The mevalonate pathway is known for the synthesis of cholesterol, but recent studies have reported that it also controls Hippo signaling, which is critical for the regulation of organ size and tumorigenesis. Here, we discover that the suppression of the mevalonate pathway inhibits the growth and proliferation of colon cancer cell lines. The results of transcriptomic and proteomic assays suggested that the mevalonate pathway controls multiple signaling pathways relevant to cell proliferation, and the results were further confirmed using western blot, PCR, and immunofluorescence assays. As cell proliferation is an energy-consuming process, we postulate that the mevalonate pathway may also control nutrient uptake to coordinate the processes of energy supply and cell proliferation. Here, we found that lovastatin, a mevalonate pathway inhibitor, suppresses glucose and amino acid uptake and lactate acid production. More importantly, mevalonic acid itself is sufficient to promote glucose uptake by colon cancer cells. In addition, we found that colon cancer tissues displayed a higher expression of mevalonate pathway enzymes, which may promote cell growth and stimulate energy uptake. Together, our findings establish the mevalonate pathway as a critical regulator in coordinating energy input and cell proliferation. Amino acids are the classical stimulus for mTORC1 activation7. In the presence of amino acids, mTOR promotes growth by stimulating the de novo synthesis of proteins, nucleotides, and lipids, and by inhibiting autophagy through the phosphorylation of ULK1 at Serine 75810. Glucose is the major source of energy for the cell. Recent studies showed that the Hippo pathway and AMP-activated protein kinase (AMPK) were activated during glucose starvation. Cellular energy stress, e.g. glucose withdrawal, induces YAP phosphorylation and cytoplasmic localization, as well as proteasomal degradation11. As a key transcription factor that induces cell growth and proliferation, YAP is also regulated by the condition of cellular energy supply.Cell proliferation and growth are under the tight control of intracellular signaling pathways and the extracellular environment, such as energy availability. How cells sense the extracellular nutrients and utilize them for growth and proliferation has been extensively studied12. HMG-CoA reductase is the target of statins, which are commonly used for lipid-lowering therapy in patients with high-cholesterol. Statins have been shown to suppress the proliferation of cancer cells15. In addition, some studies have shown that statin use slightly decreased the risk of certain types of cancer, such as colon cancer18. Interestingly, epidemiological data also showed that statin use increased the risk of diabetes22. The evidences suggest that the mevalonate pathway is involved in the regulation of cell proliferation25, and probably, to control energy homeostasis simultaneously.The mevalonate pathway is known to synthesize cholesterol. HMG-CoA synthase and HMG-CoA reductase are rate-limiting enzymes catalyzing the conversion of acetyl-CoA to mevalonic acid (MVA)3. Based on these findings, we postulate that the mevalonate pathway may function as a mediator to coordinate nutrient uptake and cell proliferation. In this study, we revealed that MVA, a key intermediate product of the mevalonate pathway, is essential for cell growth and proliferation. Transcriptome and proteome sequencing analysis showed that MVA activated multiple pathways responsible for cell growth and proliferation. MVA also promoted glucose and amino acid uptake, which orchestrates the cell proliferation. In addition, compared to the normal colon tissue, the colon carcinoma has increased the mevalonate pathway activity, represented by a higher expression of HMGCR and HMGCS1. These data establish the mevalonate pathway as a mediator that coordinates cell proliferation and nutrient uptake.Two independent studies reported that statins could significantly suppress the nuclear localization and transcriptional responses of YAP and TAZ, two transcription factors that are influenced by energy supplyWnt and YAP/TAZ are two signaling pathways that control cell growth and proliferation. Given their key roles in the pathogenesis of colon cancer, we used colon cancer cell lines to test the effect of lovastatin on cell proliferation. The results show that proliferation was significantly suppressed by lovastatin treatment in all cell lines tested, except for HT-29 Fig. . The celTo explore the underlying mechanisms by which lovastatin suppresses cell proliferation, we performed RNA sequencing and proteomics analysis. RNA sequencing results demonstrated that lovastatin treatment considerably changed the transcriptional profile of RKO cells Fig. –3. Compa28. Wnt signaling is frequently activated in multiple carcinomas, especially in colon cancer28. The canonical Wnt signaling is activated by the accumulation of β-catenin28, which translocates to the nucleus to act as a coactivator of the TCF/LEF transcriptional factors to initiate gene transcription. Of note, the KEGG enrichment analysis shows that Wnt signaling is regulated by the mevalonate pathway , together with casein kinase Iα (CKIα), phosphorylate β-catenin, which initiates subsequent β-catenin ubiquitination and degradationlls Fig. . Once β-nts Fig. . As expe30. RNA sequencing data showed that CTGF, CYR61, ANKRD1, and AMOTL2, which are all verified YAP/TAZ target genes29, were strikingly suppressed by lovastatin treatment, but upregulated by MVA supplementation. To further confirm that MVA not only regulates canonical Wnt signaling but also controls the alternative Wnt signaling mediated by the transcription factors YAP/TAZ, we performed RT-PCR and confirmed the sequencing results  to test whether the activated mevalonate pathway by LPS promotes the activity of YAP/TAZ signaling in vivo. It showed that the RNA expression of CTGF and CYR61 were upregulated in the liver by LPS treatment , ribosomal protein S6 (RPS6), and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). As expected, exogenous MVA addition recovered mTOR signaling activity in RKO and SW480 cells  uptake assay and confirmed that lovastatin suppresses 2-DG uptake, while MVA addition rescues the suppression by lovastatin  which was supplied with or without exogenous MVA. The medium was harvested after 36 h. The results show that MVA addition stimulates glucose uptake and metabolism as seen by the decreased glucose level and increased lactate level in the medium Fig. . This reNext, we performed western blot analysis and found that the protein levels of β-catenin and TAZ are lower in an energy stress condition  but can be partly rescued by MVA supplementation Fig. . This re34.The proliferation of cancer cells is much faster than their corresponding normal cells. Thus, we hypothesized that cancer cells may have a higher mevalonate pathway activity to support nutrient uptake and cell proliferation. To test this, we compared the mevalonate pathway activity of colon cancer tissue and the paracarcinoma tissue from the same patient. As expected, the RNA expression of multiple enzymes of the mevalonate pathway was higher in colon cancer tissue than in paracarcinoma tissue Fig. . WesternAs the mevalonate pathway regulates Wnt and YAP/TAZ signaling in vitro, we postulate that the upregulated mevalonate signaling may accompany activated Wnt and YAP/TAZ signaling in carcinoma tissues. Here, we found that the expression of β-catenin, which represents classical Wnt signaling activity, was higher in cancer tissues Fig. . In addiCell proliferation and growth should be tightly coordinated with nutrient uptake. However, how the cells perceive the abundance of nutrients in the external environment and then use these nutrients to support cell proliferation is not completely understood. In this study, we explored the role of the mevalonate pathway in the regulation of both cell proliferation and nutrient uptake.HMG-CoA reductase is the rate-limiting enzyme of the mevalonate pathway, which is also the target of statins. We found that lovastatin significantly suppressed the proliferation of multiple colon cell lines, while the addition of MVA back to the medium completely recovered the proliferation. The results of transcriptome and proteome analysis suggested that the mevalonate pathway significantly regulated multiple signaling pathways related to cell proliferation and growth. In addition, biological processes like cell cycle, DNA replication, mitosis, which are relevant to cell division, are all controlled by the mevalonate pathway. These results explain why the mevalonate pathway controls cell proliferation.30. Canonical Wnt signaling is mediated by β-catenin and the TCF/LEF transcriptional factors28. It was shown that lovastatin treatment increased the phosphorylation and decreased the protein level of β-catenin. Furthermore, the activity of the TCF/LEF luciferase reporter was significantly suppressed by lovastatin treatment but rescued by MVA addition. Recent studies showed that YAP/TAZ are key mediators of alternative Wnt signaling30. Here, we found that the YAP/TAZ protein was significantly suppressed by lovastatin treatment and completely rescued by MVA addition, which is consistent with previous findings. Western blot, RT-PCR, and reporter assays further confirmed the role of the mevalonate pathway in the regulation of YAP/TAZ. Of note, mice treated with LPS, which leads to an accumulation of metabolites from the mevalonate pathway in the liver, show increased protein level of YAP/TAZ and their target genes in the liver. These data build on prior reports35 and confirm that the mevalonate pathway controls YAP/TAZ in vitro and in vivo.Wnt signaling includes canonical and alternative pathways33. Given that the IGF1 receptor regulates glucose uptake, this may be a likely mechanism for the effect. Together, these data suggest that the mevalonate pathway is not just for the synthesis of cholesterol but also a key pathway for promoting cell proliferation and energy uptake.Cell growth and proliferation are energy-consuming processes and should be supported by nutrient uptake. In this study, we found that the mevalonate pathway not only supports cell proliferation but also promotes glucose and amino acid uptake. More importantly, our results indicate that MVA is sufficient to promote glucose uptake, although the mechanism remains to be further explored. Recent studies showed that mevalonate supplementation activates the IGF1 receptorCancer cells grow and proliferate faster than normal cells, and accordingly, they need more energy to support cell proliferation. Thus, we hypothesized that colon cancer tissue may have a higher mevalonate pathway activity than its paracarcinoma tissue. Indeed, we found that cancer tissues have higher expression of multiple enzymes in the mevalonate pathway. Consistent with the increased expression of the enzymes that catalyze MVA synthesis, Wnt signaling was activated, reflected by the increased protein levels of β-catenin and YAP/TAZ.3. It was reported that AMPK can phosphorylate and suppress the activity of sterol regulatory element binding proteins (SREBP-1c and -2)36, the transcriptional factors that control gene expression of enzymes in the mevalonate pathway. Except to suppress the activation of SREBPs, AMPK was shown to directly regulate the phosphorylation of HMG-CoA reductase and reduce its enzymatic activity37. Based on these and the findings in our study, we established a model depicting how the mevalonate pathway coordinates cell nutrient uptake and proliferation , geranylgeranyl pyrophosphate (G6025), farnesyl pyrophosphate (F6892) were purchased from Sigma Aldrich. Lovastatin was purchased from MedChemExpress .HCT116 was cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin; RKO, SW480, HT29, CACO2 were cultured in DMEM supplemented with 10% FBS and penicillin/streptomycin. Cells were maintained at 37 °C in the presence of 5% COWestern blot analyses were performed using standard protocols. The primary antibodies used in this study are listed: TAZ (4883S), YAP (P46937), p-YAP (13008T), phospho-GSK-3β (5558), phospho-β-catenin (2009) antibody, and the mTOR pathway antibody kit (9862) were from Cell Signaling Technology; CTGF antibody (PA5–32193) was from Thermofisher Scientific; CYR61, HMGCS1, and GAPDH antibodies were from Sangon Biotech ; Tublin antibody was from Boster . HMGCR (ab174830), β-catenin (ab32572), ENC1 (ab124902), TYMS (ab108995), DHFR (ab133546), RhoA (ab187027), RhoB (D160056) antibodies were purchased from Abcam. Western blots were developed using the Immobilon ECL Ultra Western HRP Substrate (Millipore), and images were captured using a ChemiDoc MP Imaging System (Bio-Rad).Trizol  was used for total RNA isolation. Following isolation, 500 ng total RNA was reverse transcribed with the All-in-One First-Strand cDNA Synthesis Kit  and qPCR was performed using All-in-One qPCR Mix  and Applied Biosystems 7500 Real-Time PCR detection system. The gene expression was referred to 36B4. The primer sequences for qPCR assay were as follows:Human:HMGCR For: GGACCCCTTTGCTTAGATGAAA; Rev: CCACCAAGACCTATTGCTCTG;HMGCS1 For: GGGCAGGGCATTATTAGGCTAT; Rev: TTAGGTTGTCAGCCTCTATGTTGAA;ACAT2 For: GCGGCGCGGACCAT; Rev: CCTGGACAGGAACAGCAGCTA;IDI1 For: TTTCCAGGTTGTTTTACGAATACG; Rev: TCCTCAAGCTCGGCTGGAT;FDPS For: CTTCCTATAGCTGCAGCCATGTAC; Rev: GCATTGGCGTGCTCCTTCT;MVD For: TGAACTCCGCGTGCTCATC; Rev: CGGTACTGCCTGTCAGCTTCT;PMVK For: CCGCGTGTCTCACCCTTT; Rev: GACCGTGCCCTCAGCTCAT;AXIN2 For: GCTGACGGATGATTCCATGT; Rev: ACTGCCCACACGATAAGGAG;ENC1 For: CATTTGTCAGCACCTGGAAA; Rev: TCTCATCGAGTGATGGAGTGA;BIRC5 For: AGAACTGGCCCTTCTTGGA; Rev: CAAGTCTGGCTCGTTCTCAGT;BCL2L1 For: CCCAGGGACAGCATATCAG; Rev: AGCGGTTGAAGCGTTCCT;ANKRD1 For: CGAGATAAGTTGCTCAGCACAG; Rev: GTTCAGTCTCACCGCATCATG;CTGF For: AGCTGACCTGGAAGAGAACATT; Rev: GCTCGGTATGTCTTCATGCTG;CYR61 For: AAGAAACCCGGATTTGTGAG; Rev: GCTGCATTTCTTGCCCTTT;PCNA For: CCTGCTGGGATATTAGCTCCA; Rev: CAGCGGTAGGTGTCGAAGC;36B4 For: CAACCCAGCTCTGGAGAAAC; Rev: GTGAGGTCCTCCTTGGTGAA.Mouse:CTGF For: CTGCAGACTGGAGAAGCAGA; Rev: GCTTGGCGATTTTAGGTGTC;CYR61 For: CAACCAGTGTACAGCAGCCTA; Rev: GCAGTATTTGGGCCGGTAT;36B4 For: CAACCCAGCTCTGGAGAAAC; Rev: GAGGTCCTCCTTGGTGAACA.27. RKO and SW480 were used for luciferase reporter transfection. Eighteen hours after plasmids transfection (0.15 μg/cm2), cells were proceeded to ANG II treatment. Twenty-four hours after ANG II (5 μM) addition, the cells were lysed and then the ONE-Glo™ Luciferase Assay System detection reagent  was used to measure the luminescence.8×GTIIC-luciferase reporter  and TCF/LEF luciferase reporter  assays basically followed the protocol recommended by Promega  and our previous publicationsThe Glucose Uptake Colorimetric Assay Kit  was used to evaluate the 2-DG uptake by RKO cells. Briefly, RKO cells were treated with lovastatin (5 μM) or lovastatin plus MVA (0.5 mM) for 24 h, and the medium was changed to no serum medium (with lovastatin or lovastatin plus MVA) for another 12 h, and then the 2-DG uptake assays were performed according to the manufacturer's instruction.27. Briefly, total RNA was isolated from the MEF using Trizol reagent (Invitrogen). Before library construction, RNA degradation and potential contamination were tested by agarose gel electrophoresis, and RNA integrity and quantitation were checked by Agilent 2100 bioanalyzer. Three micrograms of total RNA was sequenced by Hiseq 2500.RNA sequencing was performed as described in our previous study1. SILAC labelingl-13C6-lysine/l-13C615N4-arginine) or “light isotopic amino acids” (l-lysine/l-arginine) using a SILAC Protein Quantitation Kit  according to manufacturer’s instructions. The cells were grown for more than six generations before being harvested, to achieve more than 97% labeling efficiency. After that, the differently labeled cells were treated with lovastatin or lovastatin plus MVA for 40 h, then the cells were washed two times with cold PBS and proceeded to protein extraction.The proteomics assay and analysis were done in the PTM biolabs . RKO cells were labeled with either “heavy isotopic amino acids”  in lysis buffer . The remaining debris was removed by centrifugation at 12,000 3. Trypsin digestion4HCO3 to urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4-h digestion.For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM NH4. HPLC fractionationThe tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column . Briefly, peptides were first separated with a gradient of 8–32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into 18 fractions and dried by vacuum centrifuging.5. LC-MS/MS analysisThe tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a reversed-phase analytical column . The gradient was comprised of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23–35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350–1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control was set at 5E4.6. Database search38. Tandem mass spectra were searched against Swiss-Prot Human database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to two missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification and oxidation on Met was specified as variable modifications. FDR was adjusted to <1% and minimum score for peptides was set >40.The resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8)7. Calculation of differential proteinst-tests were used to compare expression of proteins. In general, a significance level of 0.05 was used for statistical testing, and we reported the P value or significance level any time a statistical test was performed.The quantification was mean-normalized at peptide level to center the distribution of quantitative values. Protein quantitation was then calculated as the median ratio of corresponding unique or razor peptides for a given protein. Two-sample, two-sided t-test (2-tail) while multi group analysis was assessed by the one-way analysis of variance (ANOVA) test (2-tail). The data are represented as mean ± standard error of mean (SEM) and the significance level was set at 0.05 (two-sided). Differential expression analysis of two conditions/groups  was performed using the DESeq R package (1.10.1). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P < 0.05 found by DESeq and Log2 fold change is greater than ±1 were assigned as differentially expressed.Comparisons between two groups were assessed by the unpaired Student’s S-Fig 1S-Fig 2S-Fig 3S-Fig 4S-Fig 5S-Fig 6S-Table 1S-Table 2S-Table 3S-Table 4supplementary figure legends


Fear of falling (FOF) is more common in people with Parkinson's disease (PD) than in healthy controls. It can lead to several negative consequences such as restrictions in everyday life. Moreover, FOF is a risk factor for future falls.  This study aimed to identify predictive factors of FOF  after three years, with and without adjusting for concerns about falling at baseline, in people with PD.  This study included 151 participants (35% women) with PD. At baseline, their mean (SD) age and PD duration were 68 (±9.0) and 9 (±6.1) years, respectively. The Falls Efficacy Scale-International (FES-I) was used as the dependent variable in multivariable linear regression analyses. p < 0.001). The strongest  predictor of concerns about falling was walking difficulties (β = 0.378), followed by age (0.227), problems maintaining balance while dual tasking (0.172), and needing help in daily activities (0.171). When adjusting for baseline FES-I scores, the strongest predictive factor was problems maintaining balance while dual tasking (β = 0.161), which was followed by age (0.131) and female sex (0.105).  The mean (SD) FES-I score increased from 28.1 (11.9) to 33.1 (14.0) three years later ( This study pinpoints several predictive factors of concerns about falling that are modifiable and which could be addressed in rehabilitation: perceived walking difficulties, having problems maintaining balance while dual tasking, and dependence on others in daily activities. The importance of dual tasking is a novel finding, which future studies need to confirm or refute. One should be aware of the fact that an increased age predicts concerns about falling with and without adjusting for baseline FES-I scores, whereas female sex predicts concerns about falling only when adjusting for baseline FES-I scores. Fear of falling (FOF) is considered a broad term comprised of different conceptualizations, such as concerns about falling , 2, low In order to be able to design efficient interventions for FOF in people with PD, it is important to gain an increased understanding of predictive factors of FOF. Seven previous cross-sectional studies used multivariable analyses in order to identify factors associated with FOF in people with PD , 19–23. N = 88) addressed a change in fall-related self-efficacy and presented two different models; the first showed that the total number of falls was the only significant predictor, whereas the second found PD severity to be the only significant predictor. There is a need for larger studies with a longer follow-up to establish predictive factors of FOF in PD; such knowledge would be valuable for identifying factors of importance for future intervention studies.Longitudinal studies are necessary to clarify the most important factors leading to FOF. To our knowledge, there is only one longitudinal study that addressed predictors of FOF in people with PD . That stThis study aimed to identify predictive factors of FOF  after three years, with and without adjusting for concerns about falling at baseline, in people with PD.This study is part of a longitudinal project “Home and Health in People Ageing with PD” . Baseline assessments were conducted in 2013 and a 3-year follow-up in 2016. More information about the methods and design of the larger project is described in the study protocol .n = 10), severe cognitive difficulties (n = 91), living outside Skåne County (n = 58), or other reasons that made them unable to give informed consent or take part in the majority of the data collection . The remaining 437 persons were invited to participate. Of these, 22 were unreachable, two had a revised diagnosis, and 157 declined. One person was excluded due to extensive missing data, resulting in a sample of 255 participants at baseline.Outpatient participants were recruited from three hospitals in southern Sweden (see n = 255) were considered eligible for the 3-year follow-up (±3 months). At that time, 22 persons were deceased, three had moved, and one ended up outside the follow-up window. Thus, 229 persons were invited to participate. Of these, eight were unreachable, four had a revised diagnosis, and 51 declined. One person was excluded due to extensive missing data and low data quality. In total, the 3-year follow-up had a sample of 165 persons. Since the primary focus of this study was FOF, i.e., conceptualized as concerns about falling and assessed by using the Falls Efficacy Scale-International (FES-I), we only included subjects with a total score of FES-I at both time points . At baseline, their mean (SD) age was 68 (±9.0) years, and 35.1% were female. Mean PD duration at baseline was 9 (±6.1) years. Median (q1–q3) PD severity during “on-state” was 2  [All those who completed baseline assessments and had agreed to be contacted again (= worse) . Additiop < 0.001 and mean 9 vs. 10 years PD duration, p=0.048 (independent samples t-tests). Although a trend, there was no statistically significant difference regarding sex .The included 151 participants were significantly younger and had a shorter PD duration than the 104 who were lost for follow-up: mean 68 vs. 72 years old at baseline, The project was conducted in accordance with the Helsinki Declaration and was approved by the Regional Ethical Review Board in Lund, Sweden (nos. 2012/558 and 2015/611). All participants gave their written informed consent.The data collectors underwent project-specific training. Two registered occupational therapists collected the data during baseline. Two other occupational therapists, with the help of a PhD student in physiotherapy, collected data during the 3-year follow-up. Both data collection waves comprised a self-administered postal survey and a home visit, which included structured interviews and clinical assessments. The postal survey was administered about ten days ahead of the home visit.Concerns about falling constituted the dependent variable and was assessed by using the self-administered questionnaire Falls Efficacy Scale-International (FES-I), which includes 16 items . The response options are not at all concerned (score 1), somewhat concerned score 2), fairly concerned (score 3), or very concerned (score 4) [, fairly Several independent variables were considered. Data on personal factors included age, sex, and general self-efficacy  . EnvironClinical assessments administered at the home visit addressed the severity of parkinsonian motor symptoms and cognitive functioning. The motor part of the Unified Parkinson's Disease Rating Scale (UPDRS III) is scored 0–108 points (higher = worse) . The MonSeveral self-administered questionnaires were included in the postal survey. Walking difficulties in daily life were assessed by using the Generic Walk-12  . FatigueSome rating scales and questions were administered as an interview at the home visit. The Geriatric Depression Scale (GDS-15) includes 15 items and is scored from 0 to 15 points (higher scores = more depressive symptoms) . Dichotor). There were no signs of multicollinearity , except between Walk-12G and FES-I baseline scores . Associations between the dependent variable  and independent variables were analysed in a series of univariable linear regression analyses . All 17 independent variables fulfilled this criterion and were consequently included. The estimates and p value for each independent variable were examined, and the variable with the highest p value was manually removed from the multivariable model. This step continued until all remaining independent variables had p values <0.1. Finally, the analyses were re-ran and controlled for baseline FES-I scores in order to identify predictive factors of a change in FES-I scores over a 3-year period . That is, this model identifies predictive factors given the FES-I score at baseline. Unadjusted and adjusted R2 are presented as indications of the predictive capacity of the models. Statistical significance was set to a 0.05 level.The relationships among the independent variables were studied by using Pearson's correlation coefficient .p < 0.001) at the 3-year follow-up.At baseline, 40% (61/151) of the participants reported FOF, which increased to 55%  from 28.1 (11.9) to 33.1 (14.0) three years later. This increase exceeded the measurement error , see Total and item scores of FES-I are presented in p value <0.05. Walking difficulties  had the strongest effect on follow-up FES-I scores , followed by balance problems while dual tasking .Univariable linear regression analyses are presented in R2) of the variance in FES-I scores at the 3-year follow-up. Four out of the six factors were statistically significant. The strongest predictive factor  was walking difficulties (β = 0.378) followed by age (β = 0.227), problems maintaining balance while dual tasking (β = 0.172), and needing help in daily activities (β = 0.171). The other two variables were depressive symptoms (β = 0.118) and sex (β = 0.107); both had a p value of 0.060. See The final model resulted in six independent variables explaining 57.5% (adjusted R2) of the variance in FES-I scores at the 3-year follow-up. The strongest predictive factor for a change in FES-I scores (when adjusting for baseline FES-I scores) was problems maintaining balance while dual tasking (β = 0.161), which was followed by age (β = 0.131) and female sex (β = 0.105). The fourth variable  was not significantly associated with concerns about falling . See When adjusting for FES-I scores at baseline, the final model included four independent variables explaining 62.6%  the strongest predictive factor for concerns about falling was perceived walking difficulties followed by age, problems maintaining balance while dual tasking, and needing help in daily activities and (2) the strongest predictor for a change in concerns about falling over a 3-year period (when adjusting for baseline FES-I scores) was problems maintaining balance while dual tasking, higher age, and  sex.The fact that walking difficulties was the strongest predictor for concerns about falling is in line with prior cross-sectional studies , 10, 22.Problems maintaining balance while dual tasking predicted concerns about falling and was the strongest predictor for a change in concerns about falling. This finding is in contrast to two cross-sectional studies , 22, wheThis study showed that needing help in daily activities predicts concerns about falling, which is in line with prior cross-sectional studies , 10, 20.The present study found a trend towards depressive symptoms being a predictive factor of concerns about falling. This is in line with the cross-sectional study by Jonasson et al. , which wAge predicted concerns about falling and both age and female sex predicted a change in concerns about falling. These findings are in contrast to the longitudinal study by Gazibara et al. . GazibarIn our study, a history of falls did not predict concerns about falling. This finding agrees with the longitudinal study by Gazibara et al. , which aIn order to identify predictive factors, a longitudinal study design is needed. To the best of our knowledge, this is the first study that identified predictive factors of concerns about falling and a change in concerns about falling in people with PD. We included 17 independent variables into an initial linear regression model. Although one could argue that the number of variables in the initial model was somewhat high in relation to the sample size , we founPeople with cognitive problems were included as long as they were able to give informed consent and take part in the majority of the data collection. Global cognition  did not predict concerns about falling, but one should be aware of the fact that there is a debate how cognitive impairments relate to FOF. It might be that cognitive impairments generate insensitivity to FOF, which might increase the risk for future falls , 51.All follow-up studies are at risk of having participants who dropout, this was also the case in the current study which can affect the external validity of the findings.This study pinpoints several predictive factors of FOF in people with PD, that are modifiable and which, thereby, could be addressed in rehabilitation: perceived walking difficulties, having problems maintaining balance while dual tasking, and dependence of others in daily activities. Perceived walking difficulties was the strongest predictor for concerns about falling. Problems maintaining balance while dual tasking was also an important predictor and was the strongest predictor for a change in concerns about falling after a 3-year period. This is a novel finding, which needs to be confirmed by future studies.One should be aware of the fact that an increased age predicts concerns about falling and both age and female sex predicts a change of concerns about falling over time.


Camellia sinensis (C. sinensis) population and aims to (i) identify single nucleotide polymorphisms (SNPs) on the genome level, (ii) investigate the genetic diversity and population structure, and (iii) characterize the linkage disequilibrium (LD) pattern to facilitate next genome-wide association mapping and marker-assisted selection.To efficiently protect and exploit germplasm resources for marker development and breeding purposes, we must accurately depict the features of the tea populations. This study focuses on the We collected 415 tea accessions from the Origin Center and analyzed the genetic diversity, population structure and LD pattern using the genotyping-by-sequencing (GBS) approach. A total of 79,016 high-quality SNPs were identified; the polymorphism information content (PIC) and genetic diversity (GD) based on these SNPs showed a higher level of genetic diversity in cultivated type than in wild type. The 415 accessions were clustered into three groups by STRUCTURE software and confirmed using principal component analyses (PCA)—wild type, cultivated type, and admixed wild type. However, unweighted pair group method with arithmetic mean (UPGMA) trees indicated the accessions should be grouped into more clusters. Further analyses identified four groups, the Pure Wild Type, Admixed Wild Type, ancient landraces and modern landraces using STRUCTURE, and the results were confirmed by PCA and UPGMA tree method. A higher level of genetic diversity was detected in ancient landraces and Admixed Wild Type than that in the Pure Wild Type and modern landraces. The highest differentiation was between the Pure Wild Type and modern landraces. A relatively fast LD decay with a short range (kb) was observed, and the LD decays of four inferred populations were different.This study is, to our knowledge, the first population genetic analysis of tea germplasm from the Origin Center, Guizhou Plateau, using GBS. The LD pattern, population structure and genetic differentiation of the tea population revealed by our study will benefit further genetic studies, germplasm protection, and breeding.The online version of this article (10.1186/s12870-019-1917-5) contains supplementary material, which is available to authorized users. Camellia sinensis (L.) O. Kuntze, Theaceae (C. sinensis), has been grown in the Yunnan-Guizhou Plateau in southwest China for approximately 5,000 years and is now widely cultivated all over the world [Tea is one of the most popular beverages worldwide , 2 with he world . The Guihe world , 5, wherhe world . Large sSect. Thea (L.) Dyer, and are defined as varieties grown for more than 100 years. Wild teas, including wild type and self-wild type, are valuable for scientific research and application as they have mainly undergone natural selection and were only minimally affected by artificial selection. Analyzing genetic diversity and population genetic structure is significant to depicte the domestication event and genetic relationships of tea plants. It is also helpful for expediting the development on breeding strategies [Ancient tea plants belong to rategies . Moleculrategies , nSSR [1rategies , gSSRs [rategies , SSR [10rategies , SNP [12rategies , ISSR [1rategies , EST-SSRrategies , 16, etcrategies , Guangxirategies  or acrosLD is defined as the association of allelesat different loci within a given population. Understanding the LD pattern is crucial for tea breeding –21. GBS GBS was performed on 415 tea accessions using Illumina HiSeq X ten. After the primary quality filtering step, 390.3 Gb clean data were obtained with an average of 0.94 Gb clean data per accession , observed heterozygosity (Ho) and polymorphism information content (PIC) of 415 tea accessions were 0.257, 0.247 and 0.214, respectively Table . Among tWe used STRUCTURE and PCA to analyze the genetic structure of the tea accessions. Both analyses were performed using 1,135 LD-pruned SNPs. Based on the genetic distance matrix of the 415 tea accessions, we used TASSEL v.5.2.37 to build an UPGMA tree.Camellia tachangensis F.C.Zhang, of which most were from the zones IV, III and II .The number of clusters was estimated based on the ΔK method , 36 and C.tachangensis, C.sinensis and C. remotiserrata)  Fig. d of tea The plateau criterion was also used to estimate the number of clusters –40. As s2 values were below 0.16  at approximately 35 kb. Conversely, LD declined the most rapidly in GP02—r2 = 0.08 corresponded to a physical distance of approximately 1 kb—followed by subgroup GP03–1, in which r2 = 0.08 corresponded to approximately 2 kb. The LD of subgroup GP03–2 declined below r2 = 0.08 at approximately 25 kb.LD decay in the four inferred groups was estimated  to analyze the population structure of the 415 tea varieties, and the results we obtained complemented the previous studies. STRUCTURE could effectively identify global clusters, which were subsequently validated by PCA. However, the two parameters we used to determine the number of clusters in STRUCTURE yielded different K values—the Evanno’s ΔK method identified K = 2 when analyzing the entire germplasm collection and the cryptic structure. Evanno’s method focuses exclusively on the change in slope, therefore, it estimates the uppermost level structure of the data which may cause ΔK to be artificially maximal at K = 2 in some cases, as reported previously by Campoy JA et al. . We usedWe detected the lowest genetic differentiation and genetic distance between the modern and ancient landraces. The Pure Wild Type and modern landraces exhibited the largest genetic differentiation and genetic distance, followed by that between the Pure Wild Type and ancient landraces, and that between the Admixed Wild Type and ancient landraces. These results support the notion that the evolution of tea plants was related to the historical tea cultivation in the Guizhou Plateau. The Pure Wild Type is the most primitive resource that originated in the region, and the retained species purity was owing to the isolated ecological environment. The ancient landraces and the Admixed Wild Type likely emerged in the Ming Dynasty, when local landraces, introduced landraces, and wild species were co-cultivated. The co-cultivation facilitated cross-pollination among different germplasms, which reduced the genetic distance and differentiation between the ancient landraces and the Admixed Wild Type and significantly increased the diversity of the ancient landraces and the Admixed Wild Type among all inferred groups. Most modern landraces and breeding varieties were assigned to GP03–2, reflecting a narrowed genetic basis of the modern landraces due to breeding practice.We observed the lowest genetic differentiation between GP03–1 and GP03–2, suggesting that human activities may have caused frequent gene exchange between these two subgroups. GP01 and GP03–2 showed the highest level of genetic differentiation and distance, implying that geographic isolation has restricted the gene flow among populations. This observation could also be a result of the reproductive isolation between species. According to our results, GP03–1 and GP02 exhibited a higher genetic diversity compared with GP01 and GP03–2, therefore, varieties in GP03–1 and GP02 can be used for tea improvement. As revealed by our data, the differences between species did not affect clustering, which reflected the complexity and uncertainty of the tea classification systems. Thus, it is necessary to establish a more scientific classification system. In addition, natural hybridization between tea species may be another explanation of the results mentioned above  and two subpopulations (GP03–1 and GP03–2). The ancient landrace group was found to have a more complex genetic structure than the wild and modern landraces. These data will inform the collection, conservation, and application of the tea varieties in the Guizhou Plateau.Camellia sinensis (L.) O. Ktze, 100 Camellia tachangensis (F.C.Zhang), 59 Camellia remotiserrata (Zhang) and five near Camellia taliensis (W.W.Smith) were identified  were included in this study  to isolate genomic DNA from the samples. DNA integrity was tested on 1% agarose gel, and DNA purity was tested and quantified using Qubit Fluorometer (Invitrogen).We used 5 U of SacI and MseI (NEB) and 1 × restriction buffer in a 25 μl reaction to digest 100 ng genomic DNA. After digestion, SacAD and MseAD adaptors were ligated to the digested DNA fragments; 12 samples were pooled in equal volumes and purified using the QIAquick PCR Purification Kit (Qiagen) . We thenhttp://www.plantkingdomgdb.com/tea_tree/) [-T 20 -k 30’ [The barcodes were used to de-multiplex the raw DNA reads, and a custom perl script was used to trim the adaptors. Only the reads with quality values > 5 were retained as the clean data, and then aligned to the reference genome (a_tree/)  using BW0 -k 30’ . GATK (Vr2 threshold of 0.2 by Plink (v1.9). After the filtering, 415 accessions and 79,016 SNPs were retained and used for further analysis.The SNPs were filtered according to the methods used by Hussain et al. , Chen etThe polymorphism information content (PIC) values for the SNP data were calculated using the following equation .\documenThe mean number of observed alleles per locus and the observed heterozygosity (Ho) were calculated for each group using TASSEL v.5.2.37 . Geneticr2 threshold was 0.4. PLINK was used to measure pairwise LD between multi-SNPs [r2) using PopLDdecay program1. To summarize the relationship between LD decay, we fitted a locally-weighted linear regression (loess) model to the r2 data [http://www.R-project.org/) [r2 summarizing both the recombinational and mutational history [Prior to the PCA and STRUCTURE analyses, we LD-pruned the SNPs again using Plink (v1.9)  with a wlti-SNPs , 54. The r2 data , 57 usinct.org/)  with r2  history . The LD Population structure was analyzed using the model-based Bayesian analysis implemented in STRUCTURE . The numAdditional file 1:Table S1. The quality control (QC) data of each sample. Table S2. Statistics of individual heterozygosity of 287,408 SNPs based on GBS. Table S3.. Statistics of individual heterozygosity of 79,016 SNPs based on GBS. Table S4. SNP density of scaffolds based on GBS. Table S5. The p-value of genetic diversity parameters in Table Table S6. Genetic diversity parameters of three species of tea plants in Guizhou Plateau. Table S7. Fst and pairwise genetic distance among three Species of tea plant in Guizhou Plateau (XLSX 117 kb)Additional file 2:Information of 415 tea accessions used in this study, including the accession/clone/collection, the accession name, the zone, the cultivation status, growth habits, the species, the STRUCTURE URE-based grouping (Qi ≥0.8) at K = 2, the notes, the source, and the inferred populations (XLSX 45 kb)Additional file 3:Figure S1. Graphical method allowing the detection of the number of groups using ∆K inferred population structure of the 263 Pure Cultivation Type. Figure S2. Inferred population structure of the 263Pure Cultivation Type using STRUCTURE software. Bar plot of individual ancestry proportions for the genetic clusters inferred using STRUCTURE (K = 2) and the reduced dataset. Individual ancestry proportions  are sorted within each cluster. Admixture model, independent frequencies, 30,000 burn-in iterations, 100,000 Markov Chain Monte Carlo iterations were used for this analysis. Ancient landraces (GP03–1) and modern landraces (GP03–2) are shown in yellow and green, respectively. Figure S3.. Four inferred populations of the 415tea accessions using STRUCTURE (K = 3). GP01 are shown in red, GP02 are shown in red and blue, GP03–1 are shown in blue, and GP03–2 are shown in green. (PDF 207 kb)Additional file 4:2) estimated against the genetic distance for pairs of linked SNP across all scaffolds longer than 500 kb in the 415 accessions (ALL) and four inferred groups . (PDF 220 kb)Average LD decay  The geographical position of Guizhou province in China. (B) Agriculture climate regionalization map for tea plant growth in Guizhou Plateau . Ia: Are


Neutrophil gelatinase-assoicated lipocalin (NGAL) appears to be a promising proximal tubular injury biomarker for early prediction of delayed graft function (DGF) in kidney transplant recipients. However, its predictive values in urine and blood were varied among different studies. Here, we performed the meta-analysis to compare the predictive values of urine NGAL (uNGAL) and blood NGAL (bNGAL) for DGF in adult kidney transplant recipients.We systematically searched Medline, Cochrane library and Embase for relevant studies from inception to May 2018. The summary receiver operating characteristic (SROC) curves, the pooled sensitivity, specificity and diagnostic odds ratio (DOR) were used to evaluate the prognostic performance of uNGAL and bNGAL for the identification of DGF.A total of 1036 patients from 14 eligible studies were included in the analysis. 8 studies focused on NGAL in urine and 6 reported NGAL in serum or plasma. The composite area under the ROC (AUC) for 24 h uNGAL was 0.91  and the overall DOR for 24 h uNGAL was 24.17 with a sensitivity of 0.88  and a specificity of 0.81 . The composite AUC for 24 h bNGAL was 0.95  and the overall DOR for 24 h bNGAL was 43.11  with a sensitivity of 0.91  and a specificity of 0.86 .Urine and serum/plasma NGAL were valuable biomarkers for early identification of DGF in kidney transplantation. In addition, the bNGAL was superior to uNGAL in early prediction of DGF.The online version of this article (10.1186/s12882-019-1491-y) contains supplementary material, which is available to authorized users. Delayed graft function (DGF), traditionally defined as the need for dialysis within 7 days after kidney transplantation, remains one of the major obstacles for patients to recover from transplantation during the postoperative course. It significantly prolongs hospitalization stay, increases medical costs, and even worsens patients with increased risk of chronic kidney diseases or graft loss in the first year after transplantation . In addiPreviously, the requirement for dialysis, the failure of serum creatinine (Scr) to decrease, the graft histopathology and the urine output following transplantation have been applied to identify DGF in kidney transplant recipients during a few days after transplantation . HoweverTwo investigators (Y.M.L. and Y.L.) independently searched the literature in Medline (via Pubmed), Cochrane Library and Embase (via Ovid) electronic databases from inception to May 2018. Following medical subject headings or keywords were systematically searched without language restriction: “NGAL”, “neutrophil gelatinase-associated lipocalin”, “Lipocalin 2”, “delayed graft function”, “DGF” and “kidney transplantation”. In addition, reference lists of included articles were hand-searched to identify other relevant studies. All potentially relevant records were imported into Endnote X7.7  for further management.Two reviewers (Y.M.L. and Y.L.) independently screened the titles and abstracts and further retrieved the full text of each potentially relevant study to determine study eligibility. Disagreements were resolved by consensus adjudication. Prospective and retrospective cohort studies were eligible for inclusion if they evaluated the predictive performance of urine, serum or plasma NGAL for DGF in adult kidney transplant recipients. Conference abstracts, reviews, editorials, commentaries, letters and studies without mandatory predictive variables including the area under the receiver operating characteristic curve (AUC), sensitivity and specificity were excluded.Two investigators (Y.M.L. and Y.L.) independently extracted the data from eligible articles according to previously prepared data sheet. The following information were included in the sheet: (1) Study and patient characteristics, including first author, publish year, study design, research country, sample resource and size, DGF definition, mean or median NGAL levels of DGF and Non-DGF groups, the time of obtaining specimen, cold ischemia time (CIT), expanded criteria donor (ECD) or donor after cardiac death (DCD) ratio, donor age and donor terminal Scr level. (2) Predictive values, including sensitivity, specificity, cut-off value and AUC with 95% confidence interval (CI). If multiple AUC values of different sampling time points were provided in the same study, the AUC values of 24 h and 48 h were recorded. In addition, if there were multiple cut-off values at the same sampling time point, the one showing the highest Youden index (sensitivity + specificity-1) was extracted. We evaluated the quality of the included articles via Quality Assessment of Diagnosis Accuracy Studies-2 (QUADAS-2) tool that comprises 14 questions . “Yes”, Q test was used to assess heterogeneity across studies and inconsistency index I2 was applied to estimate the degree of heterogeneity. I2 values of 25, 50 and 75% were thought to be low, moderate and high heterogeneity, respectively [2 > 50%), Spearman correlation coefficient test was performed to detect the presence of threshold effect; Sensitivity analysis was conducted to explore the source of non-threshold effect heterogeneity. Publication bias was statistically assessed by Deeks’ linear regression test. In addition, we compared the risk factors of DGF  between uNGAL and bNGAL studies to figure out whether they were significantly different between these two groups of studies. The overall levels of these risk factors in each study were calculated using available mean or median values and patient numbers from DGF and Non-DGF groups. Student’s t-test or Mann-Whitney U-test were utilized to compare continuous variables with normal distribution and skewed distribution, respectively. All statistical analyses were performed with Stata version 12.0  and SPSS version 23.0 . A two tailed P-value less than 0.05 was considered statistically significant.Based on the heterogeneity of included studies, a random effect model (DerSimonian Laird) or fixed effect model (Mantel-Haenszel) was employed to estimate the pooled sensitivity, specificity and diagnostic odds ratio (DOR) with 95% CI. Moreover, forest plots of sensitivity, specificity, DOR and summary ROC (SROC) with overall AUC value were presented. Cochran’s Our initial search yielded 810 records. After removing all duplicates, 606 records remained for further screening. By looking through titles and abstracts, the remaining 29 articles were evaluated in full-text manner. Eventually, 14 eligible articles  were accP > 0.05).Characteristics of individual study were summarized in Table The quality of studies based on QUADAS-2 tool was summarized in Additional file . True-positive (TP), false-positive (FP), true-negative (TN) and false-negative (FN) were calculated based on the provided sensitivity, specificity and patient numbers in each study. To compare the predictive performances of uNGAL and bNGAL for DGF, we controlled the sampling time at 24 h. Random effect models were utilized to pool the specificities of 24 h uNGAL (7 datasets) and 24 h bNGAL (6 datasets), sensitivity and DOR for 24 h uNGAL because the obvious heterogeneities were observed . Fix effect models were used to summarize the sensitivity and DOR for 24 h bNGAL . The composite AUC for uNGAL was 0.89  and the overall DOR for uNGAL was 17.91  with a sensitivity of 0.85  and a specificity of 0.80 . The composite AUC for bNGAL was 0.95  and the overall DOR for bNGAL was 43.11  with a sensitivity of 0.91  and a specificity of 0.86 . Pooled SROC curves and DOR of 24 h uNGAL and 24 h bNGAL were plotted in Fig.  and Fig. .AUC value, cut-off value, sensitivity and specificity of individual dataset were summarized in Table 2r = 1.00, P = 1.00). While the coefficient of 7 datasets analyzing 24 h uNGAL  indicated the presence of threshold effect. Therefore, hierarchical SROC modeling was plotted to pool the sensitivity and specificity [2 value dropped from 61.9 to 35.1%; the sensitivity was 0.88  and the specificity was 0.81   in different clinical settings, such as in cardiac surgery , contrasNGAL, also known as lipcalin-2 (LCN2), is secreted by various tissues such as kidney tubules, liver, lung and gastrointestinal tract at a low level in healthy controls . It has In kidney transplantation, uNGAL and bNGAL have been extensively studied in the prediction and diagnosis of short- and long-term renal functions . The souDetermining when to detect NGAL is important for clinicians to identify DGF effectively in kidney transplant recipients. It was reported that NGAL began to rise 2 h after the injury, peak at 8-12 h and return after 24-48 h . So we rLimitations in this meta-analysis should be mentioned here. Firstly, the inconsistence of DGF definition may alter the predictive performance of uNGAL and bNGAL for DGF in individual studies, thereby may bring some deviations. Secondly, we only included the published literatures whose predictive variables  were available or can be calculated. So the predictive performance of uNGAL and bNGAL may be overestimated due to the publication and selection bias. Thirdly, different cut-off values were used in the included studies. These discrepancies may be attributed to the differences in experimental designs including detection methods, sample size and patient baseline characteristics, which may affect the sensitivity and specificity in individual study. To find an optimal cut-off value that can be implemented in clinical settings, further investigation in large prospective cohorts are required. Importantly, it might be necessary for each research center to pre-define specific normal ranges for urine and blood NGAL in normal kidney transplant recipients before its clinical application.In conclusion, the results of the present meta-analysis demonstrated that both urine NGAL and blood NGAL appeared to be valuable biomarkers of DGF in adult kidney transplant recipients. In addition, the blood NGAL was superior to urinary NGAL in early prediction of DGF. However, further large-scale prospective cohort studies are needed to determine the optimal cut-off value for NGAL that is suitable for clinical use.Additional file 1:Figure S1. Quality assessment of the 14 included studies using QUADAS-2 tool. The assessment of risk of bias and applicability concerns for individual study (PDF 98 kb)Additional file 2:Figure S2. Deeks’ funnel plots for the evaluation of potential publication bias in prediction of uNGAL (a) and bNGAL (b) for DGF (PDF 876 kb)Additional file 3:Figure S3. Forest plots of the pooled sensitivities and specificities of uNGAL (a) and bNGAL (b) level in predicting DGF in kidney transplant recipients. The black squares in the gray squares and the horizontal lines represent the point estimate and 95% CI, respectively. The dotted lines represent the pooled estimate, and the hollow diamonds represent the 95% CI of the pooled estimate (PDF 5145 kb)


Health supplement (HS) products that are available in the Emirate of Dubai  contain chemicals that may adversely affect human health. This study aimed to investigate the prevalence of, and factors associated with HS consumption, knowledge, related adverse events, and reporting practices of adverse events amongst the general population in Dubai, UAE.n = 1203) of the Dubai population that assessed HS use and knowledge. Dependent variables were supplement use and reports of adverse events while independent variables included socio-demographic factors, knowledge, attitudes, and practice. Logistic regression analysis was performed to identify factors independently associated with HS use.A cross-sectional household telephone survey using a computer-assisted questionnaire was conducted amongst a random representative sample  reported ever using HS. Amongst ever-users, reasons for use were to improve health (66.1%), for bodybuilding (9.9%), disease prevention (6.8%), and weight management (5.3%). The majority of users purchased their HS from pharmacies (88.4%) or were prescribed HS (46.6%). Vitamins were the most commonly used HS (87.9%) followed by minerals (10.5%) and sports nutrition products (10.5%). Only 2.9% of users experienced an adverse event associated with HS use which all resolved when the HS was discontinued. Only three of those affected reported the incident. Multivariate logistic regression analysis revealed that HS use was independently associated with female gender , higher income , being a past-smoker , having an allergy , more frequent doctor visits , taking prescribed medications , and knowledge about HS .Our study provides the first population-based estimates of HS use and HS-related adverse events in the Gulf region. Adverse events associated with HS are infrequent and this may be due to the well-developed regulatory framework in Dubai and the high level of knowledge amongst consumers who mainly consume vitamins and minerals on the advice of pharmacists or healthcare professionals.The online version of this article (10.1186/s12906-019-2593-6) contains supplementary material, which is available to authorized users. In the Emirate of Dubai  dietary and herbal supplements are combined under a common definition of “health supplements” (HS) . This deGinkgo biloba has been implicated in the occurrence of epileptic seizures, and chronic use of zinc may result in anemia [Hypericum perforatum), a plant derived product that is used to treat mild and moderate depression, can induce liver enzymes and therefore has the potential to interact with many narrow therapeutic range medicines that are metabolized by the liver such as anti-depressants [Ginkgo biloba can increase the risk of bleeding when administered with anticoagulants [HS products such as minerals and vitamins are widely available over-the-counter and are often used without advice from a healthcare professional for indications including weight reduction and energy enhancement . As HS pn anemia –7. Othern anemia . When prn anemia . HS prodn anemia . Also, pn anemia . For exaressants . Other sagulants . In manyagulants . If suspagulants , 14.Globally, HS are widely consumed and a recent systematic review reported that the prevalence of regular HS use ranged from 22 to 53% amongst the general population in Canada  and Male (M) combined 40%), France , Germany , Korea , Sweden , the United Kingdom , and the United States  . At presThis section has been written in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines .n = 1203) of the Dubai population aged 16 years or older from 01 September to 31 December 2015. Dubai is the second largest of the UAE’s seven Emirates with a population of approximately 2.45 million in 2015 [A cross-sectional household telephone survey using a computer-assisted questionnaire was conducted amongst a random representative sample . The study protocol was approved by the Social Sciences Research Ethics Committee of the United Arab Emirates University. All the participants provided verbal informed consent.‘Demographic Data’ included 10 items collecting information on age, gender, marital status, nationality, occupation, health insurance coverage, monthly income, education, and self-reported body mass (kg) and height (cm). Body mass index (BMI) was calculated as body mass in kilograms divided by height in metres squared (kg/m2). Section two ‘Health and Lifestyle’ was composed of five items collecting data on allergies, frequency of doctor visits within the past 12 months, diagnosis of chronic medical conditions, prescription drug use in the past month, and smoking status. Section three ‘Health Supplements Consumption’ included 12 questions on knowledge of HS and HS use including type, frequency, duration, form, ingredients, and reasons for taking and/or discontinuation of HS, source of purchase, and number of HS products ever used. Section four “Information about Health Supplement Products” contained seven items on source of HS advice, HS prescription, and usefulness and adherence to HS label advice/information. Section five ‘Adverse Events to Health Supplement Consumption’ contained 10 questions on frequency, type, severity, timing of adverse events, and seeking medical treatment. Section six ‘Reporting Adverse Events’ was composed of four items collecting data on the reporting of ‘ever’ experiencing an adverse event and perceptions on the usefulness of an adverse event surveillance system.A computer-assisted telephone-administered questionnaire was developed to obtain relevant information related to socio-demographic and lifestyle factors (independent variables) and HS use and experience of HS-related adverse events (dependent variables). The 48-item questionnaire was adapted from previously used surveys and reviews on HS use and adverse events , 23–25. ‘Did not visit a doctor in the last 12 months’, ‘Less than monthly’, and ‘1-3 times a month/At least once a week’. A binary (Yes/No) variable was created for knowledge of HS from the question ‘Do you know what health supplements are?’  was categorized into AED < 5000, AED 5000- < 10000, AED 10000–20000, and AED > 20000 (USD 1.00 ≈ AED 3.67). Education was categorized as less than high school, high school, graduate, or postgraduate. Smoking status was categorized into non-smoker, past smoker, current occasional smoker, and current regular smoker. Frequency of visiting a doctor in the last 12 months was categorized as n = 134; 48.4% Emirati) of Arabic and English speaking participants. No changes were made to the questionnaire following the pilot study; however, pilot sample participants were not included in the final analysis; (iii) interviewers fluent in Arabic, English, and other commonly spoken languages in Dubai (e.g. Urdu) were hired and completed a training course on the specific data collection procedure; (iv) data collection was supervised and used computer-assisted telephone interviewing to ensure standardization of procedures across interviewers; and (v) although data collection was supervised, telephone interviews were repeated on a random sub-sample of 40 participants by an independent researcher for interview verification and quality assurance purposes; specifically, the independent researcher called the telephone number to verify that the interview had been conducted.The following strategies were used to minimize possible bias: (i) in an attempt to minimize selection bias and maximize the possibility of recruiting a representative sample, the study used stratified  random sampling from a sampling frame provided by the Dubai Statistics Center that contained a list of all residential addresses and telephone numbers in Dubai; (ii) the telephone-administered questionnaire was piloted on a random sample  or means (standard deviation) as appropriate. Difference between subgroups of the sample  were tested using a Chi-square test for categorical variables and ANOVA for continuous variables. Simple logistic regression analysis was performed to assess the association between HS use (outcome variable) and selected correlates (independent variables). The variables having n = 455) reported ever using health supplements and of these only 2.9% (n = 13) had experienced an adverse event  had a monthly income in the range of AED 5,000- < 10,000 (USD 1.00 ≈ AED 3.67), 319 participants (26.5%) earned between AED 10,000-20,000, 221 participants (18.4%) had an income higher than AED 20,000 while only 164 participants (13.6%) earned less than AED 5,000.The majority of respondents were employed and 41.5% (2 and nearly two-thirds (64.2%) of participants were classified as overweight or obese according to their body mass index  had completed undergraduate degrees, 22.4% postgraduate education, and 11.5% had completed high school education  followed by minerals (10.5%) and sport nutrition products  of respondents reported using HS and the majority (88%) consumed vitamins with health improvement as the predominant (66%) reason for HS use. Despite widespread use of HS amongst the Dubai population, adverse events associated with HS use were relatively rare (< 5%). Interestingly, we found that HS use was independently positively associated with female gender, higher income, being a past smoker, having an allergy, more frequent doctor visits, taking prescribed medications, and knowledge about HS. Conversely, there was a negative association between HS use and being Emirati or South Asian.n = 37,958) collected between 1999 and 2012 found that the use of HS remained stable between 1999 and 2012, with 52% of adults reporting use of any supplements in 2011–2012 [N = 1173; 56.4% female; 90.2% response rate) in 2010–11 and revealed that 71.4% of the sample used HS [N = 726; 59.8% female; response rate 93.0%) attending randomly selected community pharmacies from six Lebanese governorates [N = 534; aged 19–26 years) attending one University in Saudi Arabia [N = 419; 59.2% male; response rate 82.4%) attending two private universities in Qatar [Previous population-based surveys in the United States have shown that approximately half of the population regularly consume HS . The Nat011–2012 . Current used HS . The stun = 1474) from 2011 to 2016 in the city of Jeddah in Saudi Arabia reported that only 3.6% of poisoning caused by drug overdose were due to vitamins and the majority of these were accidental (82.7%) and in the 0–4 year old age group (84.6%) [n = 1708) of United States Royal Navy and Marine Corps personnel reported that 29% of HS users  reported at least one adverse event in the previous six month period [Of the users, only 3% reported experiencing adverse events and most of these were not serious. The low level of adverse events experienced by HS users may account for the finding that amongst all respondents, 46% were unsure if there would be any benefits from establishing a reporting system for HS-related adverse events. That said, most participants expressed the view that a reporting system would be beneficial.The published literature identifies a high risk of adverse events associated with HS use, especially herbal supplements as they have a greater chance to have been contaminated by, for example, heavy metals and pesticide residues –29. Prev (84.6%) . One reah period .The finding that adverse events, albeit uncommon, are usually unreported is noteworthy. Currently, there is no adverse events monitoring system in Dubai. Consideration should be given to establishing such a system and raising awareness amongst consumers of the importance of reporting adverse events. This will permit ongoing surveillance of HS-related adverse events and ensure that any future increases can be promptly recognized.n = 242) from one university in Saudi Arabia reported very low awareness of commonly used herbs such as St. John’s Wort, Echinacea, and Gingko biloba [In our study we found strong and significant associations between use of HS and female gender, higher income, history of allergy, being a past-smoker, more frequent to doctors and taking of prescribed medication. These associations may not be surprising as use of HS may be part of a spectrum of health behaviors where consumers are seeking treatment or substitutes in combination with allopathic medicine and prescribed pharmaceuticals. Less than half of our sample had been prescribed HS by a physician. Self-reported diagnosis of diabetes mellitus 36.2%) and cardiovascular disease (45.2%) was relatively high amongst HS users. The concomitant use of prescribed allopathic medicine with over-the-counter HS creates a challenge for physicians as HS can interfere with the pharmacokinetics and pharmacodynamics of prescribed medicine causing reduced efficacy and possibly adverse events [6.2% and o biloba . In viewo biloba .Clearly, HS can be expensive and the cost may be prohibitive to members of the Dubai population without health insurance and/or with lower incomes. We found that HS is strongly associated with female gender. This finding is well known and is thought to be explained by women’s greater health consciousness, although women also have higher utilization of allopathic health services . We founThe main strength of this study was the recruitment of a large random representative sample using trained multi-lingual researchers to conduct the telephone-based computer-assisted interview. The large representative sample permits comparisons with other studies and ensures more precise estimates with sufficient power to detect associations between HS use and potential explanatory variables. The major limitation of this study is the cross-sectional design which does not allow for the determination of temporality or causality between independent and dependent variables. Another limitation is that the sample may not be completely representative of the overall Dubai population as we used a minimum age requirement of 16 years which slightly reduces the generalizability of the findings. There is limited data available on the age, gender, and nationality structure of the Dubai population to compare the representativeness of our sample. However, estimates from 2016 indicate that our sample with a minimum age of 16 years was slightly older (mean age 39 years versus median age for the population of 30 years), contained a higher proportion of males (83% versus 70%), and a marginally higher proportion of Emiratis (11.8% versus 8.6%) . There aOur population-based household telephone survey reported that one third of the general population in Dubai aged ≥16 years use HS, mainly vitamins, on a regular basis. Moreover, approximately half of users had been prescribed HS by a physician and 60% received HS information from a pharmacist. In view of these findings and the high levels of self-reported chronic disease in our sample, the curricula of medical and pharmacology schools in the UAE needs to cover the possible interactions and adverse events associated with the concomitant use of prescribed medicine with commonly used over-the-counter HS. Despite the relatively low prevalence (< 5%) of adverse events associated with HS use, Dubai may benefit from an online, mobile app, or telephone-based reporting system as this would raise awareness amongst consumers and allow the long-term monitoring of adverse events in Dubai. As with any cross-sectional design, it is not possible to draw conclusions about the temporal or causal nature of the association between proposed independent and dependent variables. Future studies utilizing more robust longitudinal cohort designs would allow researchers to relate different sociodemographic variables with HS use, or even HS use with specific adverse events. Such studies may want to consider focusing on higher-risk individuals including children, pregnant women, and older adults with co-morbidities.For the first time in the Gulf region, we have estimated HS use and HS-related adverse events in a representative sample of the general population that included both UAE nationals and expatriates. Levels of HS use are similar to those reported in other studies but the HS-related adverse events are infrequent. The current well-developed regulatory framework for HS availability and use in Dubai may be the reason for this reassuring situation. Nevertheless, we recommend that an adverse events monitoring system is established to complement the steps that have already been taken to ensure the continued safe use of HS by the people of Dubai.Additional file 1:Health Supplement Use in Dubai Telephone Questionnaire. (PDF 579 kb)Additional file 2:Table S1 Information and advice to use HS amongst ever users of HS in Dubai, 2015 (n = 455). (DOCX 16 kb)Additional file 3:Table S2 Forms and ingredients of HS reported by ever users of HS in Dubai, 2015 (n = 455). (DOCX 16 kb)Additional file 4:Table S3 Adverse events from HS use reported by ever users of HS in Dubai, 2015 (n = 455). (DOCX 17 kb)Additional file 5:Table S4 Correlates of HS use, cross-sectional study of HS use and HS-related adverse events, Dubai, 2015 (n = 1203). (DOCX 20 kb)Additional file 6:Table S5 Crude odds ratios for HS use, cross-sectional study of HS use and HS-related adverse events, Dubai, 2015 (n = 1203). (DOCX 20 kb)


Panax notoginseng (Burk.) F. H. Chen is one of the most common herbs in China. Because of its good efficacy and little adverse reaction, Panax notoginseng has been used widely to treat cardiovascular diseases (CVDs). Panax notoginseng flower (PN-F) on rats with myocardial infarction (MI).  To investigate effects of Panax notoginseng flower decoction 500 mg/kg, n=10), and FD1000 (high-dose) group . Panax notoginseng flower decoction or Betaloc was orally administrated for two to four weeks before and after operation. Sham-operated group was used as a normal untreated group, in which animals were treated with double distilled water, once daily. HE (hematoxylin and eosin) staining, immunofluorescent assay, TUNEL assay, quantitative real-time PCR, and western blot analysis were, respectively, performed to observe morphology, count mean minimal vessels, investigate apoptotic cells, and record gene  and protein (Bcl-2 and Bax) expressions.  The proximal left anterior descending coronary artery in rats was ligated to induce acute myocardial infarction. Then, animals were randomly assigned to four experimental groups: MI control group, Betaloc control group , FD500 (low-dose) group  and proteins (Bcl-2 and Bax).  PN-F demonstrated a potential herb to treat rats with myocardial infarction through promoting angiogenesis and inhibition of apoptosis in the infarct area. Proangiogenesis improved the outcome of myocardial infarction [Myocardial infarction is a major consequence of coronary artery disease, and heart remodeling secondary to myocardial infarction is considered the first step towards heart failure . Both myfarction . Howeverfarction , 7. The Panax notoginseng (Burk.) F. H. Chen, also named Sanqi, is one of the most common herbs in China. According to TCM theory, it can dissipate blood stasis and arrest bleeding, remove swell, and relieve pain. Because of its good efficacy and little adverse reaction, Panax notoginseng has been used widely to treat cardiovascular diseases (CVDs) and has attracted more and more attention in many countries such as the United States, Japan, and Korea [Panax notoginseng (PN-F) can also be used as folk herbs or diet supplements, which can treat hypertension, vertigo, and acute faucitis in China [Panax notoginseng herb? Therefore, in the present study, we aimed to observe angiogenesis effects of PN-F herb on MI rats, figure out its possible mechanism, and provide a basis for the development of the proangiogenesis herb.nd Korea . Usuallyin China , 10. PN-in China . These sin China , inhibitin China , and enhin China . More imin China  and imprin China . Furtherin China –19. How PN-F (Lot 120801) was bought from Tong-Ji-Tang Pharmaceutical Company in Shanghai, China. PN-F was extracted twice with 10-fold water at 100°C for 20 min. The extracts were filtrated, combined, and concentrated to 1 g rude herb per ml.The chromatographic system used is of Agilent 1200 HPLC series, consisting of a binary pump (Model G1312B), a vacuum degasser (Model G1379B), an autosampler (Model G1367C), and a column oven (Model G1316B). The mass spectrometer used was an Applied Biosystems Sciex 4000 Q-Trap® mass spectrometer  combined with a Source 5000 LC/MS gas generator . Data acquisition was carried out by Analyst® 1.4.2 software on a Dell computer.All experimental procedures were performed according to the guidelines of the National Animal Welfare Law of China . Seventy male Sprague-Dawley rats (100–110 g) were purchased from Shanghai Laboratory Animals Limited Liability Company . They were anesthetized with ether, and thoracotomy was performed through the fourth infarcted space .n=10), Betaloc group , FD500 group , and FD1000 group . Panax notoginseng flower decoction or Betaloc was orally administrated once daily for two weeks before and after operation, totaling four weeks. In addition to the MI group, the sham-operated group (n=8) was also made with the same procedure but without the LAD ligation. Heart samples were collected at the 14th day after MI.The rats were randomly assigned to four experimental groups: MI group (The left anterior descending (LAD) coronary artery was identified and occluded with a 6-0 Prolene suture in the middle portion. The exposed heart was immediately pushed back into the thoracic cavity. The chest was closed to let the animals recover . Finallyμm in thickness. These slices were then stained with hematoxylin and eosin (HE) for histopathologic observation.All the rats were sacrificed after 14 days, and the hearts were removed. The LV was cut into sections and mounted, and the infarct size was calculated as a percentage of the LV. The remaining rats' heart tissues were fixed in 10% formalin buffer for histopathological and immunofluorescent analysis. After fixing, the heart tissues were dehydrated with gradient ethanol, cleared, immersed, embedded in paraffin, and sliced to 5 α-SMA antibody  for 2 h at room temperature. After washing with PBS three times (15 min/time), the samples were incubated with fluorescence-conjugated secondary antibody for 1 h at room temperature in dark place and washed with PBS three times again (10 min/time). Finally, the sections were dripped with the water-soluble DAPI (diamidino phenylindole) mounting medium, covered with a coverslip, sealed with nail polish, and stored at 4°C.Paraffin slices were deparaffinized and rehydrated according to the standard protocol. Afterwards, these tissues needed antigen retrieval. In detail, dewaxed tissues were washed with PBS  three times (3 min/time), soaked in 3% hydrogen peroxide for 10 min, and rinsed, respectively, with tap water for 30 s and PBS for 5 min. Then, the tissues were soaked in citrate antigen retrieval buffer, boiled over high heat for 8 min and medium heat for 5 min using a microwave, and cooled down under room temperature for 30 min. After three rounds of washing with PBS (3 min/time), the tissues were blocked with 5% goat serum (diluted with PBS) for 15 min under room temperature and then washed twice with PBS (3 min/time). The samples were incubated with VWF primary antibody  and α-SMA antibody showed green fluorescence, while endothelial cells which expressed VWF antibody showed red fluorescence. Five zones of the peri-infarct zone and remote area were selected. The minimal vessels were counted and pictured. Average vessel numbers of the five zones were recorded as mean MVC (microvessel count).Under the fluorescence microscope of 400 magnified fields, the smooth muscle cells which expressed Paraffin slices were dewaxed and rehydrated. The antigen of tissues was repaired according to the above standard protocol. After washing with PBS, the slices were incubated with 0.1% TUNEL  antibody  overnight at 4°C. The slices were then washed twice with PBS (10 min/time), dripped with the DAPI mounting medium, covered with a coverslip, sealed with nail polish, and stored at 4°C. Under the fluorescence microscope of 200 magnified fields, apoptotic cells expressed green fluorescence, while the nucleus showed blue fluorescence. Five zones of the peri-infarct zone and remote area were selected. The apoptotic cells were counted. Average apoptotic cell numbers of the five zones were recorded.ß-mercaptoethanol on ice for 30 min. After centrifugation at 12,500g for 20 min at room temperature, the supernatant was collected. The total RNA concentration was quantified with a spectrophotometer. The total RNA (1 μg) from each sample was reverse transcribed to cDNA in a 20 μl reaction mixture. The obtained cDNA was diluted with 980 μl cDNA buffer  to final solution (1 μg/ml). Quantitative real-time PCR was performed using an ABI 7500 Real-Time PCR system (Applied Biosystems). In a total volume of 15 μl reaction mixture, 7.5 μl cDNA templates were mixed with 7.5 μl iTaq™ Universal SYBR Green Supermix (Bio-Rad Laboratories) and 0.45 μl primers. The primers and probes used for the quantitative real-time PCR are shown in Heart tissues were lysed with RNA lysis buffer containing 0.5% μl. Then, protein was transferred to PVDF (polyvinylidene difluoride) membranes for 60 min and blocked in 5% skimmed milk for 1 hour. After washing at 80 r/min for 5 min, the membranes were placed together with primary antibodies in a shaker for 1°h and incubated overnight at 4°C. Then, the membranes were washed three times (shaked for 10 min each time) and incubated with HRP- (horseradish peroxidase-) conjugated fluorescence secondary antibody for 30 min at room temperature. After another three times of washing, immunoreactive proteins were detected by enhanced chemoluminescence . The target protein included Bax and Bcl-2, using ß-actin as the internal standard.The protein electrophoresis was performed using SDS-PAGE (polyacrylamide gel electrophoresis) for 90 min (condensed gel 10% and separate gel 5%). The volume of loading protein from each sample was 25 P < 0.05 was considered to be statistically significant.Results were expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed with one-way ANOVA  using SPSS 17.0 software for Windows. Panax notoginseng flower are listed in Panax notoginseng flower was controllable.The contents of mark ingredients in extracts of p < 0.05), indicating that PN-F significantly enhanced capillary density in the border area of MI.As shown in Histological morphology of heart tissues was observed after HE staining . In the p < 0.05), and the compensation did not appear in the sham group. Compared to the MI group, numbers of minimal vessels in the Betaloc group and FD1000 group were significantly increased (p < 0.05), indicating that PN-F might have angiogenesis effects on the infarcted area.Angiogenesis of minimal vessels in the infarct area could improve ischemia to some extent under the pathological condition of LAD artery ligation. In the study, minimal vessels in infarct zones were determined by an immunofluorescent assay after two weeks of ischemia. As shown in p < 0.05), indicating PN-F decoction might relieve injury induced by myocardial infarction.Cell apoptosis was determined by TUNEL staining after myocardial ischemia . In the p < 0.05), indicating that PN-F decoction also inhibits apoptosis in the remote area.In the remote area, apoptotic cells appeared in all groups, indicating LAD artery ligation might induce apoptosis in both the peri-infarct zone and remote area; the apoptotic cells in FD500, FD1000, and Betaloc groups were less than those in the MI control group (p > 0.05), indicating that PN-F might induce production of HIF and VEGFA genes but not obviously.The expressions of HIF-1, VEGFA, and KDR in the infarcted area were detected by qPCR after two weeks of ischemia. As shown in α) and KDR (kinase insert domain receptor), compared with the sham group, the expressions of the MI control group and FD1000 group obviously increased (p < 0.05), indicating the expressions of the FD1000 group were higher than those of the MI group, suggesting that PN-F could induce VEGFA and KDR expressions. Betaloc groups were higher than MI control groups in VEGFA (p < 0.01), but the differences were not significant in KDR (p > 0.05).For VEGFA , indicating that PN-F might inhibit cell apoptosis in the infarcted area to protect heart tissue.The expressions of the protein-infarcted area were detected after two weeks of ischemia. Results show  that, foPanax notoginseng had proangiogenic effects on healthy and vessel-lost zebrafish which were damaged by VEGF receptor kinase inhibitor [Panax notoginseng had more powerful and significant angiogenic/restorative effects than the extract from roots of Panax notoginseng [Panax notoginseng flowers (herb) on MI rats were unclear. Therefore, the present article paid attention to angiogenesis effects of flowers (herb) on MI rats.The growth of new capillaries from current blood vessels, named angiogenesis, is a complex multistep process, comprising a series of cellular events that lead to neovascularization , 23. Angnhibitor . Intriguoginseng , 17–19. β-Blockers were clinically valuable for the setting of acute MI. Many evidences showed mortality was reduced when ß-blockers were administered early [ß-blockade was a class I indication according to clinical practice guidelines [ß-blockers as positive control medicine. In order to guarantee the repeatability of the experiment, the contents of chemical structures in Panax notoginseng flowers were determined using the HPLC method.There were various methods to establish an MI animal model, including drug induction, tube placement, and LAD ligation –30. Amoned early . The useidelines . TherefoFirst, we need to observe morphologic changes after myocardial infarction. Myocardial infarct size was a strong predictor of cardiovascular events, so we observed histological morphology of heart tissues after HE staining. Results showed that the FD1000 group and Betaloc group significantly decreased infarct size, demonstrating PN-F had potential therapeutic functions on MI rats.Second, in order to figure out its primary mechanism, minimal vessels in infarct zones were determined by an immunofluorescent assay after ischemia. Results showed that minimal vessels in the Betaloc group and FD1000 group were significantly increased, demonstrating that PN-F could improve minimal vessels in the infarct area. Apoptosis of cardiomyocytes also significantly contributed to loss of cardiac function after MI, as well as the development of heart failure , 34. TheNext, we studied further molecular mechanism. Based on the above experiment, there were two prospects: growth of vessels and inhibition of cell apoptosis in the infarct area; thus, these two prospects were investigated.α was activated by hypoxia and then targeted a wide array of genes, including proangiogenesis genes  [α and VEGF expressions in cardiomyocytes [The angiogenesis properties of VEGF during vascular development or tumor angiogenesis attracted people to evaluate its role in post-MI angiogenesis . Hypoxia., VEGF) . Cardiacmyocytes . VEGF inmyocytes , 40. In On the other side, inhibition of cell apoptosis in the infarct area was also important. Both Bax and Bcl-2 protein related to apoptosis, but functions were different: Bcl-2 was specifically considered an important antiapoptotic protein , while BTo sum up, our study revealed that PN-F (herb) could relieve symptoms of MI rats. Its mechanism might be to promote growth of minimal vessels in the infarct area through stimulation of HIF-1, VEGFA, and KDR and inhibit apoptosis of cardiomyocytes through downregulation of Bax/Bcl-2.