Progress on Prevention and Treatment of Cerebral Small Vascular Disease Using Integrative Medicine

Cerebral small vessel disease(CSVD)is a senile brain lesion caused by the abnormal structure and function of arterioles,venules and capillaries in the aging brain.The etiology of CsvD is complex,and disease is often asymptomatic in its early stages.However,as CsvD develops,brain disorders may occur,such as stroke,cognitive dysfunction,dyskinesia and mood disorders,and heart,kidney,eye and systemic disorders.As the population continues to age,the burden of CsvD is increasing.Moreover,there is an urgent need for better screening methods and diagnostic markers for CsvD,in addition to preventive and asymptomatic-and mild-stage treatments.Integrative medicine(IM),which combines the holistic concepts and syndrome differentiations of Chinese medicine with modern medical perspectives,has unique advantages for the prevention and treatment of CsvD.In this review,we summarize the biological markers,ultrasound and imaging features,disease-related genes and risk factors relevant to CsvD diagnosis and screening.Furthermore,we discuss IM-based csvD prevention and treatment strategies to stimulate further research in this field.

How do Chinese medicines that tonify the kidney inhibit dopaminergic neuron apoptosis?

Wistar rats were intragastrical y perfused with Chinese medicines used for tonifying the kidney. These included 0.180 g/mL of Herba Epimedi （Epimedium）, Semen Cuscutae （Dodder Seed）, or Herba Cistanches （Desertliving Cistanche）, 0.04 mg/mL monoamine oxidase-B inhibitor selegiline, or distil ed water for 14 consecutive days to prepare drug-containing serum or blank serum. MES23.5 cells in the logarithmic phase were cultured in media supplemented with 15%drug-containing serum for 24 hours, fol owed by incubation in culture solution containing 100μmol/L H2O2 for 3 hours. 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） and flow tometry results showed that al drug-containing serums improved the survival rate of H 2 O 2-injured MES23.5 cells, inhibited pro-apoptotic FasL and caspase-3 expression, promoted anti-apoptotic Bcl-2 expression. However, drug-containing serums had little influence on Fas expression in H 2 O 2-injured MES23.5 cells. Enzyme-linked immunosorbent assay results showed that serum containing Herba Cistanches or Herba Epimedi increased the expression of nerve growth factor, brain-derived neurotrophic factor, and glial cellline-derived neurotrophic factor in injured MES23.5 cells;serum containing Semen Cuscutae only increased brain-derived neurotrophic factor expres-sion; while expression of the above neurotrophic factors remained the same in cells treated with serum containing selegiline. These findings indicate that Chinese medicines used to tonify the kid-ney can protect nerve cells by regulating the expression of apoptosis-related factors and neuro-trophic factors in MES23.5 cells.

Protective effects of kidney-tonifying Chinese herbal preparation on substantia nigra neurons in a mouse model of Parkinson's disease

The Chinese herbs Herba Epimedii, Fructus Ligustri Lucidi and Rhizoma Polygonati were injected into Parkinson＇s disease mice established via intraperitoneal injection of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride. The selective monoamine oxidase B inhibitor selegiline was used as a positive control drug. After successive administration for 4 weeks, Herba Epimedii could downregulate the expression of caspase-3 and increase the brain-derived neurotrophic factor level, as well as increase tyrosine hydroxylase activity in the substantia nigra of Parkinson＇s disease mouse models. Rhizoma Polygonaticould downregulate the expression of caspase-3 and FasL, and increase neural growth factor and brain-derived neurotrophic factor levels. Fructus Ligustn Lucidi could downregulate caspase-3 expression. Rhizoma Polygonati and Fructus Ligustn Lucidi did not produce obvious effects on tyrosine hydroxylase activity. Herba Epimedii and Fructus Ligustri Lucidi yielded similar effects on apoptosis-promoting factors to those elicited by selegiline. Herba Epimedii and Rhizoma Polygonati significantly increased the levels of neurotrophic factors compared with selegiline. Herba Epimedii significantly increased tyrosine hydroxylase activity compared with selegiline. It is indicated that the kidney-tonifying Chinese herbal preparation can downregulate the expression of apoptosis-promoting factors, increase neurotrophic factors levels in the substantia nigra and striatum, as well as increase tyrosine hydroxylase activity in the substantia nigra of Parkinson＇s disease mouse models, thereby exerting a stronger or similar neuroprotective effects compared with selegiline.

Effects of glossy privet fruit on neural cell apoptosis in the cortical parietal lobe and hippocampal CA1 region of vascular dementia rats

BACKGROUND： Glossy privet fruit inhibits neural cell apoptosis following the onset of vascular dementia. OBJECTIVE： To confirm glossy privet fruit effects on neural cell apoptosis in the cortical parietal lobe and hippocampal CAI region of rat models of vascular dementia using molecular biology techniques. DESIGN, TIME AND SETTING： The neural cell morphology experiment was performed at the Laboratory of Flow Cells and Biochemistry, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, and the Basic Room of Pathology, Academy of Chinese Medicine from December 2006 to May 2008. MATERIALS： A total of 60 Wistar rats were used to establish vascular dementia models using a photochemical reaction method. Glossy privet fruit was purchased from Fujian, China. Hydergine was co-produced by Sandoz, Switzerland and Huajin, China. METHODS： The 60 Wistar rats were randomly divided into 6 equal sized groups （n = 10）, i.e. model, blank, high, moderate and low doses of Chinese medicine, and hydergine control groups. Rats in the model group were treated with distilled water （1 mL/100 g） by gavage following model establishment. Rats in the blank group underwent experimental procedures as for the model group, except that rat models were created without illumination. Rats in the high, moderate and low doses of Chinese medicine groups, and the hydergine control group respectively received high, moderate and low doses of glossy privet fruit, and hydergine suspension （1 mL/100 g） by gavage, once a day, for 30 days. MAIN OUTCOME MEASURES： Morphology of neural cells from the rat cortical parietal lobe and hippocampal CA1 region of all groups was observed with an electron microscope. Positive neural cells in the injury site of the rat cortical parietal lobe and hippocampal CA1 region were investigated using the Fas immunohistochemieal method. Absorbance of Fas-positive neurons was detected by the MPIAS-500 multimedia color imaging analysis system. RESULTS： Neural cells were normal, and nuclei were regular in the right cortical parietal lobe and hippoeampal CA1 region in the blank group. Karyopyknosis, an integral nuclear membrane, vacuole and apoptotic bodies were presented in the model group. The quantity and morphology of neural cells were normal in all doses of Chinese medicine groups, and the hydergine control group. Compared with the model group, absorbance was reduced at the injury site of rat cortical parietal lobe and hippocampal CA1 region in the blank, high, moderate and low doses of Chinese medicine, and hydergine control groups （P 〈 0.05）. The decrease was particularly significant in the blank group （P 〈 0.01 ）, followed by the high dose of Chinese medicine group （P 〈 0.01）. Compared with the model group, the percentage of apoptosis was decreased at the injury site of the rat cortical parietal lobe and hippocampal CAI region in the blank, high, moderate and low doses of Chinese medicine, and hydergine control groups （P 〈 0.01） and this decrease was significant in the high dose of Chinese medicine group （P 〈 0.01）. CONCLUSION： Glossy privet fruit, a kidney-tonifying Chinese herbal medicine, can inhibit cell apoptosis by reducing apoptotic signals induced by cerebral ischemia/hypoxia.

Mechanisms of Dangua Fang(丹瓜方)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics

OBJECTIVE:To explore the mechanism of Dangua Fang(丹瓜方,DGR)in multi-target and multi-method regulation of glycolipid metabolism based on phosphoproteomics.METHODS:Sprague-Dawley rats with normal glucose levels were randomly divided into three groups,including a conventional diet control group(Group A),high-fat-highsugar diet model group(Group B),and DGR group(Group C,high-fat-high-sugar diet containing 20.5 g DGR).After 10 weeks of intervention,the fasting blood glucose(FBG),2 h blood glucose[PBG;using the oral glucose tolerance test(OGTT)],hemoglobin A1c(HbA1c),plasma total cholesterol(TC),and triglycerides(TG)were tested,and the livers of rats were removed to calculate the liver index.Then,hepatic portal TG were tested using the Gross permanent optimization-participatiory action planning enzymatic method and phosphoproteomics was performed using liquid chromatography with tandem mass spectrometry(LC-MS/MS)analysis followed by database search and bioinformatics analysis.Finally,cell experiments were used to verify the results of phosphoproteomics.Phosphorylated mitogen-activated protein kinase kinase kinase kinase 4(MAP4k4)and phosphorylated adducin 1(ADD1)were detected using western blotting.RESULTS:DGR effectively reduced PBG,TG,and the liver index(P<0.05),and significantly decreased HbA1c,TC,and hepatic portal TG(P<0.01),showed significant hematoxylin and eosin(HE)staining,red oil O staining,and Masson staining of liver tissue.The total spectrum was 805334,matched spectrum was 260471,accounting for accounting 32.3%,peptides were 19995,modified peptides were 14671,identified proteins were 4601,quantifiable proteins were 4417,identified sites were 15749,and quantified sites were 14659.Based on the threshold of expression fold change(>1.2),DGR upregulated the modification of 228 phosphorylation sites involving 204 corresponding function proteins,and downregulated the modification of 358 phosphorylation sites involving 358 corresponding function proteins,which included correcting 75 phosphorylation sites involving 64 corresponding function proteins relating to glycolipid metabolism.Therefore,DGR improved biological tissue processes,including information storage and processing,cellular processes and signaling,and metabolism.The metabolic functions regulated by DGR mainly include energy production and conversion,carbohydrate transport and metabolism,lipid transport and metabolism,inorganic ion transport and metabolism,secondary metabolite biosynthesis,transport,and catabolism.In vitro phosphorylation validation based on cell experiments showed that the change trends in the phosphorylation level of MAP4k4 and ADD1 were consistent with that of previous phosphoproteomics studies.CONCLUSION:DGR extensively corrects the modification of phosphorylation sites to improve corresponding glycolipid metabolism-related protein expression in rats with glycolipid metabolism disorders,thereby regulating glycolipid metabolism through a multi-target and multi-method process.

Qingjie Fuzheng granules inhibit colorectal cancer cell growth by the PI3K/AKT and ERK pathways

BACKGROUND Qingjie Fuzheng granules(QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, including colorectal cancer(CRC).However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs' anticancer effect have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of colorectal cancer cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways.AIM To better evaluate the mechanism underlying the anti-cancer effect of QFGs on the CRC cell lines HCT-116 and HCT-8.METHOD First, we measured cell viability and cytotoxicity by performing MTT and lactate dehydrogenase(LDH) assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation and analyzing Hoechst 33258 staining. Second, cell cycle and apoptosis rates were measured by fluorescence activated cell sorting, and the expression levels of survivin, cyclin D1, CDK4, p21,Bax, Bcl-2, Fas, FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blots and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were used to elucidate the specific apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3 K/AKT and ERK signaling pathwayswas examined using the western blot assay to investigate the possible mechanism.RESULTS MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by(6.90% ± 1.03%)–(59.70% ± 1.51%)(HCT-116; P < 0.05)and(5.56% ± 4.52%)–(49.44% ± 2.47%)(HCT-8; P < 0.05), and cytotoxicity was increased from 0.52 ± 0.023 to 0.77 ± 0.002(HCT-116; P < 0.01) and from 0.56 ±0.054 to 0.81 ± 0.044(HCT-8; P < 0.01) compared with the non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells.QFGs also increased the expression levels of Bax, Fas and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast, when adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3 K, AKT and ERK.CONCLUSION These results demonstrated that QFGs can inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3 K/AKT and ERK signaling pathways.

Application of Serum Pharmacology in Evaluating the Antitumor Effect of Fuzheng Yiliu Decoction(扶正抑瘤方) from Chinese Medicine

Objective： To investigate the feasibility of serum pharmacology in evaluating the antitumor effect of Chinese medicine （CM） of Fuzheng Guben （扶正固本, supporting the healthy energy and strengthening the body＇s resistance to pathogens）, the effects of Fuzheng Yiliu Decoction （扶正抑瘤方, FYD）, a typical prescription of Fuzheng Guben, on proliferation and apoptosis of hepatoma cells in vitro were observed by two methods with serum pharmacology and traditional pharmacology, respectively. Methods： HepG2 cells were treated with FYD- containing serum or crude FYD extract in vitro. The proliferation rate was determined by methyl thiazolyl tetrazolium （MTT） assay. Cell cycle and apoptosis rate was performed by flow cytometry. And the levels of interleukin-2 （IL-2） and tumor necrosis factor α （TNF-α） in FYD-containing serum were detected by radioimmunoassay. Results： FYD-containing serum remarkably inhibited proliferation and induced apoptosis of hepatoma cells at least by promoting the production of IL-2 and TNF- α in vivo. On the contrary, crude FYD extract promoted the proliferation and did not induce cell apoptosis. Conclusion： The results by serum pharmacology were accordant with those of our previous animal and clinical trials which indicates that serum pharmacology is a reasonable and feasible method for the evaluation of the antitumor effect of herbs of Fuzheng Guben.

Traditional Chinese Medicine syndrome-related herbal prescriptions in treatment of malignant tumors

OBJECTIVE： To investigate the distribution characteristics of TCM syndromes and the related herbal prescriptions for malignant tumors （MT）. METHODS： A clinical database of the TCM syndromes and the herbal prescriptions in treatment of 136 MT patients were established. The data were then analyzed using cluster and frequency analysis. RESULTS： According to the cluster analysis, the TCM syndromes in MT patients mainly included two patterns： deficiency of both Qi and Yin and internal accumulation of toxic heat. The commonlyprescribed herbs were Huangqi （Astraglus）, Nuizhenzi （Fructus Ligustri Lucidi）, Lingzhi （Ganoderma Lucidum）, Huaishan （Dioscorea Opposita）, Xiakucao （Prunella Vulgaris）, and Baihuasheshecao （Herba Hedyotidis）. CONCLUSION： Deficiency of Qi and Yin is the pri- mary syndrome of MT, and internal accumulation of toxic heat is the secondary syndrome. The herbs for Qi mainly heat-cl supplementation and Yin houris used, with earance and the assistance of detoxification. hment are herbs for

Babao Dan Inhibits Gastric Cancer Progression in vivo Through Multiple Signaling Pathways

Objective:The aim of this study was to explore the effects of Babao dan(BBD),a traditional Chinese medicine,on gastric cancer(GC)progression in vivo.Materials and Methods:A subcutaneous xenograft mouse model of GC was established using MGC80-3 cells.The terminal deoxynucleotidyl transferase-mediated dUTP:2'-deoxyuridine 5'-triphosphate-biotin nick-end labeling method was adopted to detect cell apoptosis in vivo.The expression levels of proteins associated with proliferation,apoptosis,and angiogenesis were measured by immunohistochemical staining or western blotting(WB).The activation and protein levels of p-c-Jun N-terminal kinase(JNK),p-p38,p-extracellular-regulated kinase 1/2,p-nuclear factor-κB(NF-κB),and p-STAT3 were examined by Bio-plex and WB.Results:BBD significantly inhibited tumor growth in GC mouse models with no adverse effect on body weight or organ function.It was also found that BBD significantly suppressed the proliferation of GC tumor cells,induced the apoptosis of tumor cells,and inhibited angiogenesis through inactivating with mitogen-activated protein kinase,NF-κB,and STAT3 pathways.Conclusions:BBD exerts suppressive effects on GC tumor growth by regulating multiple pathways in vivo,which may provide a novel treatment option for GC therapy.

Fuzheng Yiliu Granule(扶正抑瘤颗粒)Inhibits the Growth of Hepatocellular Cancer by Regulating Immune Function and Inducing Apoptosis In Vivo and In Vitro

Objective：To study the inhibitory effect of Fuzheng Yiliu Granule（扶正抑瘤颗粒,FYG） on hepatocellular cancer（HCC） and investigate the mechanism mediating its bioactivity.Methods：H22 tumor-bearing ICR mice were treated with FYG[3.6 g/（kg·d）]for 5 days.Tumor volume and tumor weight,percentages of CD3~＋,CD4~＋,CD8~＋,and natural killer（NK） cells in peripheral blood,tumor apoptosis and serum levels of interleukin-2（IL-2）,and tumor necrosis factor-α（TNF-α） were evaluated.FYG-containing serum was prepared from SD rats treated for 7 days[high dose 3.6 g/（kg·d）;middle dose 1.8 g/（kg·d）;low dose 0.9 g/（kg·d）].Cell cycle,cell viability,and apoptosis were evaluated after HepG2 cell line was cultured in FYG-containing serum for 48 h.The levels of IL-2 and TNF-αin FYG-containing serum were also determined.Results：FYG produced a potent antitumor effect（P0.01） and induced marked apoptosis of the tumor tissue（P0.05）.Mice treated with FYG had higher percentages of CD3~＋ and CD4~＋（P0.05）,and more NK cells（P0.01） in the peripheral blood than those in the animals treated with normal saline.Mice receiving FYG had the highest serum levels of IL-2 and TNF-α（P0.01）.High-dose FYG-containing serum significantly decreased HepG2 cell viability,inhibited cell proliferation（P0.05）,and induced apoptosis（P0.01）.In addition,the levels of IL-2 and TNF-αof highdose -containing serum were higher than the blank serum（P0.01）.Conclusion：FYG could inhibit HCC growth by regulating immune function and inducing apoptosis of tumor cells in vivo and in vitro.

Computational Pharmacology Study of Tougu Xiaotong Granule(透骨消痛颗粒) in Preventing and Treating Knee Osteoarthritis

Objective： To study the pharmacological properties of Tougu Xiaotong Granule （透骨消痛颗粒, TGXTG） in preventing and treating knee osteoarthritis （KOA） at the molecular level. Methods： The computational methods, including principal component analysis, molecular docking, target-ligand space distribution, and the predictions of absorption, distribution, metabolism, excretion and toxicity （ADMET）, were introduced to characterize the molecules in TGXTG. Results： The structural properties of molecules in TGXTG were more diverse than those of the drug/drug-like molecules, and TGXTG could interact with significant target enzymes related to KOA. In addition, the cluster of effective components was preliminarily identified by the target-ligand space distributions. As to the results of ADMET properties, some of them were unsatisfactory, and were merely regarded as references here. Conclusion： Based on this computational pharmacology study, TGXTG is a broad- spectrum recipe inhibiting many important target enzymes, which could effectively postpone the degeneration of cartilage by coordinately inhibiting the biological effects of cytokines, matrix metallopeptidase 3, and oxygen free radicals.

Effect of Xiaoaiping injection on advanced hepatocellular carcinoma in patients

OBJECTIVE: To investigate the effect of Xiaoaiping Injection (XAP) on advanced hepatocellular carcinoma (HCC) in patients. METHODS: Sixty-eight patients with advanced HCC were assigned to a control group of 36 and a treatment group of 32.The control group was treated with best supportive treatment (BST) and the treatment group was given XAP plus BST. XAP was administered daily by iv and the treatment course was lasted for 30 days for both groups.The immediate therapeutic efficacy, Karnofsky performance status (KPS) scores, and the changes in immunity indicators (CD3+, CD4+ and CD8+ T cells) were measured and compared before and after treatment. The progression-free survival (PFS) rate and overall survival (OS) rate in the 2 groups were analyzed. RESULTS: The immediate therapeutic efficacy and KPS of the treatment group were better than those in the control group (P<0.05). Patients in the treat-ment group had higher percentages of CD3 and CD4 T-lymphocytes in peripheral blood than those in the control group (P<0.05). The median survival time was 27.0 weeks in the treatment group and 24.5 weeks in the control group. The 6-months cumulative survival rates in the treatment and control groups were 33.3% and 25.0% , respectively, with no significant difference (P>0.05). The PFS was 18 weeks in the treatment group and 15 weeks in control group (P<0.05). CONCLUSION: XAP enhances the quality of life (QOL) of patients with advanced HCC, improves their immunity and extends their PFS.

Experimental Study on the Suppression of Sodium Nitroprussiate-lnduced Chondrocyte Apoptosis by Tougu Xiaotong Capsule(透骨消痛胶囊)-Containing Serum

Objective：To study the mechanism of action of Tougu Xiaotong Capsule（透骨消痛胶囊,TGXTC） ex vivo in suppressing chondrocyte（CD） apoptosis induced by sodium nitroprussiate（SNP）.Methods：Thirty New Zealand rabbits,2 months old,were randomized by lottery into five groups,six in each：the blank group treated with saline,the positive control group treated with Zhuanggu Guanjie Pill（壮骨关节丸,70 mg/kg）,and the three experimental groups,EGA,EGB,and EGC,treated with low dose（35 mg/kg）,moderate dose（70 mg/kg）,and high dose（140 mg/kg） of TGXTC,respectively.All treatments were administered via gastrogavage twice a day for 3 days.Arterial blood was collected from the abdominal aorta and drug or drug metabolites-containing serum was prepared.CDs obtained from knee joints of 16 four-week-old New Zealand rabbits were cultured to the third passage and confirmed by toluidine blue staining.SNP of various final concentrations（0,0.5,1.0,and 2.0 mmol/L） was used to induce CD apoptosis,and the dosage-effect relationship of SNP in inducing CD apoptosis was determined.Serum samples from the blank,control,and three dosages of TGXTC-treated rabbits were tested in the CD culture in the presence of SNP.Cell apoptosis was determined by Hoechst 33342 staining,viability of CDs was quantified by MTT,CD apoptosis rate was determined by annexin V-FITC/PI staining,levels of p53 and Bcl-2 mRNA expression in CDs were determined with RT-PCR,and contents of caspase-3 and caspase-9 proteins were determined by colorimetry.Results：CD apoptosis was induced by SNP at all concentrations tested and in a dose-dependent manner.The SNP concentration of 1 mmol/L and treatment duration of 24 h appeared to be optimal and were selected for the study.Serum samples from the positive control rabbits and from the two higher doses of TGXTC-treated rabbits showed reduction of SNP-induced CD apoptosis,decrease in p53 mRNA expression,inhibition of catalytic activities of caspase-3 and caspase-9,and increase in Bcl-2 mRNA expression when compared with the serum from the blank group（P0.05）.Conclusion：TGXTC-containing sera antagonized SNP-induced CD apoptosis and the molecular basis for the action was associated with up-regulation of Bcl-2, down-regulation of p53 expression,and inhibition of caspase-3 and caspase-9 catalytic activities.

Bear Bile Powder Inhibits Growth of Hepatocellular Carcinoma via Suppressing STAT3 Signaling Pathway in Mice

Objective: To evaluate the inhibitory effect of bear bile powder(BBP) on hepatocellular carcinoma(HCC) growth in vivo and investigate the underlying mechanisms. Methods: A HCC xenograft mouse model was developed by producing with huh7 cells. After 5 days following xenograft implantation, ten HCC xenograft mice were given intra-gastric administration with 10 mg/(kg·d) dose of BBP or saline for 3 weeks. Tumor growth in HCC xenograft mice was evaluated by measuring the tumor weight and volume. Cell apoptosis, proliferation or tumor angiogenesis were examined via immunohistochemical(IHC) staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL), proliferating cell nuclear antigen(PCNA) or cluster of differentiation 31(CD31), respectively. Phosphorylation of signal transducer and activator of transcription 3(STAT3) were determined by Western blot. The m RNA and protein expressions of Bcl-2, Bax, Cyclin D1 and Cyclin-dependent kinase 4(CDK4) in HCC tumor tissues were respectively determined by reverse transcription polymerase chain reaction(RT-PCR) and Western blot. The protein expression of vascular endothelial growth factor A(VEGF-A) in tumor tissues was examined by IHC staining. Results: BBP treatment led to a significant decrease on tumor volume and tumor weight in HCC mice(P<0.05) and had no effect on the change of body weight. In addition, BBP profoundly promoted cell apoptosis, inhibited cell proliferation and intratumoral microvessel density in HCC tumor tissues(P<0.05). Moreover, BBP treatment remarkably suppressed the STAT3 phosphorylation and modulated the expression of critical target genes including Bcl-2, Bax, Cyclin D1, CDK4 and VEGF-A in HCC mice. Conclusion: BBP exerts its anti-cancer activities via suppressing STAT3 signaling pathway and affecting multiple intracellular targets.

Electroacupuncture Attenuates Ischemic Brain Injury and Cellular Apoptosis via Mitochondrial Translocation of Cofilin

Objective:To investigate the potential mechanisms of electroacupuncture(EA)to prevent ischemic stroke.Methods:The method of middle cerebral artery occlusion(MCAO)was employed to establish a rat model of ischemic stroke.Seventy-eight Sprague-Dawley rats were divided into the sham group,MCAO+EA control(EC)group,and MCAO+EA(EA)group according to a random number table(n=26 per group).EA was applied to the acupoints of Baihui(DU 20)and Shenting(DU 24)5 min and 6 h,respectively after the onset of MCAO for 30 min.Rats in the sham and EC groups received only light isoflurane anesthesia for 30 min after MCAO.The neuroprotective effects of EA were evaluated by rota-rod test,neurological deficit scores and infarct volumes.Additionally,Nissl staining and immunostaining were performed to examine brain damage,rod formation,cellular apoptosis,and neuronal loss induced by ischemia.The activities of caspase-3,and expression levels of cofilin and p-cofilin in mitochondria and cytoplasm after ischemic injury were determined by Western blot.Results:Compared with the EC group,EA significantly improved neuromotor function and cognitive ability after ischemic stroke(P<0.05 or P<0.01).Therapeutic use of EA also resulted in a significant decrease of cofilin rod formation and microtubule-associated protein-2(MAP2)degradation in the cortical penumbra area compared with the EC rats(P<0.01).Furthermore,Western blot analysis showed that EA stimulation significantly inhibited mitochondrial translocation of cofilin and caspase-3 cleavage(P<0.05 or P<0.01).Additionally,brain damage(infarct volume and neuropathy),cellular apoptosis and neuronal loss induced by ischemia were remarkably suppressed by EA in the cortical penumbra of rats(P<0.05 or P<0.01).Conclusion:EA treatment after ischemic stroke may attenuate ischemic brain injury and cellular apoptosis through the regulation of mitochondrial translocation of cofilin,a novel mechanism of EA therapy.

Paradox of Using Intensive Lowering of Blood Glucose in Diabetics and Strategies to Overcome It and Decrease Cardiovascular Risks

Hyperglycemia significantly increases the risk of cardiovascular disease （CVD） in diabetics. However, it has been shown by a series of large scale international studies that intensive lowering of blood glucose levels not only has very limited benefits against cardiovascular problems in patients, but may even be harmful to patients at a high risk for CVD and/or poor long-term control of blood glucose levels. Therefore, Western medicine is faced with a paradox. One way to solve this may be administration of Chinese herbal medicines that not only regulate blood glucose, blood fat levels and blood pressure, but also act on multiple targets. These medicines can eliminate cytotoxicity of high glucose through anti-inflammatory and anti-oxidant methods, regulation of cytokines and multiple signaling molecules, and maintenance of cell vitality and the cell cycle, etc. This allows hyperglycemic conditions to exist in a healthy manner, which is called ＂harmless hyperglycemia＂ Furthermore, these cardiovascular benefits go beyond lowering blood glucose leve^s. The mechanisms of action not only avoid cardiovascular injury caused by intensive lowering of blood glucose levels, but also decrease the cardiovascular dangers posed by hyperglycemia.

Mechanisms of Dangua Recipe in Improving Glycolipid Metabolic Disorders Based on Transcriptomics

Objective:To explore the mechanisms of Dangua Recipe(DGR)in improving glycolipid metabolism based on transcriptomics.Methods:Sprague-Dawley rats with normal glucose level were divided into 3 groups according to a random number table,including a conventional diet group(Group A),a DGR group(Group B,high-calorie diet+20.5 g DGR),and a high-calorie fodder model group(Group C).After 12 weeks of intervention,the liver tissue of rats was taken.Gene sequence and transcriptional analysis were performed to identify the key genes related to glycolipid metabolism reflecting DGR efficacy,and then gene or protein validation of liver tissue were performed.Nicotinamide phosphoribosyl transferase(Nampt)and phosphoenolpyruvate carboxykinase(PEPCK)proteins in liver tissues were detected by enzyme linked immunosorbent assay,fatty acid synthase(FASN)protein was detected by Western blot,and fatty acid binding protein 5(FABP5)-mRNA was detected by quantitative real-time polymerase chain reaction.Furthermore,the functional verification was performed on the diabetic model rats by Nampt blocker(GEN-617)injected in vivo.Hemoglobin A1c(HbA1c),plasma total cholesterol and triglycerides were detected.Results:Totally,257 differentialdominant genes of Group A vs.Group C and 392 differential-dominant genes of Group B vs.Group C were found.Moreover,11 Gene Ontology molecular function terms and 7 Kyoto Encyclopedia of Genes and Genomes enrichment pathways owned by both Group A vs.Group C and Group C vs.Group B were confirmed.The liver tissue target validation showed that Nampt,FASN,PEPCK protein and FABP5-mRNA had the same changes consistent with transcriptome.The in vivo functional tests showed that GEN-617 increased body weight,HbA1c,triglyceride and total cholesterol levels in the diabetic rats(P<0.05 or P<0.01);while all the above-mentioned levels(except triglyceride)were decreased significantly by GEN-617 combined with DGR intervention(P<0.05 or P<0.01).Conclusion:Nampt activation was one of the mechanisms about DGR regulating glycolipid metabolism.

Potential Synergistic and Multitarget Effect of Herbal Pair Chuanxiong Rhizome-Paeonia Albifora Pall on Osteoarthritis Disease:A Computational Pharmacology Approach

Objective：To study the polypharmacological mechanism of herbal pair Chuanxiong Rhizome-Paeonia Albifora Pall（HP CXR-PAP） on the treatment for osteoarthritis（OA）.Methods：Chemical space was used to discuss the similarities and differences between the molecule sets of HP CXR-PAP and drugs.Docking protocol was used to study the interaction between HP CXR-PAP and OA target enzymes.The similarities and differences of HP CXR-PAP and drugs in target spaces were elucidated by network features.Results：The plots between the molecule sets of HP CXR-PAP and drugs in chemical space had the majority in the same region, and compounds from HP CXR-PAP covered a much larger additional region of space than drug molecules, which denoted the diverse structural properties in the molecule set of HP CXR-PAP.The molecules in HP CXR-PAP had the properties of promiscuous drugs and combination drug,and both HP CXR-PAP ligand-target interaction network and drug ligand-target interaction network were similar in the interaction profiles and network features,which revealed the effects of multicomponent and multitarget.Conclusion：The clue of potential synergism was obtained in curing OA disease by Chinese medicine,which revealed the advantages of Chinese medicine for targeting osteoarthritis disease.

Effect of Xuefu Zhuyu Capsule (血府逐瘀胶囊) on Angiogenesis in Hindlimb Ischemic Rats

Objective:To investigate the effect and mechanism of Xuefu Zhuyu Capsule(血府逐瘀胶囊,XZC)on pro-angiogenesis in the hindlimb ischemic model rats.Methods:A total of 100 Sprague Dawley rats were randomly divided into a model group,a regular-dose XZC group(0.48 g·kg^-1·d^-1)and a high-dose XZC group(0.96 g·kg^-1·d^-1)using random number table method.The model of hindlimb ischemic rats were made through femoral artery embolization with Bletilla microsphere age nt.XZC were give n on the first day after embolization surgery and lasted 5 days.Finally 72 models were obtained with 12 in each group for each time point.The lower Ischemic limb was amputated on the third day after embolization surgery.Histopathological characters and the number of blood vessels of granulation tissues were observed at 36 and 48 h after amputation,respectively.The main genes were obtained from microarray analysis and were validated using real-time quarttitative polymerase chain reaction.Results:The vascular number of granulation tissues at both 36 and 48 h were characterized by new and fresh vessels.The number of angiogenesis in the high-dose XZC group at 36 and 48 h was greater compared with that in the regular-dose XZC and model groups(P<0.01),and high-dose XZC at 36 h increased more vessels than that at 48 h(P<0.01).Consequently,granulation tissues from the high-dose XZC group at 36 h were chosen for microarray analysis.In all,2,085 differentially expressed genes(DEGs)were detected and 25 DEGs were determined to be directly related to an giogenesis.Four biological process terms were found including an giogenesis,regulati on of an gioge nesis,positive regulati on of an giogenesis,and positive regulation of vascular end othelial growth factor receptor sign aling pathway(P<0.05).Microarray an alysis also showed 49 pathways including 11 pathways related to an giogenesis.Conclusion:XZC promoted angiogenesis moderately and the mechanism invoIved multiple DEGs and multiple pathways.

Hydrogen peroxide preconditioning enhances the therapeutic efficacy of Wharton＇s Jelly mesenchymal stem cells after myocardial infarction

Background Exposure of cells to sublethal concentrations of hydrogen peroxide （H202） can alleviate subsequent oxidative stress-induced apoptosis. We assessed the effects of H202 preconditioning on the therapeutic potential of human umbilical cord Wharton＇s Jelly mesenchymal stem cells （WJ-MSCs） in a murine model of myocardial infarction. Methods WJ-MSCs were incubated in the media for 2 hours with or without 200 iJmol/L H202. Mice underwent left anterior descending coronary artery ligation, and received injection of phosphate buffered saline, 1×10^6 WJ-MSCs, or 1×10^6 H202 preconditioned WJ-MSCs 3 hours later via tail vein. Echocardiography was performed 0, 7, 14 and 28 days after surgery, and the mice were euthanized on day 28 for histological analysis. In vitro cytokine concentrations in the WJ-MSC cell supernatant were measured by enzyme-linked immunosorbent assay （ELISA）. The effect of WJ-MSC cell supernatant on the migration and proliferation of endothelial cells were observed by transwell migration and 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyl tetrazoliumbromide （MTT） assays. Results Echocardiographic measurements revealed a significant improvement in the left ventricular contractility of the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Histological analysis revealed increased neovascularization and reduced myocardial fibrosis in the WJ-MSCs-H2O2 group compared to the WJ-MSCs group. Pretreatment of WJ-MSCs with H2O2 increased the secretion of interleukin-6 （IL-6） into the cell culture supernatant by approximately 25-fold. The culture supernatant from WJ-MSCs-H2O2 significantly increased the migration and proliferation of endothelial cells; these effects could be blocked using an anti-IL-6 antibody. Conclusions This study demonstrates that H2O2 preconditioning significantly enhanced the therapeutic potential of WJ-MSCs, possibly by stimulating the production of IL-6 by WJ-MSCs, which may cause migration and proliferation of endothelial cells and increase neovascularization.