and HLA-F expression was associated with IL-12,MIP-1α and MIP-1β levels,indicating that HLA-F and the differential cytokines might jointly involve in the classification of CM syndromes in CHB.(Registration No.ChiCTR-RCS-13004001)

Efficacy and Safety of Yanggan Jian in Hepatitis B Virusrelated Decompensated Cirrhosis:A Randomized,Doubleblind,Controlled Trial

Background and Aims:The aim was to evaluate the efficacy and safety of Yanggan Jian(YGJ)in HBV-infected patients with decompensated cirrhosis.Methods:This randomized,double-blind controlled trial enrolled 160 patients with HBV-related decompensated cirrhosis who were already receiving or about to start antiviral therapy.Patients were randomly assigned to receive YGJ or placebo for 24 weeks,and were followed-up to 36 weeks.The primary outcome was the proportion of patients with a≥2 point reduction in Child-Turcotte-Pugh(CTP)score from baseline at week 24.Secondary outcomes were CTP class and score,serum liver function indices,mortality,incidence of hepatocellular carcinoma and variceal bleeding.Results:The proportion of patients with a CTP score reduction≥2 was significantly greater in the YGJ than in the placebo group(p=0.009);the percentage of patients with CTP class C was significantly less than that in the placebo group(p<0.05),and the YGJ group had a significantly greater mean change from baseline in CTP score at week 24(p=0.034).The improvement in measured values and change from baseline of prothrombin time,serum albumin,platelets,cholinesterase,international normalized ratio,and activated partial thromboplastin time were significantly better with YGJ than with placebo.Between-group differences in cumulative rates of variceal bleeding,hepatocellular carcinoma,death,or the frequency of any adverse event(AE),AEs related to treatment,or discontinuation because of AEs were not significant.Conclusions:YGJ significantly improved CTP scores and hepatic synthetic and reserve function in patients with HBV-related decompensated cirrhosis,and was safe and well tolerated.

Sustainable utilization of precious Chinese medicines:challenges and the road ahead

Traditional Chinese medicine(TCM)is considered as a unique treasure of Chinese civilization for its profound theoretical system and extensive experience in clinical practice for more than two thousand years.As one of the most commonly used folk medicines,Chinese medicines have made indelible contributions to well-being maintenance and disease treatment,as well as the progress of human civilization[1].

Preface for special issue on new analytical techniques and methods in drug metabolism and pharmacokinetics

Analytical technologies and approaches for drug metabolism and pharmacokinetics(DMPK)research in the pharmaceutical industry and academic research institutes have evolved rapidly over the past decade.On one hand,the discovery and development of small molecule drug candidates requires earlier and better understanding of their absorption,distribution,metabolism and excretion(ADME)in human as well as their interactions with metabolizing enzymes.

Nanopolyphenol rejuvenates microglial surveillance of multiple misfolded proteins through metabolic reprogramming

Microglial surveillance plays an essential role in clearing misfolded proteins such as amyloid-beta,tau,andα-synuclein aggregates in neurodegenerative diseases.However,due to the complex structure and ambiguous pathogenic species of the misfolded proteins,a universal approach to remove the misfolded proteins remains unavailable.Here,we found that a polyphenol,α-mangostin,reprogrammed metabolism in the disease-associated microglia through shifting glycolysis to oxidative phosphorylation,which holistically rejuvenated microglial surveillance capacity to enhance microglial phagocytosis and autophagy-mediated degradation of multiple misfolded proteins.Nanoformulation ofα-mangostin efficiently deliveredα-mangostin to microglia,relieved the reactive status and rejuvenated the misfolded-proteins clearance capacity of microglia,which thus impressively relieved the neuropathological changes in both Alzheimer’s disease and Parkinson’s disease model mice.These findings provide direct evidences for the concept of rejuvenating microglial surveillance of multiple misfolded proteins through metabolic reprogramming,and demonstrate nanoformulatedα-mangostin as a potential and universal therapy against neurodegenerative diseases.