Murraya koenigii L. Spreng, cloves of Allium sativum L., fruits of Garcinia quaesita Pierre, and seeds of Piper nigrum L. is a popular drug which has been used by indigenous practitioners in Sri Lanka for the treatment of diabetes mellitus and dyslipidemia. The acute toxicity assessment was conducted, following a single oral dose of 0.25–2.0 g/kg in healthy rats, and rats were observed up to 14 days. The hot water extract (1.0 g/kg) and the water : acetone extract  were administered to Wistar rats for 28 days in the subchronic study. Hypoglycemic and antihyperglycemic activities (dose response studies) of cold water, hot water, and water : acetone extracts of the polyherbal mixture were evaluated at the doses of 0.5, 1.0, and 1.5 g/kg in healthy and streptozotocin-induced diabetic rats , respectively. Acute toxicity study showed that the polyherbal drug did not cause any change in animals throughout the experimental period of 14 days. The administration of the hot water extract and the water : acetone extract of the polyherbal drug for 28 days did not produce changes in the selected biochemical and hematological parameters in Wistar rats (p > 0.05). The histological assessment corroborated the biochemical findings with no significant treatment-related changes in the kidney and liver. The treatment of polyherbal drug significantly lowered the serum glucose concentration compared to the diabetic control rats (p < 0.05) while it did not lead to a severe reduction of glucose concentration in healthy rats. The hot water and water : acetone extracts of the polyherbal drug showed a statistically significant improvement on total area under the glucose tolerance curve in diabetic rats (p < 0.05), reflecting dose-dependent antihyperglycemic effects of the drug. Based on the results, we conclude that the aforementioned antidiabetic polyherbal remedy is free of toxic/adverse effects at the equivalent human therapeutic dose in healthy Wistar rats and would be a safe therapeutic agent for long-term treatments.A polyherbal drug composed of leaves of  Murraya koenigii L. Spreng (Family: Rutaceae), cloves of Allium sativum L (Family: Amaryllidaceae), dried fruit rinds of Garcinia quaesita Pierre (Family: Clusiaceae), and seeds of Piper nigrum L. (Family: Piperaceae) has been prescribed by many indigenous practitioners in Sri Lanka in the treatment of diabetes mellitus and dyslipidemia [M. koenigii, cloves of A. sativum, P. nigrum, and their isolated compounds [G. quaesita, plants of this genus such as Garcinia pedunculata, Garcinia indica, and Garcinia xanthochymus have been reported for their significant antidiabetic activities in vivo [in vivo; however, it needs to be balanced with the safety particularly when the drug is being properly recommended for long-term treatments.Different types of noncommunicable diseases such as diabetes mellitus have been treated with many traditional medicines which are derived from medicinal plants . Howeveripidemia , 3. In aompounds –9. Altho in vivo –12. The A. sativum extract where the low doses of A. sativum extract (250 mg/kg body weight/day) exerted no deleterious effects on the organs of the Wistar rats while high doses (>400 mg/kg body weight/day) led to toxicity changes on selected body tissues such as the liver, kidney, and heart. Thus, Fowotade and the team highlighted the potential ability of A. sativum extract to induce morphological changes in the liver, kidney, and heart of humans upon the consumption of high doses of garlic for medicinal purposes [Despite the presence of therapeutically beneficial secondary metabolites, a number of toxic phytochemical constituents are found in plants. Chronic use of these plant-based therapeutic agents may lead to chronic adverse/toxic effects mainly renal and hepatic toxicities in humans . Emerginpurposes .Further, the clinical significance, therapeutic utility, and successful commercialization of any herbal drug would depend not only on its therapeutic efficacy but also on its lack of toxicity . TherefoWe herein report the acute and subchronic toxic effects of a polyherbal drug in healthy Wistar rats and acute hypoglycemic and antihyperglycemic effects of the drug in normoglycemic and streptozotocin-induced diabetic rats, respectively. This is the first report on the toxicity profile of the aforementioned antidiabetic drug at a preclinical level.Streptozotocin (STZ) was purchased from MP Biomedicals, France. Acetone (Pubchem CID: 180), citric acid, and sodium citrate were of analytical grade  and used without any further purification. The biochemical parameters were estimated using UV-Visible spectrophotometer . A tissue processor  and a light microscope  were used for the preparation of H&E stained tissues and histopathology assessments, respectively. A microtiter plate reader  was used to measure the absorbance at 450 nm for ELISA measurements. An automated hematological analyzer  was used in reading hematological parameters.M. koenigii, cloves of A. sativum, dried fruit rinds of G. quaesita, and dried seeds of P. nigrum were collected from the Central Province, Sri Lanka, in March 2017. The plant parts were taxonomically authenticated by comparing specimens at the National Herbarium, National Botanical Garden, Peradeniya, Sri Lanka. The voucher specimens (No: 6/01/H/03) were deposited at National Herbarium, Royal Botanical Garden, Peradeniya, Sri Lanka.The fresh leaves of The polyherbal drug was prepared by grinding an equal amount of dried plant materials (100 g each) and extracted into cold water and water : acetone (1 : 1) separately in an electrical shaker at room temperature . Hot water extract of the drug was prepared by refluxing 100 g of plant mixture (25 g of each plant) in 500 mL water at 100°C for three hours. Solvents were evaporated using a rotary evaporator at 40°C. Extracts were further dried by freeze drying to obtain a fine powder of each extract of the drug and it was stored at 0°C until further use.ad libitum [Adult male Wistar rats  purchased from Medical Research Institute (MRI), Colombo, Sri Lanka, were used in all experiments. The animals were housed in the Animal Research Facility Centre, Faculty of Medicine, University of Ruhuna, Sri Lanka, at approximate 12 h/12 h of light and dark cycle, temperature at 25°C, and at relative humidity of 65 mmHg. Pelleted food and free water were given  libitum . Adult mn = 6/group) and received distilled water. The rats of the group two and three were further divided into five subgroups for graded doses of hot water and water : acetone extract of the polyherbal drug at 0.25, 0.5, 1.0, 1.5, and 2.0 g/kg (n = 6/subgroup). After a single oral administration of the drugs, rats were observed for 24 hours and once daily for a period of 14 days. The rats were weighed and observed for mortality, salivation, diarrhea, asthenia, hypoactivity, hyperactivity, piloerection, hyperventilation, aggressiveness, yellowing or loss of hair and skin fur, drowsiness, convulsions, tremors, and dizziness.The hot water and water : acetone extract were selected for the acute and subchronic toxicity study as those extracts showed potent glucose lowering effect than the cold water extract. The acute toxicity assessment was performed according to the Organization of Economic Cooperation and Development (OECD) guideline 420 for testing of chemicals . Healthyn = 6/group). The first group served as the untreated healthy control group and received distilled water daily. The Wistar rats in the second group (n = 6/group) received the hot water extract of the polyherbal drug at 1.0 g/kg . The third group of rats was in three subgroups for three doses selected (n = 6/subgroup), and they received the water : acetone extract of the polyherbal drug at doses 0.5, 1.0, and 1.5 g/kg. The oral administration of the polyherbal drug was continued for a period of 28 consecutive days. Fasted rats (12 h) were euthanized on the 28th day with an overdose of aesthetic ether. Subsequently, blood was collected by cardiac puncture and serum was separated for the estimation of biochemical parameters. Kidney and liver tissues were excised for the preparation of H&E stained sections.Wistar rats were randomly divided into three main groups (The cold water extract was not used for the toxicity studies as it has not shown potent glucose lowering effect compared to the hot water and water : acetone extract.(1) Estimation of Biochemical and Hematological Parameters in Healthy Rats. Serum concentrations of alanine aminotransferase  [2.6.1.2) , asparta2.6.1.2) , alkalin2.6.1.2) , total p2.6.1.2) , urea [22.6.1.2) , and cre2.6.1.2)  were est2.6.1.2) , total c2.6.1.2) , high-de2.6.1.2)  were als2.6.1.2) . Hematol(2) Histopathological Evaluation of the Liver and Kidney Tissues. Liver and kidney tissues of all euthanized rats were excised and preserved in 10% buffered formalin. Small slices of the organs were cut by a microtome, stained with hematoxylin-eosin, and examined under a light microscope. Photomicrographs of the liver and kidney tissues of the H&E stained sections were recorded.(3) Measurement of Relative Organ Weight. The heart, lung, small intestine, liver, spleen, pancreas, stomach, and kidney were excised for the assessment of the relative weight of organs. The relative organ weight (ROW) of the heart, lung, small intestine, liver, spleen, pancreas, stomach, and kidney of each animal was calculated by the weight of the organ divided by the bodyweight of the animal on the day they were euthanized.(4) Measurements of Consumption of Food and Intake of Water. The consumption of food and intake of water were measured daily from the quantity of food and water supplied and the amount remaining after 24 hours. The body weight of each rat in all groups was measured during the experimental period at weekly intervals and on the day of sacrifice.n = 6/group) served as a healthy untreated group and received distilled water. Cold water, hot water, and water : acetone extracts of the polyherbal drug were administered orally to healthy male Wistar rats in groups two, three, and four, respectively. Each test group except the healthy control group (group one) was subdivided into three and received different doses of the polyherbal drug at 0.5 g/kg (n = 6/subgroup), 1.0 g/kg (n = 6/subgroup), and 1.5 g/kg (n = 6/subgroup) as a single dose. Oral glucose tolerance test (OGTT) was performed in all groups as mentioned below.Fasted (12 hours) healthy male Wistar rats were randomly divided into four groups based on their body weight. Group one (Glucose (3.00 g/kg) was orally administered to all experimental rats 30 minutes after the administration of polyherbal drug, and blood samples were collected from the tail tip at first, second, third, and fourth hours after the administration of glucose solution. The fasting serum glucose concentration was estimated using a spectrophotometric enzyme assay kit . After sDiabetes was induced in Wistar rats with an intraperitoneal injection of streptozotocin (STZ), at a dose of 70 mg/kg, dissolved in 0.1 M citric acid/sodium citrate buffer, pH 4.5 . After 7n = 6/group) served as the healthy untreated group and group two (n = 6/group) served as the diabetic untreated control group which received distilled water. Cold water, hot water, and water : acetone extracts of polyherbal drug were administered orally to diabetic rats in groups three, four, and five, respectively. Each group was subdivided into three and received different doses at 0.5 g/kg (n = 6/subgroup), 1.0 g/kg (n = 6/subgroup), and 1.5 g/kg (n = 6/subgroup). Glibenclamide powder (0.5 mg/kg dose) was dissolved in warm distilled water (37°C) and administered to diabetic rats in the sixth group (n = 6/group) which served as the positive control. The oral glucose tolerance test (OGTT) was performed in all groups at 30 minutes after single dose administration of polyherbal drug as described previously.Wistar rats were randomly divided into six groups. Group one , which suggests that the polyherbal drug might not have hepatotoxic effects and may preserve normal hepatocellular functions at the selected doses (p < 0.05). These results indicated that regular kidney functions were maintained normally upon the administration of both extracts in healthy Wistar rats.The biochemical, hematological, and histopathological assessments were conducted in evaluating the subchronic toxicity effects of the polyherbal drug. There were no significant alterations in ALT, AST, and ALP enzymes for both hot water and water : acetone extract treated rats and healthy control rats (ed doses . The hotp < 0.05). The water : acetone extract of the polyherbal drug significantly reduced the TC, LDL-C, TG, and VLDL-C by 22.3%, 20.9%, 9.2%, and 26.8%, respectively, whereas the serum HDL-C concentration was increased by 16.3% at the therapeutic dose. The results emphasize the modest activity against dyslipidemia in healthy rats, and we postulate that the potency of correcting abnormal lipid alterations would be better in an animal model with dyslipidemia rather than in healthy Wistar rats. The polyherbal drug produced a significant reduction in serum glucose concentration in hot water and water : acetone extracts treated rats compared to healthy control rats by 10.4% and 14.5%, respectively (p < 0.05).Abnormalities in the serum concentration of lipid parameters offer descriptive information on the irregularity of lipid metabolism. The concentration of serum lipid parameters in polyherbal drug treated and healthy control rats is listed in in vivo.The results of hematological parameters in polyherbal drug treated rats are shown in The histopathological assessment is crucial in evaluating toxicity-related changes of herbal drugs and has been considered as the gold standard. The photomicrographs of histopathological changes of the H&E stained kidney and liver sections of the experimental animals are shown in Figures p > 0.05)  . The heap > 0.05), a significant reduction in body weight gain was demonstrated in polyherbal drug treated rats from the second week onwards. However, there is no significant difference (p < 0.05) in consumption of food and intake of water in plant extract treated rats and healthy control rats which suggests that polyherbal drug might not interfere with the overall metabolic processes in healthy rats.The effects of polyherbal drug on body weight gain, consumption of food, and intake of water in experimental rats at weekly intervals are shown in Tables 4p < 0.05). Low total area under the curve reflects high efficacy or improvement on glucose tolerance of the particular drug. The hot water and water: acetone extracts of the polyherbal drug showed a statistically significant improvement on total area under the glucose tolerance curve (TAUC) in streptozotocin-induced diabetic rats at the doses of 1.0 and 1.5 g/kg (p < 0.05). The highest dose (1.5 g/kg) of each group resulted in the maximum antihyperglycemic effect in diabetic rats while water: acetone extract of polyherbal mixture showed higher antihyperglycemic effect than the hot water extract. Further water: acetone extract of polyherbal drug at 1.5 g/kg dose showed potent blood glucose lowering effect which is closer to the antidiabetic effect of positive control, glibenclamide.The results of the area under the glucose tolerance curve values upon the administration of cold water, hot water, and water: acetone extracts of the polyherbal drug to healthy and diabetic rats are shown in Tables in vivo studies. Indeed, absence of toxicity provides confidence for recommending polyherbal remedies in clinical practice if these are being able to prove the efficacy.Diabetes and its associated complications have become devastating health problems at present and millions of people are affected with this dilemma worldwide. Although synthetic drugs are capable of treating diabetes mellitus for a certain extent, popularity of these drugs is being overshadowing by unwanted side effects . TherefoM. koenigii, cloves of A. sativum, dried fruit rinds of G. quaesita, and seeds of P. nigrum in Wistar rats which have been used in the treatment of diabetes mellitus and dyslipidemia.Toxic or adverse effects are influenced by the rate at which the pharmacological agents enter the body and how they interact with various body tissues including vital organs such as the liver and kidney. Studies in whole animals are critical to ensure the proper use of the beneficial medicines as laboratory tools simply cannot duplicate complicated body phenomena . In the 50) of the hot water and water:acetone extracts was assumed to be greater than 2.0 g/kg, suggesting that the polyherbal drug could be generally considered as nontoxic at the human equivalent therapeutic dose. In the subchronic toxicity study, the therapeutic dose of 1.0 g/kg was used for the administration of the hot water extract, while three doses were selected as 0.5, 1.0, and 1.5 g/kg for the most active water: acetone extract, with an aim of evaluating the dose-dependent toxic effects, if any. Indeed, therapeutic dose of drug administration involves measuring drug concentrations to facilitate the clinical interpretation of the results [Toxicity assessment of hot water and water: acetone extracts of the polyherbal drug was conducted in healthy Wistar rats. Male Wistar rats were used as sensitivity of males is greater than that of females in most of the routine renal and liver toxicity parameter measurements . In the  results .The assessment of biochemical parameters reflects possible toxic effects of herbal drugs towards hepatic and renal functions. The assessment of liver and kidney functions is crucial as vital organs in the body have multifarious mechanisms to eliminate toxic substances through the liver and kidney in order to reduce their toxic effects. Even though liver and kidney are remarkable in eliminating toxins, their functions can be damaged during the toxin purgation process . In thisThus, beneficial changes in the serum concentration of glucose, TC, HDL-C, LDL-C, TG, and VLDL-C reported in this study might suggest the hypoglycemic and hypolipidemic potential of the combined plant mixture. Even though fasting serum glucose concentration and lipid parameter values are not involving directly in elucidating toxic effects, these are important in eliciting the antihyperlipidemic efficacy for a certain extent. However, detailed mechanistic approach is warranted to explore detailed antidiabetic cellular mechanisms.Evaluation of hematological parameters is used to determine the extent of the deleterious effect of the tested extract on the blood-related functions in experimental healthy rats. The results of the present study revealed that there were no significant effects on erythropoiesis, morphology, or osmotic fragility of RBC . SimilarThe histology of the liver and kidney tissues are considered as vital tools for illustrating liver and kidney damages by toxic substances . The toxA. sativum can decrease the cell size of white adipose tissue in order to suppress the body weight gain [A. sativum has improved the metabolism in high-fat diet-induced obese mice by regulating the composition of the intestinal microbiota [M. koenigii and its isolated compound, mahanimbine, have been reported with significant reduction of body weight gain [G. quaesita, shows promising effects on reduction of body weight [The body weight changes normally revealed a proper nutritional status of experimental rats. Body weight is an important anthropometric parameter in the assessment of overall nutrition . Previought gain . Allicincrobiota . Antiobeght gain . Furthery weight . TherefoM. koenigii that are responsible for hypoglycemic activity. Among them, mahanimbine, koenidine, and koenimbine have potent hypoglycemic activities which enhance insulin effect in diabetic individuals through increasing the peripheral glucose uptake or secretion by islets of Langerhans of pancreatic beta cells [A. sativum such as S-allylcysteine have allicin which has the potential to reduce the serum glucose concentration comparable to glibenclamide through insulin secretion mechanism [G. quaesita, and it could assist in generation of insulin-producing β cells [P. nigrum has potent antidiabetic activity and has the ability to inhibit the biotransformation process of compounds in the liver and gastrointestinal track during metabolic process [β cells as possible antidiabetic mechanisms based on published reports.Recent studies have shown that carbazole alkaloids are the major phytochemical class present in ta cells , 52. Morechanism , 54. Gar β cells –57. Pipe process , 59. Hen50) of the polyherbal drug was assumed to be > 2.0 g/kg as no mortality was observed in the acute toxicity study. The absence of significant alterations in biochemical parameters, hematological parameters, and relative organ weights and histopathology of H&E stained sections of the liver and kidney tissues of hot water and water: acetone extracts of polyherbal drug confirmed its in vivo safety for the first time. In addition, the results of the glucose tolerance test revealed that the polyherbal drug composed of leaves of M. koenigii (L.), cloves of A. sativum (L), dried fruits of G. quaesita Pierre, and dried seeds of P. nigrum (L) possesses potent antihyperglycemic activity in streptozotocin-induced diabetic rats and did not cause severe hypoglycemia in healthy Wistar rats.Acute toxicity study showed that the polyherbal drug did not cause any change in animals throughout the experimental period of 14 days. The lethal dose (LD


Influenza is a major cause of morbidity and mortality worldwide. Following the 2009 pandemic, there was widened interest in studying influenza burden in all regions. However, since data from the World Health Organization (WHO) Middle East and North Africa (MENA) region remain limited, we aimed to contribute to the understanding of influenza burden in Lebanon.A retrospective chart review extending over a period of 8 seasons from Jan 1st, 2008 till June 30th, 2016 at a tertiary care center in Beirut was performed. All cases confirmed to have influenza based on rapid antigen detection or/and polymerase chain reaction on a respiratory sample were included for analysis. Data on epidemiology, clinical presentation, complications, antiviral use and mortality were collected for analysis.A total of 1829 cases of laboratory-confirmed influenza were identified. Average annual positivity rate was 14% . Both influenza A and B co-circulated in each season with predominance of influenza A. Influenza virus started circulating in December and peaked in January and February. The age group of 19–50 years accounted for the largest proportion of cases (22.5%) followed by the age group of 5–19 years (18%). Pneumonia was the most common complication reported in 33% of cases. Mortality reached 3.8%. The two extremes of age (< 2 years and ≥ 65 years) were associated with a more severe course of disease, hospitalization, intensive care unit (ICU) admission, complications, and mortality rate. Of all the identified cases, 26% were hospitalized. Moderate-to-severe disease was more likely in influenza B cases but no difference in mortality was reported between the two types. Antivirals were prescribed in 68.8% and antibiotics in 41% of cases. There seemed to be an increasing trend in the number of diagnosed and hospitalized cases over the years of the study.Patients with laboratory-confirmed influenza at our center had a high rate of hospitalization and mortality. A population based prospective surveillance study is needed to better estimate the burden of Influenza in Lebanon that would help formulate a policy on influenza control. Acute respiratory infections constitute a major cause of morbidity and mortality globally . AccordiInfluenza is a major cause of both influenza-like illness (ILI) and more importantly severe acute respiratory infection (SARI) across the globe , 5. SeveInfection with influenza may manifest in a wide spectrum of illness levels ranging from asymptomatic infection to death. Different studies reported asymptomatic cases to account for 14% up to 77% of influenza infections –12. The Annual seasonal influenza vaccination can reduce the chances of getting infected and subsequently prevent spread of the virus. Vaccines are available in two forms: the inactivated influenza vaccine (IIV), which is administered intramuscularly or intradermally, and the live attenuated influenza vaccine, administered intranasally, (LAIV). Vaccination is recommended by the Centers for Disease Control, USA, annually to everyone above 6 months of age and should be given before influenza season commences, usually during early fall or late summer . HoweverGiven the epidemic nature of the illness, there has been an increasing interest in the burden resulting from its outbreaks in addition to concerns about its association with myocardial infarctions, glycemic variation in diabetic patients, and the exacerbation of other chronic illnesses , 18–27. In Lebanon, the influenza burden of disease is unknown. Since 2008, surveillance of influenza virus among ILI patients at the American University of Beirut Medical Center (AUBMC), was performed at the Center for Infectious Diseases Research (CIDR) –33. The This is a retrospective review of patients who tested positive for influenza virus at AUBMC, a tertiary care medical center located in Beirut, Lebanon, between January 1, 2008 and June 30, 2016. Samples were collected from these patients and tested for influenza A and B viruses by rapid diagnostic test and/or by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Information already collected for patients diagnosed to have influenza A or B covering all the seasons from 2008 till 2016 who were previously enrolled in three ongoing surveillance studies at the CIDR at AUBMC  using one of the rapid diagnostics test kits or were sent in sterile normal saline to AUBMC laboratory and tested within 24 h. Deep tracheal aspirates were mostly used for testing patients who were intubated.Storage: For those samples obtained from patients participating in the IRB-approved surveillance studies since 2008, a second sample was taken (after obtaining consent) and was transported in virus transport media, stored at 4 °C for no more than 24 h or at -20 °C if taken over the weekend. These samples were ultimately stored at − 80 °C until their transfer to the Department of Public Health at Niigata University, Japan for multiplex polymerase chain reaction and viral culture.Different rapid antigen detection kits were used across the different seasons. These included: Rapid FLU virus test kit Capilia™ Tauns, Japan; Quick-Ex flu A/B rapid influenza test Denka Seiken, Japan; Quick-Ex flu A/B & RSV rapid influenza test Denka Seiken, Japan; and BD Directigen™ EZ Flu A + B . Testing was performed following the manufacturer’s instructions.Polymerase chain reaction: real-time reverse-transcriptase (RT) polymerase chain reaction (PCR) using the GENEXPERT (Cepheid) platform was used at AUBMC Clinical Molecular Biology Laboratory .Only 2 patients were diagnosed by serology testing . This was during the 2009 influenza pandemic.All patients were identified retrospectively through medical records at AUBMC and through the parallel ILI surveillance studies for influenza conducted during the study period. These surveillance studies enrolled patients with symptoms fitting the ILI case definition, presenting as outpatients or inpatients, and tested them for influenza using rapid diagnosis test (RDT) and then RT-PCR. The list of remaining patients was retrieved by the medical coder by looking at the following lab codes: “8108” for rapid influenza A & B antigen detection and “8988” for Flu PCR. This was done through the Department of Pathology and Laboratory Medicine by sending a Statistical Information Request. Data from each chart was collected and recorded on a case report form (CRF). Medical records with the discharge diagnosis of influenza were also retrieved.2 requirement, intensive care admission, mechanical ventilation, complications), radiology findings, targeted laboratory results, samples dates, method of influenza detection, use of antibiotics/antivirals, hospital stay and outcomes.The CRF included information on demographic data , social history, medical history, influenza vaccination history, clinical symptoms, hospital course, severity parameters  fever > 39 degree Celsius (39 °C), and/or (2) physician-verified shortness of breath, pulmonary congestion, pneumonia, bronchiolitis, bronchitis, wheezing, croup, or acute otitis media, and/or (3) physician-diagnosed serious extra-pulmonary complication of influenza, including myositis, encephalitis, seizure, and/or myocarditis .t test. Statistical significance was considered below a type-1 error threshold  of 0.05. Following that a multivariate logistic regression model comprised of all risk factors with unadjusted p-value < 0.2  was constructed and reported.The Statistical Package for Social Sciences (SPSS) program, version 22.0 for Windows was used for data analysis . Simple descriptive statistics was used to describe patients’ demographics, morbidity and mortality. Bivariate and multivariate analyses of risk factors for mortality in cases of laboratory proven influenza in hospitalized patients was analyzed by Pearson’s Chi-square test or Fisher’s exact test (when the number of patients in a subgroup was less than 5). Continuous risk factors were analyzed with student During the study period spanning 8 influenza seasons from January 1st, 2008 till June 30th, 2016, a total of 11,288 patients presented to AUBMC with a respiratory illness for which they were tested for influenza infection. Out of all patients tested, 1829 patients (16%) had a positive test result for influenza; 542 influenza cases (29.6%) were identified during the 2009 influenza pandemic. Of the 1829 positive cases, 1529 (84%) were caused by influenza A, 242 cases (13%) by influenza B, and 55 cases (3%) had co-infection with both types (type was not reported in three cases). During the 2009 pandemic, 97.6% of the infections were caused by influenza A and 2% were co-infected. However, subtyping was done for only 35 cases during the pandemic, of which 28 cases were caused by influenza A (H1N1)pdm09.Of the 1829 cases, 916 (50.1%) cases were female and 72 cases were pregnant; 24% of female patients in the childbearing age (Table The most common presenting symptoms were respiratory (96%) with cough constituting 91.5% of these symptoms. Fever was present in 89% of cases; 78% of febrile patients with data on highest temperature recorded had high-grade fever ≥39 °C. Gastrointestinal symptoms were present in 33.7% including vomiting (47%), diarrhea (32%) and abdominal pain (29.6%). Myalgia was reported in 31% of patients. Other less common symptoms (13.6%) included conjunctivitis, otitis, neurological events, confusion and dizziness . Other reported complications were croup (3 cases), shock (20 cases) of which 20% were below 2 years and 25% were above 65 years of age, sepsis (23 cases) of which 39% were ≥ 65 years, myocardial infarction (4 cases), renal failure , acute respiratory distress syndrome , encephalopathy (6 cases), hemorrhagic pneumonia (1 case), stroke (2 cases), seizures (7 cases) and liver failure .Complications secondary to influenza infection were captured when available. Almost 24% (295/1245) of patients developed at least one complication. Pneumonia, confirmed by the presence of infiltrates or consolidation on radiological examination, was the most common complication . Chest X-ray was done in 233 of these cases and detected the presence of pneumonia in 218 cases (90.4%); the remaining 15 patients (6.6%) had a clear chest X-ray but had abnormal findings on computed tomography (CT). In eight patients, only a CT was done initially and detected the presence of pneumonia. However, the treating physician did not request an imaging study in 1095 subjects (60%). When imaging was requested, pneumonia was detected in 32.6% of cases (241/739). The highest rate of pneumonia cases was in the older age groups: ≥65 years old (27.4%) and 50–65 years (19.1%)  between different age groups. Almost half of the patients below 5 years (50.5%) and above 50 years (51.6%) had moderate to severe disease while around a third (28%) of those 10 to 50 years did .Moderate to severe influenza was encountered in 591 cases 42%) with a significant % with a Influenza detection was done using RDT or RT-PCR except for 2 cases where diagnosis was made by serology because the clinical picture was highly suggestive of influenza infection despite negative RDT and PCR. These 2 cases had an Influenza A IgG dilution titers of 1:5120 . Most of the cases were diagnosed by RDT  while 144 cases (7.9%) were detected by PCR. In 137 cases (7.5%), both RDT and PCR were used at the same time for detection. In these cases, PCR was likely requested for subtyping. Subtyping was done for 245 cases only, of which 138 cases were H1N1pdm09, 7 cases were H1N1 Brisbane, 61 cases were H3N2 and 3 cases were co-infected with H1N1pdm09 and H3N2.As for influenza B cases, 20 were Victoria lineage, 14 were Yamagata and 2 were Shanghai lineage.P-value = 1).Characteristics of patients with laboratory-confirmed influenza by virus type are illustrated in Table . There wFigure N = 1787) required hospital admission for management. Table P-value = 0.788). In total, 70 (4.0%) patients required ICU admission for management of influenza-related complications. The highest rates of these admissions were at both age extremes; 13.2% of the elderly ≥65 years and 8.0% of children < 2 years with a significant P-value < 0.001 between the different age groups. However, there was no significant difference in the mean duration of ICU stay for all age groups . Oxygen supplementation was required for 121 patients (7.5%) of which 35 cases ultimately required mechanical ventilation. There was a significant statistical difference among different age groups concerning oxygen supplementation and mechanical ventilation with the highest percentages for oxygen supplementation requirement among the elderly ≥65 years (28.3%) while the highest percentages for mechanical ventilation was among children < 2 years (4.7%).A total of 475 cases  died during their hospital stay. Influenza type A was the detected type in 15 out of the 18 mortality cases, while Influenza type B was detected in the remaining 3 cases. Eleven cases (69%) were older than 50 years and three cases were young adults. Only 4 cases were in the pediatric age group (< 5 years old) with influenza A detected in all four cases. All cases with fatal outcome had at least one comorbid condition except for one case who was one month old. This case was also co-infected with respiratory syncytial virus. Another 7 cases also had laboratory proven viral co-infections , Epstein Barr Virus (EBV) and Herpes Simplex Virus Type-1 (HSV-1); five of whom had an underlying malignancy, one had an underlying neurological disease and one was a healthy pregnant female.P-value was found for moderate to severe disease, radiologically-confirmed pneumonia, and presence of viral or bacterial co-infection. Other statistically significant risk factors included neurological disorder (0.002) and malignancy (0.043). There was no statistically significant difference in mortality among different age groups or types of influenza infection detected. However, when multivariable logistic regression was done, only the presence of a proven viral co-infection, bacterial co-infection and underlying neurological diseases were found to be independent risk factors for increased mortality  with laboratory-confirmed influenza infection. Only 102 (20.6%) of those who received antibiotics had a proven bacterial co-infection. A proven bacterial co-infection was considered in the presence of a positive culture. In fact, pneumonia cases accounted for 49% of antibiotic prescription though not all of these cases were proven to be bacterial.Antibiotics were prescribed for 494 patients (41% of those with available data N = 1193) with laboratory-confirmed influenza. Oseltamivir was prescribed in 807 subjects while zanamivir was prescribed in the remaining 14 patients. Table  and trended up gradually to reach the highest during 2016 (41%)  patients were positive for either influenza A, B or both. A total of 475 (26.6%) subjects were hospitalized. On average, hospitalization rate attributable to influenza accounted for 0.24% annually. However, this number is likely an underestimate of the true influenza-related hospitalizations rate because influenza testing is done at the request of the physician and not every patient was tested for influenza. Therefore, a population based prospective surveillance study is needed to better estimate the burden of Influenza-related hospitalizations. The average annual positivity rate  for tested patients was 14% and this is similar to the positivity rate (13.4%) reported by the Lebanese SARI Sentinel Surveillance conducted by the Ministry of Public Health for the 2015/2016 season . MoghoofAccording to the WHO Global Epidemiological Surveillance Standards for Influenza, a standard age grouping into 6 groups is suggested for analysis . We furtOur analysis showed that all severity parameters, except for hospital and ICU stay duration, were highly present within two age groups: below 2 years and ≥ 65 years. These results demonstrate that extremes of age have a more severe course of disease including a higher mortality rate. Several recommending bodies consider these age groups at higher risk for severe disease and mortality and thus recommend early initiation of antiviral therapy and emphasize the importance of vaccination , 46. UnfThe presence of at least one co-morbidity, mainly cardiovascular diseases, asthma, malignancy and diabetes mellitus, in 71% of hospitalized patients and 31% of outpatients is worrisome especially with recent data on influenza being a trigger for exacerbation of these illnesses and vice versa. Several papers have showed an association between influenza infection and myocardial infarction, chronic obstructive pulmonary disease exacerbations and diabetic crises , 48, 49.Several complications have been linked to influenza infection including both pulmonary and extra-pulmonary. Both bacterial and viral pneumonias are established complications of influenza infection and have been reported as an independent risk factor for increased severity and mortality –53. Our Our population also displayed a wide array of extra-pulmonary complications including myocardial infarction, encephalopathy, shock, renal failure and liver failure. These complications have been reported in several articles –59 and iP-value < 0.05). However, after multivariate logistic regression, only the presence of a proven viral or bacterial co-infection, pneumonia and neurological disease were independent risk factors for increased mortality. Unlike the data from Egypt [Staphylococcus aureus and Streptococcus pneumoniae being the most common pathogens identified [According to the global burden of diseases study, influenza accounted for 0.26% of all-cause mortality globally. It also estimated that 0.6 per 100,000 population died from influenza in Lebanon . In our om Egypt , we didnom Egypt . It is hentified , 63. Witentified , 65. ThoAccording to the recent guidelines by the Infectious Diseases Society of America (IDSA), antiviral treatment should be started when influenza infection is suspected or confirmed in any hospitalized patient, those with severe or progressive illness, in children ≤2 years and adults ≥65 years old, pregnant females and 2 weeks postpartum, extremely obese patients, children and adolescents receiving salicylate-containing medications, patients with chronic medical conditions including immunosuppression and residents of chronic care centers . TreatmeBoth influenza A and B co-circulated during the 8 seasons with influenza A being predominant during each season. Historically, there has been more interest in researching influenza A virus mainly because of its capability to cause pandemics and underestimating the burden of influenza B virus. However, our data shows an increased morbidity associated with influenza B evident mainly by the higher incidence of ICU admissions although with no difference in mortality incidence for both types. Earlier published data have shown an increasing burden for influenza B in the USA,  whereas In the MENA region, there is limited data on the circulation patterns of both influenza A and B. It is of paramount importance to determine the timing of influenza circulation for each region in order to decide on the best timing for vaccine delivery, aid healthcare workers in their clinical management of suspected cases in addition to increasing emphasis on prevention of infection and control of transmission . In his Our study has several limitations. The most significant limitation is the fact that this data was collected retrospectively from chart review. We could not obtain accurate data on vaccination rates in addition to other missing or incomplete data. Another limitation is that all these cases reviewed were from a single center that, despite being a major tertiary medical center, cannot reflect the actual Lebanese burden of influenza. A third limitation is the fact that we included all laboratory proven influenza cases regardless of initial presentation symptoms or reason for admission. As a result, we could not calculate influenza-attributable SARI cases and we could not compare this to other published data. Also, we did not exclude possible health-care associated infections, although they contribute to the burden of influenza. Moreover, mortality rate associated with influenza might not be accurate since we had no follow up data on outpatients and thus significant data was missing.This study is a first attempt to estimate the burden of influenza in Lebanon including a large number of patients. Despite being a retrospective review, results offer groundwork for further influenza research. Based on the results presented by our review, influenza causes a substantial number of hospitalizations annually in Lebanon with significant morbidity and mortality across all age groups. Even though we had incomplete data on vaccination, a rate of 22% is fairly low. Fortunately, there is an increased interest in influenza research and surveillance along with increased testing for ILI and SARI cases. The SARI sentinel surveillance initiated by the Ministry of Public Health since 2015 promises to provide better data on the burden of influenza on a national level thus aid in developing a better vaccination program and maybe incorporate it into the national immunization program. A population based prospective surveillance study is needed to better estimate the burden of influenza in Lebanon which would help to drive policy on influenza control.Additional file 1: Table S1. Presenting symptoms of laboratory-confirmed influenza cases according to different age groups. Table S2. Results of multivariable logistic regression for independent risk factors associated with increased mortality. Table S3. Characteristics of patients receiving antiviral therapy over the study period.


The effects of quercetin liposomes (Q-PEGL) on streptozotocin (STZ)-induced diabetic nephropathy (DN) was investigated in rats. Male Sprague Dawley rats were used to establish a STZ induced DN model. DN rats randomly received one of the following treatments for 8 weeks: blank treatment (DN), free quercetin (Que), pegylated liposomes (PEGL) and pegylated quercetin liposomes (Q-PEGL). A group of healthy rats served as the normal control. The fasting blood glucose (FBG), body weights (BWs), renal hypertrophy index (rHI), serum and urine biochemistry, renal histopathology, oxidative stress and immunohistochemical measurements of AGEs were analyzed to compare the effect of different treatments. Que and Q-PEGL significantly improved DN biochemistry and pathological changes, although the treated rats still had some symptoms of DN. The therapeutic effect of Q-PEGL surpassed that of Que. Pegylated quercetin liposomes allow maintaining higher quercetin concentrations in plasma than non-encapsulated quercetin. In conclusion the use of quercetin liposomes allows to reduce disease symptoms in a rat model of DN. Diabetic nephropathy (DN) is a microvascular complication of DM and causes long-term or end-stage renal disease2. Multifactorial interaction between lipid disorders, oxidative stress, renal hemodynamic changes, polyol activation, inflammatory pathways, and mitogen-activated protein kinase signaling pathways are involved in the pathophysiology of DN7. Therefore, strong antioxidants could potentially serve as treatments to diabetes related diseases5 Quercetin  is a potent dietary bioflavonoid found in diverse fruits, seeds, and vegetables such as legumes, apples, and chili peppers7. Pharmacological studies in humans show that quercetin has multiple biological functions including circulation system protection, anti-allergic, anti-inflammatory, anti-cancer, anti-diabetes, and cataract prevention7. Another important attribution of quercetin is to reduce aldose reductase, which is an enzyme that converts glucose to sorbitol through polyol pathway5.Diabetes mellitus (DM) is a metabolic disorder characterized by hyperglycemia due to impaired body’s ability to produce or respond to the hormone insulinin vivo absorbability9. Liposomes are the most studied particle carrier systems allowing sustained release, and have the potential of enhancing the oral bioavailability of proteins and peptides10. Liposomes are small spherical lipid vesicles, composed of phospholipids and cholesterol, characterized on size, number of lamellae, and inner/outer phases12. Liposomes have good cell compatibility, reduce drug toxicity, improve drug stability and function for a long time13, and have been used to deliver drugs including antibiotic, antifungal, and cytotoxic agents14. Coating inert biocompatible polymers, such as PEG, on the liposome surface will form a protective layer over the liposome surface, which slows down liposomes recognition and clearance, and extends liposome circulation time and provides slow release of an encapsulated drug15. Yuan et al.9 utilized liposomes for delivery of quercetin to study anti-tumoral properties. Yue et al.13 also developed coenzyme Q10-loaded liposomes to treat DN.Natural quercetin has poor water solubility, therefore, quercetin liposomes are laboratory-prepared to improve its solubility and 16. In the present study, we evaluated the effects of quercetin liposomes on STZ-induced DN rats through comparing the histopathological and biochemical alterations among different treatment groups.Streptozotocin (STZ) is used to induce diabetic kidney injury with hyperuricemia, hyperlipidemia and inflammationThe maximum concentration of quercetin in the plasma was 92.77 ± 11.21 nmol/ml for the Q-PEGL group at 60 minutes and was 104.53 ± 12.08 nmol/ml for the Que group at 5 minutes Fig. . The maxP < 0.01). The body weight of all groups increased after STZ induction until week 4, from where the body weight started to decrease in the DN group and the PEGL group, while kept increasing in the rest three groups. At the end of the treatment period (8 weeks after administration of STZ), body weights in NC group and PEGL group were significantly lower than those of the other groups (P < 0.01). No significant difference was detected between the DN and the PEGL groups (P > 0.05). The body weight of Q-PEGL rats was significantly higher than that of the other groups (P < 0.01), except that it was lower than that of the NC group (P < 0.01).The characteristics of STZ induced DN rat are summarized in Fig. At the end of the STZ injection, FBG level was significantly increased in DN model rats compared to NC rats (P < 0.01). Treatment reduced FBG level in the Que and Q-PEGL groups compared to the DN or PEGL group (P < 0.01). In addition, the FBG level in the Q-PEGL group was significantly lower than that in the Que group (P < 0.01). Compared with the Q-PEGL group, the increase of FBG induced by STZ in the PEGL, Que and DN groups was more pronounced, which peaked at week 4 and then gradually decreased. The peak of FBG in Q-PEGL group was at week 3. The FBG levels of all groups are shown in Fig. P < 0.05), but there was no significant difference between the DN and PEGL groups (P > 0.05). The rHI and 24-h urinary protein were lower in the Q-PEGL than those in the Que group (P < 0.05). The rHI and 24-h urinary protein of all groups are shown in Fig. At the end of the treatment period, the rHI and 24-h urinary protein were significantly lower in the NC group than those in the other groups (P < 0.01). In addition, the rHI and 24-h urinary protein were lower in the Q-PEGL and Que groups than those in the DN and PEGL groups (both P < 0.01). The Que and Q-PEGL groups had lower levels of BUN, Scr, TNF-α, IL-1β, AGEs and MDA compared to the DN or PEGL groups (P < 0.05). There was no significant difference in these variables between the DN and PEGL groups (P > 0.05). Q-PEGL significantly decreased BUN, Scr, TNF-α, IL-1β, AGEs, and MDA (P < 0.05) compared to Que. NC group had higher levels of SOD and GSH-Px than the other groups (P < 0.01). Que and Q-PEGL groups had higher levels of SOD and GSH-Px (P < 0.05) compared to the DN and PEGL groups, and the effect of Q-PEGL was greater than Que (P < 0.05). There was no significant difference in these variables between the DN group and the PEGL group (P > 0.05).Figure P < 0.01 for both). The mesangial indices in the Que and Q-PEGL groups were significantly lower than that of the DN group .Images of PAS-stained kidney tissue sections from selected experimental groups were obtained by using an optical microscope, as shown in Fig. P < 0.01, for all), no significant difference was found between the DN and PEGL groups (P > 0.05). Interestingly, Que and Q-PEGL groups showed considerably decreased immunofluorescence labeling around the capsule and on the tubular basement membrane. The reduction of AGE expression in the Q-PEGL group was more pronounced than that in the Que group (P < 0.05).The kidney AGE expression at week 8 by immunohistochemistry is shown in Figs. 16. Quercetin is an active compound for anti-cancer, anti-bacterial, anti-allergic and analgesic, and improves DN through various signaling pathways, making it a promising compound for the management of these diseases19. The T2DM recovery from oxidative stress by quercetin was observed in single cell alkaline gel electrophoresis5. In this study, we showed quercetin carried by a synthesized PEGL was beneficial in reducing the deterioration of DN in rats.Diabetic hypertension and unresolved inflammation in DN lead to renal fibrosis and kidney failure. Currently, the mainstays of DN treatment are glycemic control and blood pressure lowering using durgs to block the renin-angiotensin system. Despite emerging clinical candidates under investigation for the treatment of DN, there is a lack of successful new treatments for DN. Some innovative treatment make use of traditional anti-diabetic drugs (insulin and exenatide), cytokines, metabolic modulation, nucleotide and quercetin12. However, because quercetin has the disadvantage of low solubility in vivo and short cycle time, we synthesized a PEGL carrier to load quercetin to improve its biological activity in vivo21. The mass ratio of the composition of the Q-PEGL compound quercetin/lecithin/cholesterol/polyethylene glycol 4000 was 6:13:4:1 in weight ratio9, and the mass of the chloroform/methanol was very small. To ensure similar load of quercetin, Que (50 mg/kg) and Q-PEGL (200 mg/kg) were administered intragastrically in our study. In addition, PEGL (150 mg/kg) and Q-PEGL (200 mg/kg) were also administered intragastrically. Our results showed that Q-PEGL sustained a higher concentration of quercetin in plasma and kidney tissue compared to the free Que group after a given time point. The peak of quercetin appeared at 60 minutes, while the result of Yuan et al.9 was at 30 minutes, probably due to the Q-PEGL in our study was intragastrical administered instead of intravenous injection.Liposomes are non-toxic, biocompatible, biodegradable, and have the following advantages as carriers in drug delivery: first, liposomes can reduce toxicity; second, hydrophilic and lipophilic agents can be incorporated into liposomes; third, liposomes control the drug release; and fourth, the liposome delivery system increases bioavailability23. The rat DN model induced by STZ increased FBG and proteinuria in our study. Previous studies have shown that quercetin or quercetin liposomes were effective in controlling FBG, BWs, rHI, biochemistry and pathology in STZ induced diabetic animals22, which is consistent with our observation. Quercetin can reduce the plasma glucose level because it inhibits intestinal glucose absorption while enhances tissue and organ glucose levels;, quercetin increases insulin secretion by β-cells and protect DM against oxidative stress7. Administering quercetin to INS-1 pancreatic β-cells potentiated insulin secretion through enhanced glucose metabolism and protected β-cells against oxidative damages24. In STZ induced DN models of our study, quercetin liposomes or free quercetin were able to prevent body weight loss, decrease the renal hypertrophy index, lower blood glucose levels, and reduce the 24-h urinary protein. The effect of quercetin liposomes was stronger than quercetin alone. In addition, FBG peaked at 3 weeks and 4 weeks in the Q-PEGL and the Que group respectively, and in both groups, it decreased gradually with time. This difference in the peak time can be explained by the slow release of PEGL carrier. Increased kidney weight caused by diabetes indicates renal hypertrophy, a common finding in the early stages of diabetes26. In this study, interstitial fibrosis, mesangial contraction, and structural remodeling contributed to the decrease in renal hypertrophy, and improved renal function reduced the 24-h urinary protein.STZ is a natural compound that can induce DN through selectively destroying islet β cells of animals, because DN is usually associated with reduced viability and dysfunction of pancreatic β cells27. Kuo et al.28 suggested that the interaction between ROS production and the anti-oxidant pathways can contribute to the progression of diabetic nephropathy. SOD, MDA, TNF-α and IL-1β are the most common indicators of oxidative stress. GSH-Px is one of the key enzymes that scavenge free radicals in the body. These factors can reflect the body’s antioxidant capacity. Literature shows that being an intracellular antioxidant, GSH-Px protects DNA, proteins and other biomolecules from ROS. AGEs are products of non-enzymatic glycosylation of proteins under chronic hyperglycemia, and levels of AGEs reflect the severity of renal injury. AGEs are thought to be a mechanism of action in the pathogenesis of clinical complications of diabetes, and accelerating synthesis and tissue deposition of AGEs leads to the microvascular alterations in DN29. Furthermore, in this study, DN model has shown that Que and Q-PEGL have the antioxidant capacity and scavenge free radicals, as evidenced by decreased levels of MDA, TNF-α, IL-1β, AGEs and increased activity of SOD, GSH-Px. Our results also revealed that Que and Q-PEGL significantly attenuated the increase in Scr, BUN levels, and reduced histopathological damage of kidneyin DN rats.The oxidative stress is considered to be important in the development of diabetic nephropathy30. In our study, pathological changes including glomerulosclerosis, interstitial fibrosis, and tubular atrophy due were observed, and these observation are consistent with previous studies in rats31. Xu et al.32 and Chen et al.33 have also reported that quercetin can improve the shape of the kidney, delay the progression of DN, and prevent diabetes-induced oxidative stress.Long-term hyperglycemia increases secretion of inflammatory and fibrotic factors by renal cells, leading to cell hypertrophy and proliferation, and renal interstitial fibrosis21. Our results showed that quercetin liposomes and quercetin decreased the glomerular mesangial matrix expansion index and AGE levels, and the effect of quercetin liposomes was more significant. Gomes et al.19 showed that quercetin treatment can significantly improve renal structural changes, including glomerulosclerosis index and kidney/body weight normalization. The PAS and immunohistochemistry results showed that the pathological changes in the Q-PEGL group were better than those in the Que group. However, the relevant indicators for the PEGL group were similar to the DN group. This suggested that PEGL did not delay the progression of DN or prevent diabetes-induced oxidative stress. The study has limitations. We did not verify the solubility of Que and Q-PEGL, and the dose used in the study was based on previous study, which could introduce potential bias to the results.During hyperglycemia, the formation of hyperglycosylated AGEs in the glomerular basement membrane collagen is increased, which is extensively cross-linked with collagen, altering its spatial structure, increasing vascular permeability, mesangial proliferation and basement membrane thickening. The presence of AGEs in the kidneys of normal control rats indicates that AGEs occur even in healthy kidneys. Low levels of AGEs are probably resulted from enzyme-mediated detoxificationIn vivo study exhibited that the developed formulation had renal protective effects in STZ-induced DN rats by reducing oxidative stress, attenuating AGE expression, and delaying the progression of DN. In addition, the positive effect of quercetin liposomes on diabetic nephropathy is greater than that of quercetin alone. Therefore, quercetin liposomes can be used as a valuable therapeutic agent in DN.In conclusion, the liposomes-loaded quercetin was successfully prepared using ratio of a mixture of quercetin/lecithin/cholesterol/polyethylene glycol 4000 and used for drug delivery by the intragastric administration route. High-purity lecithin of soybean, cholesterol, polyethylene glycol 4000, glucosidase, streptozotocin and quercetin were purchased from Sigma . Rat TNF-α and IL-1β ELISA kits were purchased from Elabscience Biotechnology Co. . The rat AGEs ELISA kit was purchased from Cusabio . All the other chemicals were purchased from Sigma . The SOD assay kit was purchased from Sigma-Aldrich .The MDA and GSH-PX assay kits were purchased from Elabscience Biotechnology Co. . All the other reagents used in the present study were of analytical grade and were purchared from Nanjing Jiancheng Bioengineering Institute .ad libitum before and during the experiments. The animals were euthanized by injection of pentobarbital sodium (200 mg/kg) after the experiments. All study protocols including diabetes induction and sacrifice operation were approved by the ethics review board of the Institutional Animal Care and Use Committee at the Guilin Medical University. The animal experiment followed the guide for the care and use of laboratory animal published by the US National Institutes of Health (NI H Publication updated in 2011).A total of 88 male Sprague-Dawley rats, weighing 190~220 g, were purchased from the Animal Laboratory of Guilin Medical University . The animals were maintained under standard laboratory conditions with a 12-h light:12-h dark cycle, at a temperature of 25 ± 2 °C, throughout the study. Animals were fed with standard rat chow and tap water 9. The dried lipid films were sonicated in a 5% glucose solution in a constant container, then concentrated and lyophilized. The PEGL was prepared in the same manner as the Q-PEGL, which did not contain quercetin in the mixture. According to the calculation reported by Mukhopadhyay et al.17, the final Q-PEGL and PEGL are small unilamellar liposomes with a size range of 128.8 ± 18.05 nm. In addition, the Que encapsulation efficiency and load capacity were 87.1 ± 2.7% and 58 ± 7%, respectively. Q-PEGL and PEGL were concentrated, lyophilized under vacuum for 5 hours and stored at −20 °C. The final product has good solubility and can be used directly or intraperitoneally dissolved in saline8.Pegylated quercetin liposomes (Q-PEGL) and empty pegylated liposomes (PEGL) were prepared at the pharmaceutical laboratory of Guilin Medical University . Briefly, a mixture of quercetin/lecithin/cholesterol/polyethylene glycol 4000 (6:13:4:1 by weight) was dissolved in a chloroform/methanol  solution, and then evaporated to dryness in a rotary evaporator18. Three days after STZ administration, the FBG was determined in a tail vein sample using blood glucose meter . Rats with FBG levels above 16.7 mM for 3 consecutive days were considered DM13. DM rats were fed with standard rat chow and tap water ad libitum for 4 weeks. All DM rats were transferred to metal metabolic cages and 24-hour urine samples were accurately collected.The DM modle induction was as follows: forty-eight rats were fasted overnight and then given a single intraperitoneal injections of 60 mg/kg STZ dissolved in 10 mM citrate buffer (pH 4.5)13. Rats were treated intragastrically with free Que (50 mg/kg) or Q-PEGL (200 mg/kg) dissolved in 0.9% sodium chloride. Dose of 200 Q-PEGL and 50 Que mg/kg/day allows similar Que load between groups for the comparison according to our estimation. At each time point , three rats were sacrificed and their blood was collected from the abdominal aorta. The plasma was obtained by centrifugation . The kidney tissue was excised, weighed and homogenized. Glucosidase was added into the plasma samples and incubated for 2 hours at 37 °C. Plasma and homogenized kidney tissue were extracted with ethyl acetate, and the supernatant was collected and evaporated to dryness. The dry residue was dissolved in methanol for high-performance liquid chromatography9. The level of quercetin in plasma or kidney is expressed as nmol/mL.A 24-hour urinary albumin excretion rate of >30 mg/24 h was observed at week 4, indicating the successful construction of DNn = 8 per group), and treated intragastrically with quercetin liposomes , free quercetin , liposomes dissolved in 0.9% sodium chloride , and blank treatment (DN group). Eight non-diabetic rats received sodium chloride and served as a normal control (NC group). Treatment of Q-PEGL, Que or PEGL was started since day 3 after STZ injection, at 9:00 a.m.–10:00 a.m. each day, and lasted for 8 weeks.There were 5 groups of rats. The DN model preparation was described above. DN rats were randomly divided into 4 groups  staining and immunohistochemical assays. The left kidneys were stored at −80 °C for biochemical evaluation.Rat urine samples were collected weekly with a metabolic cage during drug treatment and were purified by centrifugation at 3000 Body weights and fasting blood glucose levels were recorded each week before and after STZ induction, including drug intervention period. FBG was measured by a blood glucose meter  in samples obtained from the tail vein. It was about 2 mins from blood collection to obtaining results. The renal hypertrophy index (mg/g) was calculated as follows: renal index = kidney weight (mg)/body weight (g). 24-h urine protein was determined on an AU2700 automatic biochemical analyzer .g for 10 minutes and the supernatant was then used to assess the renal function. The levels of BUN and Scr were determined on an AU2700 automatic biochemical analyzer . The levels of TNF-α, IL-1β and AGEs were measured according to the manufacturer’s instructions of the immunoassay kit. The assay employs the ELISA techniques.Serum was centrifuged at 3600 g for 10 minutes at 4 °C and the clear supernatant was collected. The SOD value (U/mg) was measured by the xanthine oxidase activity assay kit; the MDA level (nmol/mg) was measured by the thiobarbituric acid method (MDA colorimetric assay kit); and the GSH-Px value (U/mg) was measured using a colorimetric assay kit according to the manufacturer’s instructions .Approximately 100 mg of kidney tissue was homogenized in 5% phosphate-buffered saline using a homogenizer. The kidney homogenate was then centrifuged at 15000 A portion of the right kidney was trimmed into small pieces and stored in phosphate buffered 10% formaldehyde solution for 24 hours. After embedding these pieces in paraffin, a thin slice of 5 mm thickness was cut. Glomerular damage was assessed in PAS stained sections. Ten glomeruli randomly selected from each group were quantified in PAS stained sections using image analysis software (Image-Pro plus 6.0) to assess the percentage of PAS-positive areas in the glomerulus, which was expressed as mesangial index.The other part of right kidney tissues was fixed, dehydrated, paraffin embedded, and cut into paraffin slices (4 mm). Tissue sections were deparaffinized, rehydrated, washed in water and subjected to antigen retrieval  in 0.1 M citrate buffer (pH 6.0). After blocking, the selected sections were incubated with AGEs (1:100) for 1 hour. After washing with PBS, the sections were incubated with goat anti-rabbit IgG (1:100) for 20 minutes. Finally, the nuclei were stained with DAB, washed with PBS and mounted in a fluoromount solution. Incubation with immunoglobulin of the same species at the same concentration with no primary antibody was used as a negative control. Five fields of view were digitized using a 400× magnifying objective. The sum of IOD of all fields was collected and analyzed by Image-Pro Plus 6.0.P < 0.05 was considered as statistically significant for a two-tailed test.Data were analyzed by the statistical software IBM SPSS version 23 . Values are presented as the means ± standard deviation. For comparisons in multiple groups, one-way analysis of variance (ANOVA) was used and post hoc analysis was done using Bonferroni test.


AminoIndex™ Cancer Screening (AICS (lung)) was developed as a screening test for lung cancer using a multivariate analysis of plasma-free amino acid (PFAA) profiles. According to the developed index composed of PFAA, the probability of lung cancer was categorized into AICS (lung) ranks A, B, and C in order of increasing risk. The aim of the present study was to investigate the relationship between the preoperative AICS (lung) rank and surgical outcomes in patients who underwent curative resection for non-small cell lung cancer (NSCLC).Preoperative blood samples were collected from 297 patients who underwent curative resection for NSCLC between 2006 and 2015. PFAA concentrations were measured. The relationship between the preoperative AICS (lung) rank and clinicopathological factors was examined. The effects of the preoperative AICS (lung) rank on postoperative outcomes were also analyzed.p = 0.0002). The multivariate analysis identified preoperative AICS (lung) rank C  as a significant predictor of postoperative recurrence, particularly in patients with early-stage disease or adenocarcinoma.The AICS (lung) rank was A in 93 patients (31.3%), B in 82 (27.6%), and C in 122 (41.1%). The AICS (lung) rank did not correlate with any clinicopathological factors, except for age. Based on follow-up data (median follow-up period of 6 years), postoperative recurrence was observed in 22 rank A patients (23.7%), 15 rank B (18.3%) and 49 rank C (40.2%). In the univariate analysis, preoperative AICS (lung) rank C was a worse factor of recurrence-free survival (Preoperative AICS (lung) rank C is a high-risk predictor of postoperative recurrence in patients undergoing curative resection for NSCLC.The online version contains supplementary material available at 10.1186/s12885-020-07575-w. Metabolite profiling remains a relatively underrepresented field of biomarker development for identifying and characterizing various types of cancer. Metabolic changes in patients with cancer may reflect differences in the concentration of a single metabolite or alterations in the constituents of an entire metabolic pathway –5. MetabBased on these findings, we developed a novel cancer screening test for the early detection of various types of cancers using AminoIndex™ technology, which scores health conditions and the probability of disease by a multivariate analysis with plasma-free amino acid (PFAA) concentrations included as a variable , 2. We dHowever, the AICS (lung) test has not yet been sufficiently examined in clinical practice for lung cancer. Recently, we reported that the majority of lung cancer patients with high preoperative AICS (lung) values exhibited a reduction in AICS rank after curative resection, but that the absence of postoperative reduction in AICS rank was strongly associated with tumor recurrence . TherefoTo investigate the clinicopathological and prognostic significance of the AICS (lung) rank among patients with lung cancer on a large scale, the relationship between the preoperative AICS (lung) rank and surgical outcomes was retrospectively investigated in 297 patients who underwent curative resection for non-small cell lung cancer (NSCLC). To the best of our knowledge, this is the first study to report the prognostic importance of the AICS rank in surgically resected patients with cancer.In total, 297 patients who underwent curative resection for NSCLC at the Osaka International Cancer Institute  or Kochi University School of Medicine  between November 2006 and September 2015 were enrolled. Patients were excluded if they had a history of other malignancies at the time of surgery for NSCLC. The clinicopathological characteristics of the enrolled patients are summarized in Table Postoperative adjuvant therapy was administered to 61 patients: adjuvant chemotherapy to 58, adjuvant radiotherapy to 2, and adjuvant chemoradiation therapy to one. The regimen for adjuvant chemotherapy was as follows: oral UFT (Tegafur-Uracil) for 15 patients with stage I disease, for 2 with stage II disease, and for 1 with stage III disease. Gemcitabine (GEM) or vinorelbine (VNR) monochemotherapy was administered to one patient with stage I disease and 5 with stage II, respectively. Platinum (cisplatin or carboplatin)-based combined chemotherapy was administered to 4 patients with stage I disease, 6 with stage II disease, and 23 with stage III disease. Combined chemotherapy using GEM and VNR was administered to one patient with stage III disease. Most regimens were performed as described previously , 13. AdjThe time of initial recurrence was assessed based on the onset of clinical symptoms, the detection of blood test abnormalities , or the detection of recurrent lesions on imaging, and the recurrence-free survival (RFS) period was defined as the period between surgery and the time of initial recurrence. When a tumor recurred, the initial recurrence site was evaluated and generally categorized into three patterns: local, distant, and multiple.Patients underwent preoperative blood sampling within 1 week prior to surgery. Preoperative blood samples (5.0 mL) were collected from a forearm vein on the morning after overnight fasting using tubes containing ethylenediaminetetraacetic acid, and were immediately placed on ice. Plasma was prepared by centrifugation at 3000 rpm at 4 °C for 15 min and stored at − 80 °C until analyzed. Plasma samples were deproteinized using acetonitrile at a final concentration of 80.0%. PFAA concentrations were measured using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry with pre-column derivatization, which have been described previously , 14.AICS (lung) values for patients with NSCLC were classified into the following three groups: rank A, B, or C. The AICS (lung) test detects lung cancer from plasma concentrations of serine, glutamine, alanine, histidine, ornithine, and lysine. AICS (lung) values range between 0.0 and 10.0, with 5.0 producing 80.0% specificity and 8.0 producing 95.0% specificity. A high AICS (lung) value was associated with a greater probability of lung cancer. Rank A was considered to be normal (0.0–4.9), rank B was relatively high (5.0–7.9), and rank C was high (8.0–10.0). Rank C indicated a high risk of lung cancer , 8, 9.P-values were calculated using a one-way ANOVA for means or Fisher’s exact test for comparisons of rank. P < 0.05 was considered to indicate a significant difference. All statistical analyses were conducted using GraphPad Prism  and R software .RFS and overall survival (OS) curves were calculated using the Kaplan Meier method, and differences were examined by the Log-rank test. Cox’s proportional hazards regression model was used to perform a multivariate analysis of factors associated with RFS and OS. This retrospective study was performed in accordance with the Declaration of Helsinki, and the study protocol was approved by the Ethics Committees of the Osaka International Cancer Institute   (1404015008) and the Kochi University School of Medicine  (ERB-000486). All participants gave their written informed consent for inclusion in this study. All data were analyzed anonymously.According to the measurement of preoperative AICS (lung) values, 93 31.3%) patients were categorized as rank A, 82 (27.6%) as rank B, and 122 (41.1%) as rank C (Table .3% patiep = 0.003) in rank C. Regarding the surgical mode, standard surgery (lobectomy or more) for lung cancer was more frequently selected for rank C (p = 0.053). No significant differences were observed in gender, the smoking status, preoperative CEA level, p-stage, or treatment method among patients with each rank.The relationship between each rank and clinicopathological factors was summarized in Table p = 0.0014). Based on this result, the following comparison was performed in two groups: rank A + B vs. rank C.The median follow-up period in the present study was 6.0 years, ranging between 0.1 and 11.5 years. There were 86 (30.0%) patients with tumor recurrence, and 42 (14.1%) died (Table p = 0.000113). Figure p = 0.0026) and p-stage II , but not in p-stage III . Regarding the histological type, only adenocarcinoma showed a significant difference in RFS . According to the univariate analysis of RFS in the present study, significant variables were the smoking status (non vs. current/ex), surgical mode (limited vs. standard), stage (I vs. II-IV), histological type , treatment method , and preoperative AICS (lung) rank (rank A + B vs. rank C) .Forty-two 14.1%) patients died. Death was not associated with the AICS rank  abnormalities in 297 patients with NSCLC, who underwent curative surgery in our two institutes. The preoperative AICS (lung) status in patients with resectable NSCLC was observed on a relatively large scale: 93 with rank A 31.3%), 82 with rank B (27.7%), and 122 with rank C (41.1%). This distribution was similar to that in our previous study . No rela.3%, 82 wIn the multivariate analysis, three variables  were identified as significant high-risk predictors of tumor recurrence in the present cohort. As described previously , the p-sWe recently reported that most patients preoperatively diagnosed with rank C potentially exhibited a reduction in the AICS (lung) rank if they were cured after surgery . FurtherIn contrast to the results obtained for RFS, AICS (lung) abnormalities only slightly affected OS. We speculated that this discrepancy was mainly due to some therapeutic effects after recurrence. However, typical biomarkers for adenocarcinoma, such as EGFR mutations and ALK abnormalities, were not adequately examined because of the old times of some enrolled patients. The potential relationship between AICS (lung) abnormalities and these therapeutic biomarkers needs to be examined in future studies.There were some limitations that need to be addressed. Although the number of enrolled patients was relatively large, the predictive value of AICS (lung) abnormalities for tumor recurrence was not satisfactory due to the retrospective nature of the analysis. Since its effect on tumor recurrence was limited in patients with early-stage disease and adenocarcinoma, the number of patients was too small to assess its significance. Moreover, the biological mechanisms and condition of AICS (lung) abnormalities in human cancer currently remain unknown. Several amino acids may exert promoting or inhibitory effects on the proliferation of cancer cells . AlanineThe present study showed that preoperative AICS (lung) rank C was a high-risk predictor of postoperative recurrence in NSCLC patients undergoing curative resection. Although AICS was originally regarded as a screening modality for the risk of cancer, the AICS abnormalities may have a biological role in increasing the grade of malignant characteristics in cancer patients.Additional file 1.