To propose a combination of blood biomarkers for the prediction of hospital-acquired pneumonia (HAP) and for the selection of traumatic brain-injured (TBI) patients eligible for corticosteroid therapy for the prevention of HAP.total&free, as a surrogate marker of the pro/anti-inflammatory response balance, were measured in samples collected before the treatment initiation. Endpoint was HAP on day 28.This was a sub-study of the CORTI-TC trial, a multicenter, randomized, double-blind, controlled trial evaluating the risk of HAP at day 28 in 336 TBI patients treated or not with corticosteroid therapy. Patients were between 15 and 65 years with severe traumatic brain injury  and were enrolled within 24 h of trauma. The blood levels of CRP and cortisoltotal&free and CRP blood levels upon ICU admission were not significantly different between patients with or without HAP. The cortisoltotal/CRP ratio upon admission was 2.30 [1.25–3.91] in patients without HAP and 3.36 [1.74–5.09] in patients with HAP (p = 0.021). In multivariate analysis, a cortisoltotal/CRP ratio > 3, selected upon the best Youden index on the ROC curve, was independently associated with HAP . The HR for HAP with corticosteroid treatment was 0.59  in patients with a cortisoltotal/CRP ratio > 3, and 0.89  in patients with a ratio < 3.Of the 179 patients with available samples, 89 (49.7%) developed an HAP. Cortisoltotal/CRP ratio > 3 upon admission may predict the development of HAP in severe TBI. Among these patients, corticosteroids reduce the occurrence HAP. We suggest that this ratio may select the patients who may benefit from corticosteroid therapy for the prevention of HAP.A cortisol Traumatic brain injury (TBI) is the leading cause of mortality and disability among young patients throughout the world. It is a major health and socioeconomic problem , 2. HospDuring the early post-traumatic period, the release of danger-associated molecular pattern by injured cells results in systemic inflammatory response syndrome (SIRS) which is characterized by increased levels of CRP and cytokines –8. ElevaIt is true that the administration of corticosteroids in a condition that is at risk of secondary infection may seem inappropriate. However, we have shown that low dose of hydrocortisone prevents the occurrence of hospital-acquired pneumonia in multiple trauma patients . The antWe aimed to assess the predictive values of the cortisol/CRP ratio  for the development of HAP in TBI patients. We also evaluated if this ratio may help to select patients who would benefit from corticosteroid therapy to prevent HAP.This study is a sub-study of the Corti-TC trial  (NCT 010n = 330 patients), we included the 179 patients with available blood samples.In the Corti-TC trial, inclusion criteria were age between 15 and 65 years, severe traumatic brain injury , and enrolment within 24 h of trauma , 17. ExcFor the purpose of the Corti-TC trial, patients received either hydrocortisone (200 mg per day tapered) and fludrocortisone (50 μg tablet once per day) or double placebo for 10 days. Before receiving study drug, adrenal function was assessed with a short corticotropin test. Treatment was stopped if patients had no adrenal insufficiency which was defined as basal blood cortisol concentration of less than 150 μg/L (413 nmol/L) or a maximum increase of less than 90 μg/L (248 nmol/L) in the 60 min after a short corticotropin test.In the Corti-TC study, the primary outcome was hospital-acquired pneumonia on day 28 of follow-up in patients with or without corticosteroid therapy. In this sub-study, the primary outcome was also the rate of hospital-acquired pneumonia in severe traumatic brain-injured patients.Blood samples were collected in the first 24 h after trauma, before any administration of the Corti-TC treatment. Sera were frozen − 80 °C upon dosage. Concentration of CRP and plasma cortisol concentrations  were investigated in sera collected in patients from the Corti-TC study with available samples.All biochemical measurements of CRP, Transcortin, and total and free cortisol were performed at the laboratory of Clinical Biochemistry, Nantes University Hospital. The laboratory is licensed according to the ISO 15189 accreditation standard for clinical laboratories.Serum CRP and total cortisol were determined in single measurement with an electrochemiluminescence immunoassay (C-Reactive Protein Gen.3 and Elecsys Cortisol II respectively) on the Cobas e602-module of the automated cobas®8000 system . The lower limits of detection (LoD) of the CRP and cortisol assays were 0.3 mg/L and 0.54 μg/L respectively. Each CRP and cortisol runs were validated by measuring two levels of quality control material prior to starting the experiment.g for 30 min at 37 °C. Free cortisol was determined in the ultrafiltrate as previously describe for total cortisol.Samples for serum-free cortisol determination were prepared by equilibrating 500 μL of serum at 37 °C for 15 min in Centrifree 30,000 molecular weight cut-off Centrifugal filters  before centrifugation at 1500Serum Transcortin concentrations were measured in duplicate using Human Corticosteroid Binding Globulin ELISA  according to the manufacturer’s instructions. Each series of assays (1 ELISA plate) was validated by two internal controls. The lower limit of detection (LoD) was 0.01 μg/mL.All care provided to patients with severe TBI followed the international guidelines in effect at the time of randomization, including respiratory management, temperature management, stress ulcer prophylaxis, nutrition, fluid therapy, glucose management, intracranial pressure monitoring, and management  as done 4 colony-forming units per milliliter (CFU/mL) for a broncho-alveolar lavage or nonbronchoscopic sample or 103CFU/mL for a protected specimen brush. Respiratory samples were always obtained before starting any new antibiotic treatment. HAP was defined as pneumonia that occurred 48 h after admission  and values were compared using the Wilcoxon-Mann-Whitney test. Categorical data were described as total/CRP) ratio was based on logistic regression between HAP and  ratio. The value corresponding to the largest Youden index was selected as the cut-off. Variables that were associated with HAP at the 0.15 level in univariate analysis were included in a multiple logistic regression. Then, variables that were non-significant at the 0.05 level  were removed one by one. Multiple logistic regressions were performed in the global sample, in the placebo group and in the corticosteroid group.The cut-off for distinguishing low versus high (cortisolp = 0.004). Patients were equally drawn from the intervention and control arms and they do not differ in other important ways from the original study population in terms of age, sex, severity, and comorbidities (data not shown).Of the 179 analyzed patients, 89 (49.7%) developed a HAP and 90 (50.3%) did not. Table p = 0.06 and p = 0.26 respectively, Table p = 0.89). The blood level of CRP was 61 [35–98] mg/L in patients without HAP as compared to 53 [33–82] mg/L in patients with HAP (p = 0.20). These results demonstrate that taken separately, blood levels of cortisol and of CRP cannot predict HAP. We thus aimed to investigate the prognostic value of a ratio combining markers of CIRCI and inflammatory response.Despite a trend for higher total and free blood levels of cortisol upon ICU admission, there was no significant difference between patients with or without HAP  . The cut-offs of cortisoltotal/CRP ratio > 3 was selected because it provides the best sensitivity/specificity balance . The positive likelihood ratio was 1.66 for a cortisoltotal/CRP ratio > 3. In the entire population, patients with a ratio total cortisol/CRP > 3 (n = 88) developed more HAP than those with a ratio < 3 [50 (62.50%) vs 39 (39.39%) respectively p = 0.002]  p = 0.004) (Table p = 0.007).In an attempt to simplify the use of the results in clinical practice, the cortisol< 3 [50 6.50% vs 3< 3 [50 6.50% vs 3total/CRP has the potential to select patients who will benefit from corticosteroid therapy, and those whose risk of HAP will not be decreased by the treatment. We thus compared the relative risk of HAP in the low and the high ratio groups, according to the treatment received (placebo or corticosteroid therapy). In high-risk patients (ratio > 3), the probability of HAP at day 28 was higher in the placebo group vs corticoid group  with an hazard ratio of 0.59  , there was no difference between placebo and treatment groups for the development of HAP )  in particular by the use of low-dose glucocorticoids , 12 or tIn our study, the correlation between total cortisol and CRP levels could be explained by the early secretion of interleukin-6 (IL-6) following trauma. Indeed, IL-6 is a pro-inflammatory cytokine known to have HPA-activating activity independent of ACTH. Thus, human IL-6 increases plasma concentrations cortisol in mice  and duriIn association with appropriate antibiotherapy, methylprednisolone administration was also associated with a faster reduction in blood IL-6 and CRP levels in the first 24 h of treatment of community-acquired pneumonia . Before A recent meta-analysis of corticosteroids in pneumonia found that hydrocortisone was not useful in this context, and only prednisone or methyl prednisolone was beneficial . Howevertotal/CRP ratio (> 3) have a higher susceptibility to develop HAP and for these patients, the introduction of low dose of corticosteroids is able to reduce this susceptibility. This effect of corticosteroid therapy is not found in patients with a ratio < 3. There are two means by which low-dose corticosteroids could decrease the rate of secondary pneumonia. Firstly, through an anti-inflammatory effect (patients with a low cortisol/CRP ratio) that decrease the excessive inflammatory response and therefore the compensatory CARS response (immunosuppression). The initiation of corticosteroid therapy may for example reduce IL-6-dependent HPA stimulation, limiting the anti-inflammatory response and thus the prevalence of HAP in highly inflammatory patients. Secondly, corticosteroids may also directly enhance immunity (patients with a high cortisol/CRP ratio). Indeed, low-dose hydrocortisone improves the phagocytic abilities of neutrophils, decreases the blood concentration of anti-inflammatory cytokines such as interleukin-10, and increases the blood concentrations of interferon γ and interleukin-12, cytokines enhancing immunity and involved in the host defense against infections [Here, taken separately, pro- and anti-inflammatory biomarkers (CRP and cortisol respectively) failed to predict the development of HAP. However, patients with high cortisolfections . Glucocofections  allowingfections , 42. We fections , 15. MorThe main strength of this ancillary study is the data from a randomized, multicentre, double-blind, controlled trial. This is the first study discriminating head injured patients at risk for developing pneumonia by using an easy-to-use anti- and pro-inflammatory factor ratio in common practice. Another strength of our study is the safety of low-dose corticosteroid use in trauma patients. Indeed, by closely monitoring patients’ natremia and glycemia, there are no serious adverse events recorded in the two large randomized trials we have conducted on the field , 12. Thetotal/CRP > 3 ratio upon admission may predict the development of HAP in patients with severe traumatic brain injury. In high-risk patients (ratio > 3), the administration of corticosteroid therapy reduces the occurrence of HAP. This ratio could be used to select patients eligible for corticosteroid therapy in prevention of HAP in patients with severe traumatic brain injury.A cortisolIn severe traumatic brain-injured patients, the imbalance between the pro- and anti-inflammatory mechanisms leads to increased susceptibility to hospital-acquired pneumonia. We show here that cortisol and CRP blood levels together as surrogate markers of the CARS/SIRS balance upon ICU admission can accurately predict the development of hospital-acquired pneumonia and define the subgroup of patients who may benefit most from “low-dose” corticosteroid therapy.Additional file 1: Figure S1. Correlations between transcortine blood level and cortisoltotal blood level in the entire population. Figure S2. Receiver Operating Characteristic (ROC) curve of the risk of Hospital Acquired Pneumonia in the entire population for the CRP blood levels. Figure S3. Comparison of the cortisoltotal/CRP ratio in the entire population. Results are given in median +/- SD. Figure S4. Receiver Operating Characteristic (ROC) curve of the risk of Hospital Acquired Pneumonia in the entire population for the cortisol/CRP ratios. Figure S5. Main characteristics of patients treated with corticosteroids compared to those treated with placebo amongst the subset with ratio >3.


Controls (n = 227) were T2D patients without listed eye complications despite high risk (T2D duration ≥10 years and fasting blood glucose >169 mg/dl). Replication was assessed in African Americans enrolled in the ARIC study. We identified 4 significant loci: WDR72, HLA-B, GAP43/RP11-326J18.1, and AL713866.1. At WDR72 the most strongly associated SNPs were rs12906891  and rs11070992 . rs11070992 replicated in African Americans (p = 0.04). Variants in this gene have been associated with diabetic retinopathy, glycemic control, revascularization, and kidney disease.Proliferative diabetic retinopathy (PDR) is a sight-threatening complication of diabetes that is associated with longer duration of diabetes and poor glycemic control under a genetic susceptibility background. Although GWAS of PDR have been conducted in Europeans and Asians, none has been done in continental Africans, a population at increased risk for PDR. Here, we report a GWAS of PDR among Africans. PDR cases ( The latter is characterized by the growth of abnormal new blood vessels on the retina. Neovascularization and contraction of the accompanying fibrous tissue can distort the retina and lead to tractional retinal detachment, producing severe and often irreversible vision loss. In addition, the new blood vessels may cause pre-retinal or vitreous hemorrhage and neovascular glaucoma, which can also cause vision loss. By 2020, the prevalence of DR is expected to almost double to ~7.2 million within the USA alone.2 Notably, the prevalence of PDR varies considerably by ethnicity with estimates as high as 8.99% in Africans and 2.67, 1.29, and 5.10%, respectively in Chinese, South Asian and Hispanic individuals7 African ancestry individuals within the US also display higher DR prevalence and more severe DR compared to non-Hispanic Whites.8Diabetic retinopathy (DR) is a well-known complication of type 1 (T1D) and type 2 diabetes (T2D). It is the most frequent cause of new cases of blindness in the working-age population9 and optimizing glycemic control is the main recommendation to reduce the risk or slow the progression of retinopathy.10 There is mounting evidence indicating a large genetic contribution to the development and severity of DR as well.12 Although the Family Investigation of Nephropathy and Diabetes (FIND)-Eye study reported a broad-sense heritability for DR of 27% overall,13 estimates for PDR have been reported to be as high as 52%.14 Furthermore, even though those with T1D may have difficulty controlling their glycemia, many individuals living with T1D for 50 years or more never develop PDR.16Together, these observations suggest that genetic factors may influence a diabetic individual’s risk of developing DR and progression to PDR.Epidemiologic studies suggest that the severity of DR closely correlates with glycemic control and diabetes duration,22 Despite African ancestry individuals displaying high prevalence of PDR, only one GWAS for PDR has been conducted in African Americans, and this study failed to identify any significantly associated loci.23Knowledge about the specific genetic risk factors for DR has come from genome-wide association studies (GWAS) Table  includinp < 0.05). The effects of population stratification on our data were assessed through visualization of Q-Q plots (λ = 1.015) using the full set of markers  in the discovery sample  located in intron 1 of WDR72 on chromosome 15. Two other WDR72 variants (rs11070992 and rs67619978), in linkage disequilibrium (LD) with rs12906891, which have the r2 in AADM and ARICAA were 0.384/0.381 and 0.518/0.504, respectively, were also genome-wide significant. In AADM the LD of rs11070992 and rs67619978 is high, r2 = 0.98 and the r2 of rs11070992 and rs3081219 is 0.87. Population frequency data for rs3081219 is not available. Analysis conditioned on rs11070992 showed only one peak in this region .Replication in ARIC-AA was attempted for each of the variants that were genome-wide significant in the discovery sample of Africans. Of these, 2 were AL713866.1), which was more statistically significant using T2D controls, though the effect was reduced . We also tested the lead SNPs from the PDR association for an association with fasting blood glucose among AADM participants ; one of the variants tested reached nominal significance  and HLA-B*45:01 (p = 0.017) were nominally significantly associated with PDR in the AADM data.The HLA-B variant that was genome-wide significant in the analysis of Africans (rs1065386), produces a missense change at position 87 of the amino acid sequence that is found on several classical HLA-B alleles, including HLA-B*07:02, HLA-B*07:05, HLA-B*14:01, and HLA-B*35:01, among others. We tested the classical HLA-B alleles that include the rs1065386 variant allele for an association with PDR in our data Table . HLA-B*0p = 0.0013/0.02) near NPY2R/LOC729902, which was reported in three different cohort studies2. In AADM, we replicated rs918519  near LOC285626, which was recently reported in White Australian individuals.22 While our replication supports these previous findings, neither of these associations were GWAS significant in the original publication, and they would not reach this threshold with meta-analysis with our data.We also investigated previously reported top associations for PDR in our data, though few of these associations reached genome-wide significance in the original publications  in African Americans.27Despite evidence for a genetic contribution to proliferative diabetic retinopathy, GWAS have not been very successful in identifying loci for PDR. Many of the results in previously published GWAS of PDR did not reach genome-wide significance  is a protein-coding gene that has been associated with HbA1c level and poor glucose control.28 Notably, WDR72 mRNA is expressed in the retina.28 Diabetic kidney disease (DKD) and DR are both microvascular complications of T2D and share pathophysiological mechanisms and common risk factors.25 Some DKD studies require the presence of DR in controls, highlighting the correlation between these outcomes.25WDR72 is associated with eGFR and is highly expressed in the kidney epithelium, suggesting that genetic variation at these loci may have relevance for both microvascular complications.29 An important paralog of WDR72, WDR7, is associated with retinitis pigmentosa 4, a highly expressed protein in the retina (https://www.genecards.org/cgi-bin/carddisp.pl?gene=WDR7). Additionally, WDR72 is critical to ameloblast vesicle turnover during enamel maturation.30 Enamel matrix derivative induces proliferation, viability, and angiogenesis of human microvascular cells.31 These observations suggest WDR72 may be involved in proliferative angiogenesis of the microvasculature in the eyes.33 In terms of classical HLA alleles, the HLA-B alleles showing significant association with PDR in the present study are HLA-B*07:02 (p = 0.0155) and HLA-B*45:01 (p = 0.0171). These are not the same classical HLA-B alleles reportedly associated with DR in T1DM: HLA-B4932 and HLA-B62.33HLA-B  is a protein-coding gene which is expressed in the retina and the pancreas. HLA-B was also associated with DR in African and Caucasian in T1DM.GAP43  is a protein-coding gene which is expressed in the retina and pancreas. GAP43 is also an axonal regenerative marker which is related to diabetic peripheral neuropathy, while axons and blood vessels share molecular signals for purpose of navigation, regeneration, and terminal arborizations.34 The function of rs72740408 located near RNA gene AL713866.1 is still unknown.NYP2R) and rs918519 (near LOC285626). Neuropeptide Y Receptor subtype 2(NPY2R) is a G protein-coupled receptor for NPY, a neurotransmitter released by endothelial cells implicated in ischemic angiogenesis.35 There is substantial genetic, biologic, and functional data supporting a role for neuropeptide Y signaling in diabetic retinopathy.39 rs918519 (35 kb upstream of LOC285626) has been previously associated with PDR.22 This gene encodes an uncharacterized, long non-coding RNA. The nearest protein-coding gene is IL12B (Interleukin 12B), a further 33 kb upstream. IL12B is expressed in the retina (The Ocular Tissue Database40) and has been implicated in both T1D43 and T2D.44We also replicated previous reported PDR associations with rs1902491 . In that study, Shtir et al.A potential limitation of this study is the small sample size which is partly due to the strict definition of controls to include only those at high risk that did not develop PDR; however, we believed this approach which resulted in “cleaner phenotypic classification” enhanced our ability to identify GWAS significant loci for PDR. When we evaluated the identified associations in an analysis that used all diabetics as controls instead of our extreme control design, we see a diluted association for all but one variant, consistent with the less carefully defined controls adding uncertainty to the analysis, as some of the controls may have become cases with sufficient length of T2D diagnosis or poor glucose control. It is anticipated that a greater number of subjects, using this strategy to define control subjects, will yield even more loci. Our discovery sample consisted of continental Africans, while our replication set was African Americans. While it would have been ideal to conduct replication in Africans, as well, we are unaware of other datasets of Africans in which such an analysis could be conducted. Thus, it is uncertain whether our inability to replicate some of our findings was a result of false positives or other factors including small replication sample size, or differences in genomic  or environmental context between Africans and African Americans.In all, this GWAS of PDR among Africans identified 4 genome-wide significant loci, one of which replicated among African Americans. Identified loci highlight pathways of known significance to diabetic retinopathy, including glycemic control, and revascularization.The AADM study was approved by the Institutional Review Board (IRB) at the National Institutes of Health, and the ARIC study was approved by the IRB of all participating institutions, including the IRBs of the University of Minnesota, Johns Hopkins University, University of North Carolina, University of Mississippi Medical Center, and Wake Forest University. Both studies were conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all human participants.46 Demographic information was collected using standardized questionnaires across the AADM study centers. Anthropometric, medical history, and clinical examination parameters were obtained by trained study staff during a clinic visit. Blood samples were drawn after an overnight fast of at least 8 h. The definition of T2D was based on the American Diabetes Association (ADA) criteria.47 Biochemical parameters were measured with a Beckman-UniCel Dxc 800 biochemistry analyzer , including fasting plasma glucose, total cholesterol, triglycerides, LDL and HDL cholesterol.Study individuals were drawn from the AADM study, a large, ongoing genetic epidemiology study of T2D and related traits in Africans.48 A diagnosis of PDR was made only with neovascularization in any field or retinal detachment with a diagnosis of diabetes retinopathy.All AADM study participants underwent a thorough ophthalmological examination. The eye examination was part of a comprehensive physical examination of each participant in the study. Each participant had the following ocular examinations: visual acuity; ocular alignment and motility; pupil reactivity and function; visual fields; intraocular pressure; slit lamp examination of the cornea, iris, lens, and vitreous; and dilated fundus examination. Previous experiences of the ophthalmologists in the 5 centers demonstrate a better than 80% concurrence between their clinical and photographic assessment of the presence and absence of lesions associated with diabetic retinopathy. To assure reproducibility of the assessment and classification of ocular complications between the 5 centers, the absence and/or presence of hemorrhages, microaneurysms, cotton wool spots, neovascularization, cataracts, retinal detachment, maculopathy, and glaucoma in each subject’s eyes was recorded, along with other ocular abnormalities.Patients with PDR  were selected as cases, irrespective of glucose level and duration of diabetes.While most genetic studies of PDR to date have used a general sample of T2D patients as the control group, this approach runs the risk of misclassifying T2D patients without a long history of disease or with good glycemic control as controls as they might develop disease if challenged with poor glucose control or with a longer time since diagnosis. To minimize such misclassification bias, we selected as controls only those patients with fasting blood glucose of at least 169 mg/dl  and with a duration of diabetes of at least 10 years but without diabetic retinopathy (NDR). We reasoned that using such “super-controls” will improve the power of the study through more precise phenotype definition, albeit at the cost of a reduction in sample size.A total of 5231 African individuals from the AADM study were genotyped on high density GWAS arrays .p < 10-6, and minor allele frequency (MAF) < 0.01), imputation was performed using the African Genome Resources Panel available from the Sanger Imputation Service (https://imputation.sanger.ac.uk/).49 The African Genome Resources Imputation Reference Panel comprised 4956 individuals, including all 2504 from the 1000 Genomes Project Phase 3, ~2000 individuals from Uganda , and ~100 individuals from each of a set of populations from Ethiopia , Egypt, Namibia (Nama/Khoe-San) and South Africa (Zulu), yielding 9912 haplotypes for 93,421,145 SNPs. Pre-phasing was performed with EAGLE version 2.0.550 and imputation was performed using software Positional Burrows-Wheeler Transform (PBWT).51 The resulting imputation dataset was filtered for MAF ≥ 0.01 and info ≥ 0.3. A total of 18,433,741 SNPs was tested. Coordinates are given based on the hg19 genome build.After technical quality control and appropriate sample- and SNP-level filtering , using imputed dosages and adjusting for genetic relatedness, sex, age, and the first two principal components. Within EPACTS, we performed single variant EMMAX association analysis. Genome-wide significance was declared to be p < 5 × 10−8. Q-Q and Manhattan plots were generated using R 3.4.2 (http://www.r-project.org).Association analysis was performed using the EPACTS 3.2.6  pipeline (p < 5 × 10−8). To evaluate our control selection strategy, we also ran analyses as above, but included as controls all AADM participants with T2D, but no PDR, regardless of duration of T2D or presence of hyperglycemia (n = 1724). To determine whether these variants were associated with PDR through an association with glucose, we also ran our main analysis model with the addition of glucose as a covariate. Additionally, we tested these variants for an association with glucose, separately in AADM participants with (n = 2048) and without T2D . We further evaluated a finding in the HLA-B region by conduct HLA imputation using the HIBAG algorithm52 and show that the missense change is on several classic HLA-B alleles.Secondary analyses were conducted to further characterize the top SNPs of our main analysis . The resulting imputation dataset was filtered for MAF ≥ 0.01 and info ≥ 0.3.The replication study was conducted in the Atherosclerosis Risk in Communities (ARIC) study accessed through dbGaP (phs000280.v2. p1). ARIC was designed as a multi-ethnic study of atherosclerosis and recruited individuals in Forsyth County, NC; Jackson, MS; the suburbs of Minneapolis, MN; and Washington County, MD. It is a population-based study of 15,792 individuals aged 45–64 years at their first examined in 1987–1989.n = 3137).56 Imputation and association analysis were performed as described above. We adjusted for genetic relatedness, sex, age, and the first two principal components. METAL57 was used to perform inverse variance-weighted fixed-effect meta-analysis of the discovery and replication samples.Cases and controls were defined as in the discovery study. Replication was assessed in 19 cases and 50 controls obtained from the ARIC African Americans study  were plotted using Locus Zoom (http://www.locuszoom.org). Conditional analysis was conducted using PLINK 1.9.58Association results for PDR at a 250-kb region surrounding rs11070992(Demographic and clinical characteristics were compared using chi-squared tests for dichotomous traits and t-tests for continuous traits. R-studio was used for preparation of the phenotype data and annotation.59 which uses genotyped SNPs to generate a genetic covariance matrix followed by eigen decomposition.To minimize the potential effect of population structure, we adjusted all analyses by principal components obtained from the R package SNPRelate,Further information on research design is available in the Supplementary MaterialsReporting Summary Checklist


Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly, little is known about how quiescent cells respond to environmental challenges. The aim of the present study is to compare stress responses of cycling and quiescent mesenchymal stem cells (MSC). Human endometrial mesenchymal cells (eMSС) were employed as adult stem cells. eMSC quiescence was modeled by serum starvation. Sublethal heat shock (HS) was used as a stress factor. Both quiescent and cycling cells were heated at 45°C for 30 min and then returned to standard culture conditions for their recovery. HS response was monitored by DNA damage response, stress-induced premature senescence (SIPS), cell proliferation activity, and oxidative metabolism. It has been found that quiescent cells repair DNA more rapidly, resume proliferation, and undergo SIPS less than proliferating cells. HS-enforced ROS production in heated cycling cells was accompanied with increased expression of genes regulating redox-active proteins. Quiescent cells exposed to HS did not intensify the ROS production, and genes involved in antioxidant defense were mostly silent. Altogether, the results have shown that quiescent cells are more resistant to heat stress than cycling cells. Next-generation sequencing (NGS) demonstrates that HS-survived cells retain differentiation capacity and do not exhibit signs of spontaneous transformation. Human MSC as promising cell therapy candidates are under intensive investigation. Their differentiation abilities, immunomodulatory effects, and homing properties offer potential for augmenting regenerative capacity of many tissues. Mesenchymal stem cells are fibroblast-like adherent cells, which can be isolated from various tissues, such as bone marrow, umbilical cord, adipose tissue, peripheral blood, spleen, and skin . CurrentA major impediment to the development of MSC-based therapies, however, is poor cell survival at the site of injury. Generally, the harsh environment of injured tissue is associated with oxidative stress, chronic inflammation, fibrosis, extracellular matrix degradation, and immune rejection . This isCells exposed to stress may respond differently: undergo differentiation, senescence (SIPS), apoptosis, or necrosis. The choice depends on the cell type and stress strength. Mild stress may improve differentiation of stem cells , 13. The+ T cells and augmented secretion of IFN-γ by these cells. It also causes the secretion of inflammatory cytokines, such as TNF-α and IL-1, alters the migration of Langerhans cells, and provokes lymphocyte homing into secondary lymphoid tissues. Heat-shocked MSC can inhibit tumor growth and enhance tumor cell death [β1, and FOXP3 mRNAs. It had no effect on IL-17A and IFN-γ secretion and reduced CXCL12 [Heat stress  is one of the well-studied types of stress. It can affect a variety of cell types. Hyperthermia can accompany therapeutic procedures, such as stem cell-based therapy and cancer treatment. Hyperthermia changes the blood circulation and oxygen supply reduces the ATP level and increases anaerobic metabolites and activity of DNA repair proteins. It has various effects on the immune system, such as increased peripheral blood mononuclear cell proliferation, increased cytotoxic activity of CD8ll death . Hyperthll death , 16. It ll death  and ostell death , 19. Sevll death , 21. Proll death , 23. Enld CXCL12 . In our d CXCL12  which isIn a living body, stem cells may long reside in the dormant state entering the cell cycle in response to local signals of damage and other regeneration needs. Quiescence is the prevailing state of many cell types under homeostatic conditions. Proliferating cells in culture can be induced into quiescence by mitogen withdrawal under serum deprivation . Serum d2, and 95% humidity. For microscopic experiments, cells were grown on glass coverslips. Cells from the 5–7th passages were used for experiments.We used MSC isolated from desquamated endometrium of menstrual blood (eMSC) of healthy donors . The cel3 cells/cm2. The next day, the medium was changed for serum-free medium for 30 h.For accumulation of quiescent cells, eMSC that reached the monolayer were harvested using 0.05% trypsin-EDTA solution  and plated at a density of 10 × 10Before HS, quiescent cells were added with serum for 2 h to heat quiescent and proliferating cells under equal conditions. Both quiescent and proliferating cells were exposed to sublethal HS at 45°C for 30 min  in parafμg/mL DAPI. Then the coverslips were mounted on ProLong Gold antifade reagent . The primary antibodies used were mouse monoclonals against Hsp70 [γH2AX , and rabbit polyclonal against Ki-67 . Goat anti-mouse IgGAlexa 488  and goat anti-rabbit DyLight 567  were applied as secondary antibodies. Images were captured using Leica TCS SP5 confocal microscope  equipped with solid-state lasers for excitation  and HCX PL APO CS 40x oil immersion objective . Adobe Photoshop software was used to view and acquire images.eMSC grown on coverslips were fixed with 4% formalin in phosphate-buffered saline (PBS) for 15 min, permeabilized with 0.1% Triton X-100, blocked with 1% bovine serum albumin for 30 min, treated with primary antibodies for 45 min, washed with 0.1% Tween 20, treated with secondary antibodies for 45 min, washed with/0.1% Tween 20, and counterstained with 1 The cells lysed by incubation in RIPA Buffer  and cocktail of protease inhibitors ) for 15 minutes on ice were scrapped off from the plates and centrifuged for 20 minutes at 15000 g. The proteins were denatured by addition of electrophoretic buffer (40 mM Tris (pH 6.8), 10% SDS, 20% 2-mercaptoethanol and 40% glycerol) to the supernatant and further incubation at 100°C for 5 minutes. Protein concentration was measured by Bradford's method using ovalbumin for construction of the calibration curve. Proteins were separated electrophoretically in 10% polyacrylamide gel with subsequent transfer onto a HybondC extra nitrocellulose membrane . For detection of protein bands, the following primary and secondary antibodies  were used: anti-phospho-pRb , anti-cyclinA2 (4656S), anti-GAPDH (2118S), GAR-HRP (7074S), and GAM-HRP (7076S). The peroxidase activity of GAR-HRP and GAM-HRP conjugates was detected by enhanced chemiluminescence (ECL) reaction . Chemiluminescent signal was recorded by exposure to an X-film from CEA RP NEW . For densitometric analysis of protein bands, ImageJ software was used.μM H2DCFDA staining solution in the dark for 20 min at 37°C, then harvested with 0.05% trypsin-EDTA solution, suspended in a fresh medium and immediately analyzed with CytoFLEX flow cytometer . Cells were detected by size and granularity using FSC/SSC dot plot, and cell debris was gated out. Mean fluorescence intensity from 10,000 cells was acquired.For the measurements of intracellular ROS level, fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate  was used. H2DCFDA was dissolved in DMSO to obtain a 10 mM stock solution which was further diluted in PBS before use to obtain staining solution. Cells were incubated with 5 μg/mL saponin , 250 μg/mL RNase A , and 50 μg/mL propidium iodide  were added to each sample tube. After incubation for 60 min at room temperature, samples were analyzed with CytoFLEX flow cytometer . Mean fluorescence intensity from 10,000 cells was acquired. Cell cycle analysis was performed using CytExpert v. 2.0 software .Cells were harvested with trypsin-EDTA solution and suspended in fresh medium. 200 μg/mL propidium iodide  was added to each sample tube. After incubation for 5 min at room temperature, samples were analyzed with CytoFLEX flow cytometer . Mean fluorescence intensity from 10,000 cells was acquired. Cells were gated by size and granularity using FSC/SSC dot plot, and cell debris was excluded from the analysis.Cells were harvested with trypsin-EDTA solution and suspended in fresh medium. 50 μL. Expression of target genes was normalized to GAPDH or actin gene. Primers and reaction conditions are presented in To analyze gene expression, total RNA was isolated with RNeasy Micro Kit (QIAGEN) according to the manufacturer's instructions. RNA was quantified in the NanoDrop ND-1000 Spectrophotometer . cDNA was obtained by reverse transcription of 500 ng RNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer's instructions. It was subsequently amplified with specific primers, using DreamTaq™ PCR Master Mix (2X) (Thermo Fisher Scientific) with CycloTemp amplificator. The electrophoresis of amplified products was performed in 2% agarose gel with TAE buffer and ethidium bromide. 100 kb DNA ladder  was used as molecular weight markers. Amplified products were visualized in UV light (302 nm) with a transilluminator and registered with a digital Canon camera. For qRT-PCR, cDNA was amplified with specific primers, using EvaGreen® dye (Biotium) and DreamTaq™ PCR Master Mix (2X) (Thermo Fisher Scientific) in the Bio-Rad CFX-96 real-time system , according to the kit's enclosed protocol. The volume of RT and PCR reactions was 20 β-galactosidase  were detected with senescence β-galactosidase staining kit  according to manufacturer's instructions and quantified microscopically by counting X-Gal-positive cells among not less 500 cells in random fields of view.Cells expressing senescent-associated Cells that survived HS were subcultured for 6 passages and subjected to NGS. NGS was done by parallel measurement of three biological samples: unheated cells and cells heated in quiescent and proliferative stages. Sample preparation for NGS and sequencing on the Illumina platform were performed in «Genotek» company . RNA was extracted using PureLink RNA Mini Kit . cDNA libraries were prepared using NEBNext® mRNA Library Prep Reagent Set for Illumina® (New England Biolabs). Quality control of prepared libraries was made using Bioanalyzer 2100 (Agilent Technologies). Sequencing of cDNA libraries was done on HiSeq 2500 (Illumina) in Rapid Run Mode with a read length of 100 nt.The differential gene expression and gene modules enriched in differentially expressed genes were done similarly to previous works . The biop < 0.005 and q < 0.15. The data on key transcription factors (TFs) regulating mesenchymal stem cell differentiation was taken from [To obtain GO biological processes and NCBI BioSystems pathways related to differentiation, we searched for gene modules containing in their titles terms related to various types of mesenchymal stem cell differentiation. We selected gene modules containing in titles terms “differentiation”, “osteo”, “adipo”, “neuro”, “BDNF”, “oligodendro”, “chondro” with significance level ken from .To evaluate cell transformation potential, we used the gene set provided by . The autt-test was used to determine the statistical significance of differences between two groups; one-way ANOVA with post hoc Tukey HSD test was used to determine the significance of differences among groups. The null hypothesis was rejected at the 0.05 level of significance.Experiments were performed in triplicate. The results are expressed as mean ± SD. Student's eMSC cultivated in serum-free medium for 30 h stopped proliferation and accumulated in the G0/G1 stage . Most ceoC for 30 min was stressful for both quiescent and proliferating eMSC. The level of HSP70 is an indication of cell stress response. Measurement of the level and localization of Hsp70 protein showed that heating at 45β-Gal positive. It is commonly believed that the key features of SIPS in MSC cultures are SA-β-Gal staining and modified morphology.The most common MSC stress response is premature senescence . We founβ-Gal-positive cells was slightly lower in quiescent than in proliferative cultures exposed to HS.The number of SA-γH2AX foci, a DNA damage marker  and oncogenes (ONGs). The data presented in Cell differentiation potential was assessed by GO biological processes and BioSystems molecular pathways enriched for genes related to differentiation  that theCell response to stress factors is under intensive examination. Much of the current knowledge of cell stress responses is based on experiments with cycling cultured cells. Less is known about the effects of stress on cells in distinct phases of the cell cycle. Chinese hamster ovary cells of the middle and late S phases are more susceptible to stress than cells in mitosis, early S, G1, and G2 phases of the cell cycle . HeLa ceStem cell response was also basically examined with proliferating cells in culture. In a living body, stem cells remain in the quiescent state for prolonged periods of time, entering the cell cycle in response to local signals of damage and other regeneration needs. Very few studies have explored stress response of MSC in the quiescent state. It has been demonstrated that quiescent MSC are more resistant to anoxia and metabolic stress than cycling cells . Genome-In our experiments, we have modeled cellular quiescence to compare the stress response of the quiescent and proliferating mesenchymal stem cells. Experiments have been performed on eMSC from desquamated endometrium of menstrual blood. It is considered that these cells are derived from shed endometrium. The cells are multipotent, capable for self-renewal, express CD13, CD29, CD44, CD73, CD90, and CD105 markers, and are negative for the hematopoietic markers CD34 and CD45 . The intQuiescence is a reversible cell cycle arrest during which the cells reside in the G0/G1 phase. They can enter the proliferative stage in response to growth signals. Different approaches are applied to mimic MSC quiescence in culture . CoatingThese cells were subjected to HS. HS is one of the most conserved cellular stress responses. It is characterized by transcription and accumulation of heat shock proteins (HSP). Chaperones have been defined as proteins that bind to and stabilize an otherwise unstable conformer of another protein. With controlled binding and release of the substrate protein, they facilitate its correct fate. Among various HSPs, HSP70, with a molecular weight of 70 kDa, is known to be the major molecular chaperone. In unstressed cells, HSP70 exists in the cytoplasm. HS as well as other stressful factors exhibits robust enhancement of HSP70 expression and its relocation from the cytoplasm into the nuclei . During γH2AX) foci are required for recruiting and anchoring of DDR participants in sites of DNA damage. γH2AX foci are easily detected by fluorescent microcopy that allows using them as a reliable DDR marker.It is commonly believed that triggering of the senescence program with various stresses is initiated by DNA damage , 35. HeaγH2AX foci. Their maximal production was registered after 24 h recovery, and afterwards, it started to decline. After 72 h of cell cultivation under standard conditions, only few cells heated in quiescence had γH2AX foci but the foci were still identified in cells exposed to HS in the proliferative phase. ATR (another DDR marker) is weakly expressed in intact cells of both types. Its expression was increased during 24 h in heated cycling cells and then dropped. In cells heated at the quiescence state, ATR expression was not activated by HS. The difference in ATR expression in two cell types is, probably, attributed to the different pattern of the cell cycle distribution in cultures subjected to HS. The difference is mostly concerned to the cell number in the S phase. In quiescent cells that underwent HS, 4% of the cells stayed at the S phase whereas HS-treated proliferating culture had 14% cells in the S phase. Most reports on ATR function link ATR to the important role during the S phase [Quiescent and proliferative cells differed in DDR response elicited by HS. The cell heating was accompanied with generation of  S phase –50. Cultβ-Gal staining, and exhibited higher expression of p21 protein. Cells that survived HS gradually restored the normal phenotype under normal culture conditions. Quiescent-heated cells did it more rapidly than cycling cells. By 72 h after HS, most cells did not display SIPS features whereas cells exposed to HS in the proliferative state still retained SIPS phenotype.In our recent experiments, HS induced preliminary senescence (SIPS) in eMSC. It was accompanied by modified cell morphology, cell cycle arrest, and enhanced expression of p21 protein, an inhibitor of cyclin-dependent kinases . Now, weROS are well known to be implicated in various important cellular processes including signaling, regulation of homeostasis . HoweverIn our experiments, HS enhanced the ROS level in cycling but not quiescent cells. In growing cells, HS evoked enhanced ROS production and expression of catalase and SOD1, PRDX4, GPX, and TXN2 genes. Supposedly, in quiescent cells, the system of the oxidative defense copes with the task to keep the ROS amount at the threshold level more rapidly than in growing cells. It is supported by unperturbed expression of catalase, SOD1, GPX, and TXN2 controlling the synthesis of redox-active proteins.Spontaneous malignant transformation of MSC during prolonged in vitro expansion is of concern. It is currently believed that normal human MSC do not undergo spontaneous transformation in vitro during their passaging. Less is known about tumorigenic potency of stress that survived MSC. We performed mRNA sequencing and assessed expression of 50 TSGs and 49 ONGs  to examiThe progeny of cells heated in the quiescent or proliferative stages retained the capacity for differentiation common for MSC. HS-survived cells heated in quiescence exhibited even higher differentiation potential compared to the progeny of cycling cell subjected to HS. Earlier, we reported that human eMSC expanded after HS were able to differentiate into adipocytes and osteoblasts as well as transdifferentiate into neuronal cells . It was Collectively, we demonstrate the higher stress tolerance of quiescent human mesenchymal stem cells compared to cycling cells. In organism, stem cells face numerous challenges and should be protected from premature exhaustion to ensure tissue maintenance. It can be speculated that transplantation of cell accumulated in the quiescent state by serum starvation suggests a new, easy to implement, and safe strategy to improve transplanted cell survival and effectiveness of stem cell therapy.


Hoffmann’s syndrome (HS) is a rare manifestation of hypothyroidism myopathy that presents with weakness, stiffness, and eventually pseudohypertrophy of muscles, especially calf muscles. We report a case of a 28-year-old male who presented with the history of generalized weakness with swelling in lower limbs and gradual progressive facial puffiness for the past few years. Physical examination of our patient showed diffuse bilateral pseudohypertrophy of deltoid and calf muscles with positive Gowers’ sign (GS). Laboratory results of low serum thyroid hormones and muscle biopsy report confirmed the diagnosis of HS. Pendred syndrome (PS) is a genetic disorder leading to congenital bilateral sensorineural hearing loss with mild hypothyroidism. On account of his congenital bilateral sensorineural hearing loss and negative serum anti-thyroid peroxidase antibodies (anti-TPO Ab), PS was declared as the cause of HS in this case. Our patient showed excellent response to levothyroxine therapy with progressive improvement in his symptoms. We outlined this case due to its rarity. A diverse array of presentations of hypothyroidism is seen in routine clinical practice. The symptoms of patients with hypothyroidism vary from mild fatigue to fatal myxedema coma . The incA 28-year-old male, deaf and dumb since birth, presented to the medical department (MD) of Dr. Ruth KM Pfau, Civil Hospital Karachi (CHK) in November 2018 for the evaluation of generalized upper and lower limb weakness along with progressive facial puffiness , his pulse was 82 beats/min, blood pressure (BP) was 110/70, respiratory rate (RR) was 16 breaths/min, and he was afebrile. There was no thyroid enlargement. Lower limbs showed nonpitting edema up to the ankles. Upon neurological examination, bulk showed diffuse bilateral pseudohypertrophy of the calf muscles  of 11.7 gm/dl, raised serum cholesterol of 310 mg/dl, raised TSH of 35.2 μIU/ml [Normal (N) = 0.4-4.0], decreased levels of thyroxine (T4) of 0.14 μg/dl (N = 4.5-12.5), low plasma triido thyronine (T3) of 26.2 ng/dl (N = 84-172), anti-thyroid peroxidase antibody (anti-TPO Ab) <10 (N = <35), elevated creatine phosphokinase (CPK) of 1108 U/L (N= 85-170), creatine kinase-muscle and brain (CK-MB) of 126 U/L, elevated lactate dehydrogenase (LDH) of 930 U/L (N = 225-450), and raised serum aldolase of 9.3 U/L (N = 0-7). Blood glucose, liver function tests, blood urea, and serum creatinine were not remarkable. Abdominal X-ray revealed bilateral renal cortical cysts; however, no calculus or hydronephrosis was seen in both kidneys. Evaluation of cardiovascular and respiratory systems did not show any significant abnormality. Electromyography (EMG) and nerve conduction study (NCS) were normal. A biopsy was taken from his right deltoid muscle which showed mild changes in fiber size and local fibrosis. The findings were suggestive of myopathy. Immunohistochemistry was not done due to financial constraints. A diagnosis of HS was made based on clinical manifestations, laboratory investigations, and confirmatory muscle biopsy report. We further suspected PS as the cause of hypothyroidism in this patient based on his symptoms of congenital deafness and thyroid dysfunction. A tone audiometry was carried out which showed bilateral sensorineural hearing loss. A diagnosis of PS was finally established by performing a perchlorate discharge test which showed an abnormally rapid loss of radioactive iodine from the thyroid gland after the administration of perchlorate.The patient was initially treated with 100 μg levothyroxine daily which was gradually increased to 150 μg after two weeks. The patient was assessed after four weeks. His swelling resolved and TSH levels went down to 13 μIU/ml . The patient is under regular follow up with improvements in his symptoms.In 1897, HS was first elucidated by Hoffmann in an adult with muscle stiffness and hindrance in relaxation of his muscles following thyroidectomy . This suUsually primary hypothyroidism leads to thyroid insufficiency, Hashimoto's thyroiditis being the main culprit. Contrary to this, a suspicion of PS as the cause of hypothyroidism was made in this case because of the associated sensorineural hearing loss since birth along with the negative serology for anti-TPO Ab. Immunohistochemistry could not be done due to the nonavailability of funds.Levels of CPK, being the best biochemical indicator of myopathies , were drPatients are often misdiagnosed as the clinical picture and biochemical studies might challenge the physician to differentiate this syndrome from polymyositis or muscle dystrophies. EMG, NCS, and muscle biopsy are useful tools for the establishment of diagnosis. In this case, EMG and NCS were normal. Muscle biopsy revealed pale muscle fibers with variation in their size and focal areas of fibrosis without any inflammatory infiltrate, excluding polymyositis. This patient experienced difficulty in walking presumably because of frail contractions of the involved muscles. This weakness of muscular contraction associated with hypothyroidism is caused by muscle fiber transformation from type 2 fast-twitching to type 1 slow-twitching and abnormal oxidative enzymatic function . The exaThe treatment comprises exogenous thyroid hormone, in the form of levothyroxine, the dosage being 100-200 µg/day. Cardiovascular status of this patient was found to be normal as it is vital to rule out any cardiovascular risks prior to starting treatment due to an increased risk of acute coronary insufficiency, more common in elderly. A typical surge in symptoms can be seen in some cases with severe myopathic manifestation after the initiation of treatment, possibly due to raised metabolic demand induced by thyroxine . In thesHoffmann’s syndrome is a rare clinical presentation of a very common disorder of endocrine system, hypothyroidism. This case is even more rarer as it indicates PS as one cause of hypothyroidism leading to HS if left untreated. The positive aspect of this unusual entity is that it carries favorable prognosis with complete reversal of symptoms on timely made diagnosis followed by prompt initiation of THRT. As in this case, the patient showed gradual but progressive recovery from myopathy and reduction in elevated levels of TSH and muscle enzymes. Clinicians should be well aware of this rare manifestation of hypothyroidism to prevent the associated morbidity by avoiding misdiagnosis and subsequent delay in management.


It decreases left ventricular end-diastolic and end-systolic diameter, and recovers the reduced fractional shortening in TAC. In addition, it suppresses the expression of atrial natriuretic peptide, brain natriuretic peptide, skeletal α-actin, and β-myosin heavy chain. Administration of gallic acid decreases perivascular fibrosis, as determined by Trichrome II Blue staining, and reduces the expression of collagen type I and connective tissue growth factor. However, administration of losartan, carvedilol, and furosemide does not reduce cardiac dysfunction and fibrosis in TAC. Moreover, treatment with gallic acid inhibits fibrosis-related genes and deposition of collagen type I in TGF-β1-treated cardiac fibroblasts. These results suggest that gallic acid is a therapeutic agent for cardiac dysfunction and fibrosis in chronic heart failure.Gallic acid is a trihydroxybenzoic acid found in tea leaves and some plants. Here, we report the effect of gallic acid on cardiac dysfunction and fibrosis in a mouse model of pressure overload-induced heart failure and in primary rat cardiac fibroblasts, and compare the effects of gallic acid with those of drugs used in clinics. Gallic acid reduces cardiac hypertrophy, dysfunction, and fibrosis induced by transverse aortic constriction (TAC) stimuli  Heart failure is a common terminal clinical feature in various heart diseases, including hypertension, myocardial infarction, and valvular disease. There are several animal heart failure models, including that of transverse aortic constriction (TAC), left anterior descending ligation, and the aged spontaneously hypertensive rat. TAC mimics human aortic stenosis, and it induces cardiac hypertrophy and heart failure in animals2. In addition, TAC induces inflammatory and fibrotic response3.Heart failure is a major health concern worldwide, and it is a leading cause of morbidity and mortality in the United States and in other developed countries. For example, an estimated 6.5 million Americans over 20 years of age experienced heart failure, on the basis of data from NHANES 2011 to 20144. Cardiac fibroblasts are converted to myofibroblasts, an activated form, in response to pathological stimuli5. Collagen type I is a representative ECM expressed in cardiac fibroblasts, the epicardium, adventitia of large vessels, and valve interstitial cells. Transforming growth factor β1 (TGF-β1) activates fibroblasts6 and enhances the synthesis of ECM proteins7. Connective tissue growth factor (CTGF), a cysteine-rich growth factor, is increased by TGF-β1 and implicated in ECM protein synthesis8.Heart failure is characterized by interstitial fibrosis, chamber remodeling, and reduced ventricular compliance. Activated cardiac fibroblasts lead to excessive deposition of extracellular matrix (ECM) proteins such as collagen type I and fibronectin, and cardiac fibroblasts are major contributors to the progression of heart failure9. This has led many researchers to develop new and safe drugs to improve outcomes in patients with heart failure.Diuretics, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers (ARBs), and beta-blockers are clinically used to treat heart failure. Recently, selective sinus node inhibitors ivabradine and LCZ696  are attracting attention as therapy for heart failure. Despite effective therapies targeted at improving or treating heart failure, the 5-year mortality rate of this condition ranges from 45% to 60%10. In animal experiments, several phytochemicals such as curcumin and resveratrol (from grapes) have been demonstrated as a novel potential therapy for heart failure12. Gallic acid is a 3,4,5-trihydroxybenzoic acid found in tea leaves and some fruits. Recently, there has been increasing evidence that gallic acid confers several benefits on vascular calcification13, cardiac hypertrophy and fibrosis14, hypertension15, and oxidative stress16. Heart failure is associated with cardiac hypertrophy and hypertension; hence, we hypothesized that gallic acid can improve heart failure. Here, we investigated the effect of gallic acid in a mouse model of TAC-induced heart failure and in cultured rat fibroblast cells. We compared the effect of gallic acid with those of the ARB losartan, beta-blocker carvedilol, and diuretic furosemide. Our findings demonstrate that gallic acid can be a novel therapeutic agent to treat cardiac dysfunction and fibrosis in heart failure.Chinese herbal medicine has also been used for the treatment of chronic heart failureth edition, 2011). CD-1 male mice  were anesthetized using an intraperitoneal injection of ketamine (120 mg/kg) and xylazine (6.2 mg/kg). Mice underwent either sham operation or TAC. TAC was performed as follows. The endotracheal tube of the mice was connected to a rodent ventilator. After exposure of the aortic arch, the thymus was removed. The transverse aortic arch was ligated (using a 7-0 silk suture) between the brachiocephalic and left common carotid arteries with an overlaying 27 G needle. The sham mice underwent the same operation, except for ligation of the aorta. The TAC procedure was confirmed by echocardiography. After 8 weeks, TAC resulted in heart failure. The experimental rats were divided into six groups: sham group (n = 12), TAC group (n = 16), TAC + gallic acid , TAC + losartan , TAC + carvedilol , and TAC + furosemide . Gallic acid, losartan, carvedilol, and furosemide were dissolved in DMSO. The dosage of gallic acid (100 mg/kg/day) used for in vivo experiments was chosen because of the lack of toxicity and tolerable concentration in previous studies17,18. After drug administration for 2 weeks, the mice were sacrificed.All animal procedures were approved by the Animal Experimental Committee of Chonnam National University Medical School (CNU IACUC-H-2017-84), and carried out according to the Guide for the Care and Use of Laboratory Animals  with a 13-MHz linear array transducer. Mice were anesthetized using tribromoethanol  before the procedures. M-mode (2-D guided) images and recordings were acquired from the long-axis view of the left ventricle at the level of the papillary muscles. The thickness of the anterior and posterior wall was measured from the images, whereas the left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured from the M-mode recordings. Fractional shortening (FS) was calculated as FS (%) = (LVEDD − LVESD) × 100/LVEDD.i; Nikon Corp., Tokyo, Japan).The paraffin-embedded tissues were cut into 3-µm thick sections, deparaffinized with xylene, and then rehydrated with different grades of alcohol. Trichrome II Blue staining kit  was used on the BenchMark Special Stains automated slide staining instruments. Trichrome II Blue staining kit is a modification of Masson’s Trichrome Stain. Briefly, heart sections were incubated with Bouin’s solution to intensify the final coloration. Cytoplasm and muscle were stained with Trichrome Red, containing Biebrich scarlet and acid fuchsin, while nuclei were stained with iron hematoxylin. After application of Trichrome Mordant, the collagen was stained with Trichrome Blue II, which contains aniline blue. Trichrome Clarifier, an acetic acid solution, was applied to create a more delicate and transparent shade of color in the heart tissue section. To determine the development of vessel in the hearts of mice in the TAC group, the circumference of the vessels was measured using the NIS Elements Software  was purchased from Sigma . Carvedilol (S1831), losartan potassium (S1359), and furosemide (S1603) were purchased from Selleck Chemicals .2PO4, 5.5 mM glucose, 5 mM KCl, 0.8 mM MgSO4) at 37 °C on a shaker at 120 rpm for 2 h. After digestion, the cells were centrifuged at 1,000 rpm for 5 min. Fibroblasts were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) plus 1× antibiotic-antimycotic mixture. Cells were incubated at 37 °C in 5% CO2. Fibroblasts were used from passage one (P1) to two (P2).Primary rat neonatal cardiac fibroblasts were isolated from 1-day-old Sprague–Dawley rat pups (30 pups/preparation). Atrium was removed from the hearts and minced to pass through a 10 mL pipette tip. The heart tissue was digested with collagenase II and pancreatin in 1× ADS buffer , and 1 μg of RNA was used for the reverse transcription reaction with TOPscript RT DryMIX . mRNA levels were quantified with the SYBR Green PCR kit (Enzynomics). The PCR primers used in this study are shown in Table 3VO4, and 5 mM NaF) containing a protease inhibitor cocktail . Proteins were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in TBST buffer  for 1 h at 25 °C. The membranes were incubated overnight at 4 °C with primary antibodies against collagen type I, fibronectin, CTGF, SMA, ANP, BNP, and GAPDH. They were then incubated with the anti-rabbit or anti-mouse horseradish-peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were visualized using Immobilon Western detection reagents (EMD Millipore). The Bio-ID software was used to quantify protein expression .Total protein from heart tissues was extracted using a lysis buffer  for 3 h and then with gallic acid (100 μM) for further 9 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, blocked with 3% normal goat serum, and incubated overnight with anti-collagen type I antibody  at 4 °C. They were then probed with goat anti-rabbit IgG secondary antibody, Alexa Fluor 568 conjugate . DAPI  was used for nuclear staining. The cells were then observed under a fluorescence microscope . To compare the fluorescence intensity of collagen stain between the groups, the normalized mean intensity was calculated by an automated “measure tool” using the NIS Elements AR 3.0 program .Immunocytochemistry was performed as described previouslypost hoc test using GraphPad Prism version 5, and a value of P < 0.05 was considered statistically significant.All data are expressed as means ± standard errors (SE). Differences between data were analyzed by one-way analysis of variance (ANOVA) with the Bonferroni To investigate whether gallic acid can ameliorate heart failure, we evaluated cardiac function by echocardiography in mice in the TAC group. We evaluated heart function every 2 weeks and confirmed reduction of FS and increased LV lumen diameter at 8 weeks after the TAC operation  and western blotting. ANP, BNP, and skeletal α-actin are markers of cardiac hypertrophy and heart failuretin Fig.  mRNA levtin Fig. . Treatme21. We observed that β-MHC mRNA levels were significantly increased in TAC hearts compared to sham hearts  is another marker for heart failure. Alpha myosin heavy chain (α-MHC) changes to β-MHC in response to cardiac pathological conditions2, epicardial vessels were statistically larger in the hearts of mice in the TAC group than in those in the sham group  registry showed that an increase in LVESD is associated with cardiovascular death24. Our valuable finding is, to the best of our knowledge, the first evidence that administration of gallic acid ameliorates heart failure and cardiac fibrosis. Similarly, Ramezani-Aliakbari et al. reported that gallic acid improved left ventricular dysfunction in a rat model of alloxan-induced diabetes mellitus25. Advanced glycation end products (AGEs) are implicated in cardiovascular diseases in diabetics. AGE-induced cardiac fibrosis and remodeling was prevented by gallic acid administration26. Furthermore, pretreatment with gallic acid improved doxorubicin-induced electrocardiographic abnormalities and cardiac damage27. Antioxidant effects of gallic acid mitigated diazinone-induced cardiovascular dysfunction in a rat animal model28 and cyclophosphamide-induced cardiorenal dysfunction29. Gallic acid was reported to have cardioprotective effects following exposure to various substances such as aluminum oxide30, lindane31, and isoproterenol32.The present study demonstrates that gallic acid improves cardiac dysfunction and fibrosis in a mouse model of pressure overload-induced heart failure and in primary rat cardiac fibroblasts. In this study, heart failure and fibrosis manifested within 10 weeks of TAC in mice. The lumen of the hearts of mice in the TAC group was significantly enlarged, leading to a decrease in FS, indicating cardiac dysfunction. Heart failure is highly fatal owing to the associated high mortality, rendering its treatment important. Clinical drugs, including ARBs, beta-blockers, and diuretics did not improve cardiac dysfunction and fibrosis in mice in the TAC group. Interestingly, gallic acid improved cardiac dysfunction, as determined by echocardiography. We were unable to explain the exact mechanism by which gallic acid reduced the increased left ventricular diameter in the hearts of mice in the TAC group. Left ventricular diameter is much more important than left ventricular mass with regard to cardiac mortality, and it is useful to assess the risk of sudden cardiac death17. In the present study, gallic acid significantly reduced the levels of heart failure markers including ANP, BNP, skeletal α-actin, and β-MHC in the hearts of mice in the TAC group. In contrast, losartan, carvedilol, or furosemide did not decrease the mRNA and protein expression of ANP, BNP, and skeletal α-actin. In our previous study, we observed that gallic acid prevents isoproterenol-induced cardiac hypertrophy and fibrosis14. In addition, gallic acid reduced left ventricular hypertrophy in rats with spontaneous hypertension16, and it diminished left ventricular remodeling in NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertension15. These observations imply that gallic acid regulates cardiac remodeling. In our experimental conditions, losartan and carvedilol were not effective for cardiac dysfunction, which may be attributed to the low doses of losartan (3 mg/kg/day) and carvedilol (1 mg/kg/day). For example, Wang et al. reported that losartan (13.4 mg/kg/day) attenuated the development of cardiac hypertrophy and heart failure in TAC for 4 weeks33. This study used higher-dose losartan than that used in our study. In addition, losartan was considered to have a therapeutic effect on the heart because the drug administration period of this study was twice as long as that of ours. Hampton et al. reported that a high dose of carvedilol (30 mg/kg/day) improved cardiac performance34.Recently, we reported that gallic acid attenuates pulmonary fibrosis in a mouse model of TAC-induced heart failure2) in the hearts of mice in the TAC group may be attributed to the supply of nutrition and oxygen through blood to the heart muscle. These hearts also had many small vessels.Cardiac fibrosis and excessive accumulation of ECM proteins are the major hallmarks of pathological cardiac remodeling in heart failure, and cardiac fibrosis therapy may be involved in the recovery of cardiac dysfunction. In the present study, treatment with gallic acid reduced cardiac fibrosis in a mouse model of pressure overload-induced heart failure. Gallic acid reduced the deposition of collagen in the perivascular regions in the heart tissues of mice in the TAC group. Our TAC model had no clear interstitial fibrosis. Mice with chronic heart failure induced by 10 weeks of TAC had larger vessels than those of sham mice, as determined by Trichrome II Blue staining and cardiac vessel area. The presence of many large vessels (>8,000 μm5. Cardiac fibroblasts get converted to activated cardiac myofibroblasts in response to pathological stress. SMA is a marker of activated myofibroblasts. In our study, SMA mRNA and protein levels were significantly increased in vivo and in vitro. We clearly demonstrated that treatment with gallic acid attenuates fibrosis marker genes in TGF-β1-treated rat cardiac fibroblasts. ICC showed a definitely reduced expression of collagen type I. Our previous report demonstrated that gallic acid suppresses cardiac fibrosis in a hypertension model of L-NAME through downregulation of histone deacetylase 215. In addition, gallic acid reduced cardiac fibrosis in response to isoproterenol through downregulation of phospho-Smad3 and its binding activity to collagen promoter14. In the current study, the regulatory mechanism by which gallic acid reduces fibrosis may be attributed to the reduction of Smad3 protein levels induced by TAC. This result is consistent with a previous report that the protein level of Smad3 was increased at week 8 after TAC35.Treatment with gallic acid significantly reduced the mRNA and protein expressions of collagen type I and CTGF in the hearts of mice in the TAC group. In contrast, administration of losartan, carvedilol, or furosemide did not have any beneficial effect on the development of fibrosis, which may be attributable to the use of low-dose losartan, carvedilol, and furosemide in the TAC model. Among MMPs, gallic acid decreased MMP2 mRNA level in the hearts of mice in the TAC group. In addition, treatment with gallic acid significantly reduced p8 mRNA levels in response to TAC. Cardiac fibroblasts play an important role in the process of fibrosis, and they are responsible for homeostasis of the ECM37, cancer40, cardiac hypertrophy and fibrosis14, pulmonary fibrosis17, vascular calcification13, and hypertension16. In the present study, we added a novel therapeutic effect of gallic acid for heart failure.Thus far, gallic acid has been reported to have pleiotropic beneficial effects on diabetesin vivo and in vitro. Thus, we suggest that gallic acid could be a novel therapeutic agent for treatment of heart failure with fibrosis.In summary, we demonstrated that administration of gallic acid improves cardiac dysfunction and fibrosis in a mouse model of pressure overload-induced heart failure. Gallic acid showed better efficacy against cardiac dysfunction and fibrosis than losartan, carvedilol, and furosemide did, and reduced the risk of heart failure and fibrosis Supplementary Figures


Fatty liver is associated with obesity, type 2 diabetes, hyperlipidemia, hypertension, and metabolic syndrome. While there are no approved drugs for the treatment of nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis, strategies to ameliorate fatty liver often target these related diseases. We sought to determine if any medications approved by the US Food and Drug Administration to treat diabetes are helpful in reducing weight and improving steatohepatitis in patients with NAFLD.We conducted a systematic review of published and unpublished studies evaluating the comparative effectiveness and harms of diabetes medications for the treatment of NAFLD. We searched MEDLINE, EMBASE, Cochrane Database of Systematic Reviews, and the Cochrane Central Register of Controlled Trials through 3rd quarter, 2019 using terms for included drugs and indications.We screened 1591 citations and included 18 trials of diabetes drugs to treat NAFLD. Studies of metformin found no difference from placebo in steatosis, fibrosis, NAFLD activity score, or resolution of NASH. While weight and glucose control were improved with metformin, it did not substantially impact liver disease. Studies of pioglitazone in NASH patients found benefits in liver function, liver fat, and NASH resolution, though significant increases in weight may be cause for concern. Evidence for other thiazolinediones was more limited and had somewhat mixed results, but findings were generally consistent with those for pioglitazone: liver fat and function and glucose measures improved, but weight also increased. We found some evidence that liraglutide improves liver fat, liver function, and HbA1c and is effective at resolving NASH and reducing weight. Exenatide performed less well but also resulted in significant reductions in liver fat and weight.Consistent with existing clinical practice guidelines, which recommend lifestyle intervention and treatment for comorbidities related to fatty liver disease as first-line treatment, trial evidence supports the efficacy of some diabetes drugs  in patients with NAFLD or NASH, though weight gain with some diabetes drugs may warrant caution. Larger trials are needed to better characterize the efficacy and harms of diabetes pharmacotherapy in these patients. Nonalcoholic fatty liver disease (NAFLD), the accumulation of excess fat in the liver (steatosis) not resulting from excessive alcohol consumption or another secondary cause, is a growing public health issue associated with the global epidemics of obesity and type 2 diabetes . NAFLD rFatty liver is associated with obesity, type 2 diabetes, hyperlipidemia, hypertension, and metabolic syndrome. While there are no approved drugs for the treatment of NAFLD or NASH, strategies to ameliorate fatty liver often target these related diseases .We performed a systematic review to determine if any medications approved by the US Food and Drug Administration (FDA) to treat diabetes are helpful in reducing weight and improving steatohepatitis in patients with NAFLD in the setting of many new drugs in development seeking an indication for NAFLD or NASH. The original review, which was commissioned by the Drug Effectiveness Review Project and is not publicly available, also evaluated the success of weight loss drugs, dyslipidemia drugs, diet, and exercise in weight loss and improvement of NAFLD. Here, we focus on the evidence for diabetes medications, which was the most well-studied intervention area.What is the comparative efficacy and effectiveness of FDA-approved drugs that are used off-label to treat nonalcoholic fatty liver disease?What are the comparative harms of FDA-approved drugs that are used off-label to treat nonalcoholic fatty liver disease?We sought evidence to answer the following questions:We followed systematic review methodology and procedures developed specifically for the Drug Effectiveness Review Project (DERP)  and thatWe searched MEDLINE, EMBASE, Cochrane Database of Systematic Reviews, and the Cochrane Central Register of Controlled Trials through 3rd quarter, 2019, using terms for included drugs and indications  who received an FDA-approved diabetes drug to treat NASH/NAFLD. We excluded any drug developed specifically for the treatment of NAFLD, including the FDA-approved obeticholic acid as well as drugs in development specifically for treatment of NALFD , as well as studies that only evaluated different doses of the same drug (dose-ranging studies). Benefit outcomes of interest included changes in alanine aminotransferase test (ALT), aspartate aminotransferase test (AST), liver fat, liver fibrosis, and resolution of NAFLD; weight loss ; long-term health outcomes ; and HbA1c or other glucose outcomes. Relevant harms included serious adverse events and withdrawals due to adverse events. We required studies to have at least 30 patients per treatment arms of interest unless the trial included liver histology, the gold standard, at the conclusion of the trial; in which case, we reduced the required sample size to 20 per treatment arm.One reviewer screened citations and a second reviewer assessed excluded citations. Two reviewers independently evaluated full-text articles by applying the inclusion criteria and resolved disagreements by consensus.Information on population characteristics, interventions, subject enrollment and discontinuation, and results for effectiveness and harm outcomes were abstract by one reviewer. The second reviewer verified abstracted data.Study quality was assessed independently by two reviewers according to the DERP’s methods , focusinWe graded strength of evidence according to the Agency for Healthcare Research and Quality’s (AHRQ) guidance for the Evidence-based Practice Center Program . SimilarN < 100 per treatment arm) and rated fair quality, primarily due to unclear blinding, unclear allocation concealment, and high attrition. Five trials were rated good quality [We screened 1591 citations and included 39 trials (in 41 publications) in the primary report; here, we report only the evidence pertaining to diabetes drugs    quality , 22. SomTwelve individual randomized trials in NAFLD patients with , 8 and wN = 3258) [N = 132) enrolled NAFLD patients with type 2 diabetes. All RCTs in the IPD meta-analysis were 26-week trials and treated patients with liraglutide 0.6 mg, liraglutide 1.2 mg, liraglutide 1.8 mg, or placebo. The IPD meta-analysis found that in patients with an elevated ALT (N = 1387), liraglutide 1.8 mg reduced ALT to a significantly greater degree than placebo, but the effect was lost after correcting for patient weight loss (P = 0.21) or after correcting for improvement in HbA1c (P = 0.63). Findings were similar for decrease in hepatic fat. In a sub-study (n = 149), there was a trend for reduction in liver fat (measured with CT scanning) with liraglutide 1.8 mg (P = 0.07), but the effect was lost after adjusting for weight loss (P = 0.90) or HbA1c (P = 0.73). While the incidence of serious adverse events was similar between liraglutide and placebo , study withdrawal due to adverse events and gastrointestinal disorder was significantly more likely in patients treated with liraglutide .An IPD meta-analysis of 6 RCTs  = 3258)  and 2 ad = 3258) , 8 (N =   and 2 aN = 132) conducted in China enrolled patients with NAFLD and diabetes and reported efficacy outcomes only [P < 0.01) [P = 0.03, our analysis). Body weight was also significantly reduced with exenatide compared with intensive insulin  but there was no differential treatment effect on HbA1c .A trial of exenatide (mes only . Twelve  < 0.01) . At the P < 0.01) [A small RCT conducted in China found no difference between sitagliptin 50 to 100 mg compared with diet and exercise on the liver function tests AST and ALT after 1 year of treatment, although sitagliptin treatment was associated with greater reduction in HbA1c  .P = 0.009); however, treatment with pioglitazone was also associated with significant gain in weight .Two RCTs enrolled patients with histologically confirmed NASH and prediabetes or diabetes as determined by an abnormal glucose tolerance test , 19. In P = 0.008), AST , and ALT . The proportion of participants who experienced improvement in hepatic fat was greater in the pioglitazone group , while there were no differences between treatments in fibrosis score. Pioglitazone treatment was also associated with weight gain compared with placebo . Study withdrawals due to adverse events were few and not different between groups.In the second trial, 55 patients were counseled to follow a hypocaloric diet and then randomized to treatment with pioglitazone 45 mg or placebo for 6 months . PioglitP = 0.04) but not significantly different between groups in other baseline characteristics. The trial reported decreased BMI from baseline for 12 months for metformin  and for rosiglitazone plus metformin . However, we believe that the values for rosiglitazone plus metformin to be in error as the correlation between baseline and 12 months would have to be greater than 0.96, which is extremely high and not likely correct. Postprandial glucose was decreased in all groups. Post-treatment liver biopsy was performed on 55% of patients and NAFLD activity score favored treatment with rosiglitazone (P = 0.01) and combination therapy (P = 0.03). Three individuals left the study, and two due to adverse events (one patient could not tolerate metformin and one stopped treatment due to hypertriglyceridemia).One trial randomized 64 patients with NAFLD to treatment with rosiglitazone 4 mg/day, metformin 1700 mg/day, or combination therapy for 12 months . All patNine trials enrolled NAFLD patients without diabetes and included metformin , 22, 23,N = 52) of liraglutide compared with placebo conducted in the UK in adults with NASH  allowed enrollment of patients with diabetes if the diabetes was well-controlled and stable (33% had diabetes) [P = 0.24), fibrosis score , ALT , or AST . However, significantly more patients on placebo had worsening fibrosis . There was greater weight loss with liraglutide  and greater improvement in HbA1c . There were no differences between treatments in study withdrawals due to adverse events, risk of serious adverse events, or risk of gastrointestinal disorders.A small trial (iabetes) . More paN = 48) conducted in Norway randomized patients with NAFLD to treatment with metformin 2500 mg (3000 mg if body weight > 90 kg) or placebo for 6 months [P < 0.001) as was reduction in HbA1c  [One small trial (6 months . Twenty-= 0.001) .n = 30), there was no difference among patients receiving metformin (n = 30) in change in BMI , though ALT (− 10.1 vs. − 0.6 IU/L) and AST (− 6.4 vs. − 0.9 IU/L) were significantly reduced among those taking metformin. No adverse events were reported.A second good-quality trial assessed 3 months of metformin (500 mg/day) use among Iranian adults with probable NAFLD by liver sonography . CompareP = 0.04), steatosis , NAFLD score , and resolution of NASH . Liver function tests, fasting serum glucose, and insulin resistance were also significantly improved with pioglitazone versus placebo. However, weight gain was increased with pioglitazone compared with placebo . There was no difference between groups in change in liver fibrosis. Twelve patients experienced serious adverse events, with fewer events in the pioglitazone group .Three trials randomized patients without diabetes to pioglitazone or placebo , 13, 22.P = 0.05). Pioglitazone was also associated with greater improvement in HbA1c compared with placebo , as well as greater improvement in ALT . However, as above, pioglitazone treatment was associated with increase in body weight versus placebo . Withdrawals due to adverse events (18%) were similar between groups.In the second RCT, 74 patients with NASH were randomized to 30 mg of pioglitazone or placebo for 12 months [n = 30 in each group) among Iranian adults with probable NAFLD by liver sonography [2; P = 0.46), ALT  and AST  were significantly reduced among those receiving pioglitazone. No adverse events were reported.A third good-quality trial compared pioglitazone (15 mg/day) with placebo (nography . While BP < 0.001). Change in BMI favored metformin and rosuvastatin over pioglitazone , whereas change in AST favored pioglitazone and metformin over rosuvastatin . Adverse events were not reported.In a randomized trial conducted in India, 98 patients were allocated to metformin, rosuvastatin, or pioglitazone . At 24 wA second trial that randomized 80 NAFLD patients to treatment with metformin 1000 mg or pioglitazone 30 mg for 4 months was conducted in Iran . Eight pN = 60) conducted in India compared 6-month treatment with pioglitazone 30 mg to treatment with pentoxifylline 1200 mg in patients with biopsy-proven NASH [P = 0.003, both comparisons) but were not different from improvements with pentoxifylline. Improvements in liver fat and fasting blood sugar were also improved from baseline with pioglitazone  but were also not significantly different from improvement with pentoxifylline. Although patients gained weight with pioglitazone, the increase (2 kg) was not statistically significant (P = 0.31). No participant left the study due to adverse events.One randomized trial (ven NASH . All patP = 0.01), along with increased normalization of transaminases  but an increase in weight . There were no differences between groups in NAFLD Activity Score or HbA1c levels between treatments, although surrogate markers of insulin sensitivity were improved with rosiglitazone. Three patients treated with rosiglitazone experienced painful, swollen legs requiring dose adjustment, or discontinuation of treatment.One RCT randomized 63 patients with NASH to rosiglitazone (4 mg/day for 1 month then 8 mg/day for 11 months) or placebo for 12 months  and founP = 0.014). Liver fat and fibrosis stages were similar between groups. Post-treatment liver biopsies showed no differences between groups on changes in NAFLD score, steatosis, fibrosis, or resolution of NASH (46% of 26 patients treated with rosiglitazone versus 36% of 28 patients treated with rosiglitazone and metformin). Liver function tests were improved in all treatment groups as was fasting serum glucose and insulin levels. The addition of metformin did not significantly help with weight gain versus treatment with rosiglitazone alone . Twelve patients, at the recommendation of their physicians, terminated the study due to 12 different adverse events, some likely unrelated to treatment .One RCT randomized patients with NASH to 4 mg of rosiglitazone or to 4 mg of rosiglitazone plus 1000 mg of metformin or to 4 mg of rosiglitazone plus 50 mg of losartan for 48 weeks . SeventeManagement of NAFLD involves treating the liver disease itself as well as associated metabolic comorbidities, including diabetes, obesity, and hyperlipidemia. A recent clinical practice guideline from the American Association for the Study of Liver Diseases (AASLD)  recommenConsistent with the AASLD recommendation against metformin use in NAFLD patients, studies of metformin found no difference from placebo in steatosis, fibrosis, NAFLD activity score, or resolution of NASH. While weight and glucose control were improved with metformin, treatment with metformin did not substantially impact liver disease.Studies of pioglitazone in NASH patients found benefits in liver function, liver fat, and NASH resolution, though significant increases in weight may be cause for concern. A recent systematic review and network meta-analysis by Sridharan et al. was consistent with our findings : pioglitWhile the AASLD guidelines recommend against using GLP-1 agonists in NASH patients, we found some evidence that liraglutide improves liver fat, liver function, and HbA1c and is effective at resolving NASH and reducing weight. Exenatide performed less well but also resulted in significant reductions in liver fat and weight.The strengths of our study include the use of systematic review processes to identify all relevant studies that meet pre-defined inclusion criteria, assessment of the internal validity  of included studies, and overall evaluation of the strength of evidence using an established approach. Limitations of the present review include restriction to English-language publications and restriction to randomized trials, which may have limited generalizability to real-world populations. Larger studies with longer follow-up are needed to better quantify the long-term benefits and harms of diabetes medications to treat NAFLD, since the disease state itself, as well as many of the common metabolic conditions associated with NAFLD, are chronic conditions with long natural histories. Additionally, longer and larger studies may provide further information on clinical health outcomes and uncommon adverse effects.Consistent with existing clinical practice guidelines, which recommend lifestyle intervention and treatment for comorbidities related to fatty liver disease as first-line treatment, trial evidence supports the efficacy of some diabetes drugs  in patients with NAFLD or NASH, though weight gain with some diabetes drugs may warrant caution. Larger trials are needed to better characterize the efficacy and harms of diabetes pharmacotherapy in these patients.Additional file 1. Search strategies.