The prevalence of metabolic-related disorders, such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (DM2), has been increasing. Therefore, developing improved methods for the prevention, treatment, and detection of these two conditions is also necessary. In this study, our primary focus was on examining the role of chronic inflammation as a potential link in the pathogenesis of these diseases and their interconnections. A comprehensive search of the PubMed database using keywords such as “non-alcoholic fatty liver disease”, “type 2 diabetes mellitus”, “chronic inflammation”, “pathogenesis”, and “progression” yielded 177 relevant papers for our analysis. The findings of our study revealed intricate relationships between the pathogenesis of NAFLD and DM2, emphasizing the crucial role of inflammatory processes. These connections involve various molecular functions, including altered signaling pathways, patterns of gene methylation, the expression of related peptides, and up- and downregulation of several genes. Our study is a foundational platform for future research into the intricate relationship between NAFLD and DM2, allowing for a better understanding of the underlying mechanisms and the potential for introducing new treatment standards. NAFLD refers to a condition characterized by hepatic steatosis, which involves the accumulation of fat in at least 5% of hepatocytes in the liver. This condition occurs without any identifiable secondary causes such as alcohol use or hepatitis C virus infection . NAFLD hAs NAFLD progresses, it may advance to a more severe condition known as non-alcoholic steatohepatitis (NASH). In NASH, additional components such as liver damage, inflammation, and fibrosis become evident, indicating a more pronounced level of liver injury. The diagnosis of NASH can be made based on several critical indicators, including the initial statement of at least 5% liver steatosis (fat accumulation in liver cells). Additionally, varying degrees of hepatic fibrosis , hepatocyte ballooning degeneration (swelling and damage to liver cells), and lobular inflammation (inflammation in the lobules of the liver) are considered significant factors in confirming a diagnosis of NASH ,4,5,6,7.NAFLD, a metabolic disorder, connects with other metabolic defects such as obesity, dyslipidemia, insulin resistance, and diabetes mellitus (DM). Nevertheless, emphasizing that this phenomenon occurs even in individuals with average weight is crucial. Among the reasons for the development of NAFLD in lean people high alcohol intake, lipodystrophy, polycystic ovary syndrome, deficiency of growth hormone, or diets rich in fructose and fat can be specified. Nevertheless, NAFLD can be caused by drugs such as amiodarone, tamoxifen, or methotrexate and even may be related to genetic disorders, for example, lysosomal acid lipase deficiency ,11,12. TIn the literature, there is an extensive exploration of the connections between NAFLD and the risk of several cancers, such as colorectal, kidney, uterine, and bladder cancer. However, the highest risk is observed for hepatocellular carcinoma. The possible mechanism of the elevated risk of cancer development in NAFLD subjects may be related to chronic inflammation and visceral adiposity, but this mechanism is still under research . AdditioA recent proposal suggests using metabolic-dysfunction-associated fatty liver disease (MAFLD) instead of NAFLD, as it provides a broader perspective and emphasizes the connection between this condition and metabolic dysfunction. Clinicians can establish the diagnosis of MAFLD when liver steatosis coexists with at least one of the following criteria: overweight, diagnosis of DM2, or the presence of at least two out of five metabolic syndrome risk factors ,23. DM comprises a group of metabolic diseases that exhibit chronic hyperglycemia. These conditions stem from defects in either insulin secretion, insulin action, or both . The worldwide prevalence of DM in 2021 (in adults aged 20–79 years) was estimated at 10.5% (536.6 million people) regardless of its type. It is crucial to note that the prevalence of all types of DM is on the rise. According to projections, by 2045, approximately 783.2 million individuals, which accounts for 12.2% of adults aged 20–79 years, are expected to be living with DM, including all types of this disease . DM2 accInflammation is a complex physiological response to the antigens such as foreign organisms, particles from another individual, cancer cells, or even cells of the host. The response to a foreign organism’s antigens is crucial for the body to stay healthy. Nevertheless, disorders of the immune system and its over-reactivity to self-antigens, which the body normally distinguishes from foreign ones, lead to an excessive inflammatory reaction and autoimmune diseases . During Additionally, there is a significant correlation between adipose tissue and low-grade inflammation, particularly in individuals who are obese. This relationship highlights the link between excess body fat and chronic, low-level inflammation . Studies have indeed demonstrated that in individuals with morbid obesity, proinflammatory genes are more expressed in subcutaneous adipose tissue than visceral adipose tissue. This observation suggests that the subcutaneous fat layer, which lies just beneath the skin, may significantly influence inflammation in cases of severe obesity . Indeed, research has provided evidence of a strong connection between visceral adipose tissue and inflammation . A notabThe relationship between NAFLD and DM2 is complex and can be defined as bidirectional. DM2 development can be promoted via NAFLD, and on the other hand, DM2 is recognized as an NAFLD-promoting factor ,39. InsuThe interplay between NAFLD, DM2, and chronic inflammation is key to understanding the complicated pathogenesis of these conditions. What follows this is the introduction of new diagnostic and therapeutic standards, which can improve patients’ conditions and decrease costs related to complications of these diseases.The demonstration of the connections between the chronic inflammatory process and pathogenesis of NAFLD and DM2 was set as a major aim of this review.Studies published in English were included in this analysis.The PubMed database according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  was searIn relation to publication date, the evaluated papers were limited to those published since 2000; more than 50% of searched papers have been published since 2019. Mainly, we searched clinical trials and randomized controlled trials. The final search was conducted on 25 March 2023. In order to find matching articles, we used a combination of Medical Subject Heading (MeSH) terms and specific search terms in variations, including “non-alcoholic fatty liver disease”, “type 2 diabetes mellitus”, “chronic inflammation”, “pathogenesis”, and “progression”. The relevant works fulfilling the following inclusion criteria were selected: studies in English that focus on chronic inflammation, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and type 2 diabetes mellitus, and their pathogenesis and relationships. All the selected studies were then downloaded and analyzed.Initially, titles, abstracts, or full texts were reviewed; subsequently, full copies were analyzed. In relation to the prepared and tested template, data on NAFLD and DM2 pathogenesis in the context of the inflammatory process and the interrelationships between them were extracted from the articles. Analyses were performed using Microsoft Office 365, CiteSpace (v.6.1. R2), and VOSviewer (v.1.6.18) to conduct a bibliometric and visual analysis of all data.The final assessment and verification were carried out by experienced researchers .As the pathogenetic factors leading to the expansion of both NAFLD and DM2 are similar, including dyslipidemia, obesity, genetics, environment, lifestyle factors, and insulin resistance, it is not surprising that they often coexist . As NAFLThe pathogenesis of DM2 and NAFLD is intricate and not fully understood; however, in the development of both, many common factors can be found. Similar causes of the pathogenesis of these diseases include, e.g., genetic predispositions ,50, enviMetabolic disorders such as NAFLD have complex pathogenesis. In fact, metabolic syndrome, insulin resistance, obesity, alterations in lipid and glucose metabolism, high caloric intake, increased hepatic de novo lipogenesis, alterations in the gut microbiota, inflammatory processes, and oxidative stress are all associated with NAFLD ,59. GeneMany risk factors are known to influence the complex pathogenesis of DM2 . These iOxidative stress, endothelial dysfunction, and inflammation are proposed to be involved in NAFLD pathogenesis . In addiThe role of inflammation in exacerbating NAFLD state was previously mentioned. In the liver tissue, we can highlight macrophages named Kupfer cells, which are involved in the inflammatory process. These cells are responsible for the release of chemokines and cytokines in response to liver damage. Kupffer cells are the largest population of resident tissue macrophages in the liver . The funCa3.1 and KCa2.3, which are components of a heterotetrameric voltage-independent potassium channel. Furthermore, it has been observed that oxidative stress upregulates these proteins, suggesting their involvement in the development of liver disease [Under normal conditions, there is a balance between Kupffer cell types, and their net effect is beneficial. However, when this balance is disturbed, due to the imbalanced secretion of IL-1β, IL-6, IL-12, IL-18, TNF-α, and chemokine (C–C motif) ligand 2 (CCL2) chemokine, liver function is impaired, and NAFLD develops ,75. In N disease .Duan et al.  conducteIt was also observed that in comparison to healthy subjects, patients diagnosed with NAFLD had elevated levels of soluble vascular cell adhesion molecule-1 (sVCAM-1), which is closely associated with endothelial dysfunction. SVCAM-1 is correlated with high-sensitivity C-reactive protein (hs-CRP) levels, which means that inflammation is associated with endothelial dysfunction . In anotOxidative stress emerges as a pivotal factor of significance in liver function. Alkhouri et al.  proposedAccumulating evidence suggests the involvement of hepatic B lymphocytes in the development of NAFLD and its progression to NASH . IndividExtensive molecular investigations have provided compelling evidence establishing a strong association between gene expressions and the progression of NAFLD to NASH, ultimately leading to hepatic cirrhosis. Notably, specific genes such as IL-1β and TGF-β1, matrix metalloproteinase 9 (MMP9), and matrix metalloproteinase 14 (MMP14), as well as ligands of chemokines, including CCL2 and chemokine (C–X–C motif) ligand 1 (CXCL1), have been meticulously identified and associated with these pathological processes. Crucially, the regulation of these genes is governed by the NOD-like receptor protein 3 (NLRP3) inflammasome (NLRP3i). The activation of these genes triggers a cascade of events involving inflammatory processes, lipid metabolism regulation, and remodeling of the extracellular matrix . NLRP3i GCKR) rs780094, patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409, and peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α gene (PPARGC1A) rs8192678 genes. Some genetic variants of the listed SNPs have been shown to confer susceptibility to NAFLD [In recent years, single-nucleotide polymorphisms (SNPs) are being studied for their role in an increased risk of NAFLD development. These SNPs include glucokinase regulatory protein  in NAFLD, especially mi-RNA155, which is involved in the inflammatory response, and its upregulation was observed in the liver of NAFLD subjects .The importance of gut microbiota in human health is well established, and alterations in the microbiome have been linked to the pathogenesis of NAFLD and DM2. As indicators of this interplay, we can point to the breakage of the intestinal barrier, the translocation of microbiota, and inflammation . Studiesdim was higher in NAFLD patients’ sera. The authors suggest that there may be some association between NK-cell function and NAFLD progression [It is known that obesity is a factor that induces systemic inflammation and reduces anti-inflammatory pathways. One of these pathways is the heat-shock transcription factor-1 (HSF1)–70 kDa family of heat-shock protein (HSP70) (HSF1-HSP70) axis, and a reduction in this pathway was observed in the liver and adipose tissue of NAFLD-diagnosed patients. Consequently, obesity exacerbates the development of NAFLD . It has gression . The patAs the importance of IR was discussed in previous sections, the molecular mechanisms of IR interplay with inflammation will be the theme of this section. In a previous study, Cifarelli et al.  confirmeAccording to the analyzed data, it can be concluded that poor vascularity in the system may lead to similar changes. The hypothesis about poorer tissue oxygenation and its connection with IR is confirmed by the fact that intermittent hypoxia adversely affects insulin sensitivity. Hypoxia may play a role in obstructive hypoventilation syndrome (OHS). A study performed among obese subjects with OHS revealed that continuous positive airway pressure (CPAP) therapy improved metabolic parameters, insulin sensitivity, and the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) scores . It has been proven that improvement in insulin resistance results in the regression of blood pressure, vascular inflammation, and enhancement of glycemia and lipid management. Moreover, these changes were associated with a decrease in expression in TNF-α and thioredoxin-interacting protein (TXNIP), which are known as mediators of IR. Additionally, decreases in MCP-1 and IL-8 levels are postulated as effects of improved insulin sensitivity ,106. ResIn the pathogenesis of DM2, pancreatic β-cell dysfunction can be distinguished during its course through the detection of a gradual decrease in the number of β cells ,112,113.In terms of DM2 pathogenesis, the role of IL-6 remains unclear. Interesting conclusions were drawn in a study highlighting the influence of IL-6 on the mechanism of glucagon-like peptide 1 (GLP-1) secretion and action. GLP-1 takes part in insulin excretion through its potentializing effects on β cells. While under stable conditions, it equally increases and lowers blood sugar levels, in subjects with an imbalance of insulin secretion and IR, one of the effects may dominate over the other ,124. DauA link between inflammation and insulin secretion by the pancreas has been shown. Reducing the concentration of hs-CRP results in an improvement in this process and reduces the risk of developing DM2. Importantly, the change in hs-CRP level was also associated with weight loss . In a 2-Taking into account the importance of the inflammatory process in the pathogenesis and development of DM2, and the standards of diabetes treatment that rather focus on controlling serum glucose levels, research on maintaining or restoring β-cell function is justified to introduce new standards of treatment . The patThe role of the inflammatory process in the pathogenesis of DM2 and NAFLD was highlighted in previous sections; nevertheless, the interplay between inflammation, DM2, and NAFLD should be discussed in more detail.PPARGC1A gene, which encodes the PGC-1α protein, has a pivotal role in mitochondria functions and also affects antioxidant management and inflammatory processes. Its expression is affected by many factors, including physical activity [PPARGC1A have been associated with the risk of DM2 [It is known that the activity . Variantk of DM2 . An altek of DM2 ,135. PGCk of DM2 . It was k of DM2 . PGC-1α k of DM2 . Increask of DM2 .The IFN-γ-induced protein (IP-10), functionally categorized as a proinflammatory chemokine, was proposed as a diagnostic biomarker for NAFLD and DM2. Correlations between IP-10 and insulin resistance were also found . BarchetIt is known that obesity is associated with many serious complications, and the pathogenesis of NAFLD and DM2 is no different. The fat tissue secretion of inflammatory markers such as IL-6 may lead to the exacerbation of NAFLD and DM2 . In one As insulin resistance is associated with chronic inflammation, it has been established that IR leads to the pathogenesis of DM2 and NAFLD . In DM2,It was revealed that alteration in the gut microbiota leads to chronic inflammation in both DM2 and liver diseases, including NAFLD, which is associated with damage to the intestinal barrier . PositivDue to the role of inflammatory processes in the development and progression of NAFLD and DM2, researchers more closely investigated these processes as a potential point of treatment. The common denominator in NAFLD and DM2 anti-inflammatory therapy may be aspirin, for which beneficial outcomes in both diseases were observed ,156. AspAs in the pathogenesis of NAFLD, the importance of M1 and M2 Kupfer cells has been shown, and therefore researchers have sought to investigate their functions to treat this disease. Oxy210—a semi-synthetic oxysterol—is a substance that inhibits macrophage polarization. The amelioration of NASH and a decrease in white adipose tissue inflammation and levels of inflammatory cytokines in the liver and blood are also the results of Oxy210 use .In DM2 individuals treated with hemodialysis, the anti-inflammatory and antidiabetic effects of linagliptin, one of the dipeptidyl peptidase-4 (DPP-4) inhibitors, and monotherapy outcomes were observed. When compared to pretreatment values, the amounts of prostaglandin E2 (PGE2), IL-6, and glycated albumin (GA) considerably dropped when linagliptin treatment began, while the amounts of GLP-1 dramatically increased . StudiesDue to the association of major metabolic components with lipid metabolism, medications improving these parameters are commonly used. Apart from their favorable effect on lipid profile parameters, these types of drugs are used to decrease inflammation. Krysiak et al.  assessedMetformin is a first-line pharmacological agent for treating DM2. Metformin’s regulation of mitochondrial respiratory complex activity and homeostatic processes such as autophagy and mitophagy prevent the generation of cellular and mitochondrial reactive oxygen species (ROS) and the release of mitochondrial DNA, resulting in improved cellular health and lower proinflammatory cytokine production  . MetformLa Grotta et al.  investigCa2.3- and KCa3.1-mediated signaling. The researchers proposed modafinil to treat liver diseases. The overexpression levels of KCa2.3 and KCa3.1 in murine models of liver disease and human-origin models treated with TGF were inhibited by modafinil. These findings indicate the antifibrotic action of MF in a mouse model of NASH and highlight the significance of KCa2.3 and KCa3.1 in the development of NASH [A study using a rodent model revealed the efficacy of modafinil treatment in ameliorating NAFLD states. Modafinil is a substance that acts through cAMP alleviation in osteoblasts and fibroblasts. As cAMP is involved in the inflammatory process, it was revealed that modafinil may have anti-inflammatory and antifibrotic effects. The cited study revealed that these effects of modafinil are related to a decrease in K of NASH . Another drug that showed an effect in DM2-diagnosed patients with NAFLD treatment was diacerein. Diacerein is a drug mainly used in osteoarthritis treatment. It exhibits an anti-inflammatory effect by acting on IL-1 pathways. Diacerein reduced liver fibrosis compared with the placebo group. However, while diacerein is an anti-inflammatory drug in diabetic patients with NAFLD, it had no effect on cytokine levels compared with placebo .In a study that aimed to block IL-1β with a human-engineered monoclonal antibody (gevokizumab) in patients with DM,2 it was observed that levels of IL-6 and TNF-α were significantly decreased. Moreover, after treatment, C-peptide showed an increased level, and the normalization of HbA1c was observed . Noe et Summarizing the information presented here, the importance of inflammatory processes in the pathogenesis of NAFLD and DM2 should be emphasized. In light of our review, the relationships between NAFLD and DM2 are complex, and a better understanding of them requires further research, including studies on the inflammatory molecular processes of gene upregulation and downregulation, the expression of corresponding peptides, gene methylation patterns, and altered pathway signaling. Moreover, environmental factors undoubtedly play a key role in the pathogenesis of these diseases. Therefore, treatment aimed at the inflammatory process may be part of the management of both diseases. It is worth emphasizing that NAFLD and DM2 often occur together, one inducing the other and vice versa; therefore, the implementation of prevention methods will work in both cases. Fortunately, many drugs targeting inflammatory pathways are already being tested in clinical trials and, although there are currently no guidelines stating which medication works better in different clinical scenarios, there is hope for better management of NASH, DM2, and transitional states, namely NAFLD and insulin resistance. Undoubtedly, the best current solution is lifestyle change, which has been proven to influence many pathological pathways.


A multiple regression analysis modeled the correlation between resin composition, flavonoids, and TEAC DPPH values, yielding a significant regression equation  = 22.629; p < 0.000; d = 2.299). Therefore, evaluating physicochemical parameters and biological activities provides a promising framework for predicting propolis’ quality and antioxidant properties, thus suggesting its potential as a functional and bioactive compound for the food industry.The antioxidant, anti-inflammatory, and antimicrobial characteristics of propolis, a bioactive compound collected from hives, have prompted its use in the food sector in recent times. This study investigated the physicochemical characteristics, phenolic profile, and antioxidant capacity of 31 propolis extracts collected from Northern Spain. The physicochemical composition  was within the allowable regulatory limits. The analysis of bioactive compounds enabled the identification of 51 constituents: flavonoids  and phenolic acids . The mean value of total polyphenols was 42.72 ± 13.19 Pinocembrin–Galangin Equivalents/100 g, whereas a range between 1.64 ± 0.04 and 4.95 ± 0.36 Quercetin Equivalents (QE) g/100 g was found for total flavonoids content. The determination of bioactivities revealed significant antioxidant capacity using DPPH . Resin content in propolis samples was positively and significantly correlated with both polyphenols (rho = 0.365;  Most researchers describe propolis as a resinous or sticky substance that bees collect from various plants, mix with waxes, and use as a construction material in their hive ,2,3. In A. mellifera) uses these “resins” to clean, insulate, and reinforce the hive, covering holes and cracks, reducing bites, thus maintaining homeostatic balance, inhibiting microbial growth, controlling airflow, waterproofing the walls against humidity, and creating some protection against external invaders, such as the greater wax moth [Galleria mellonella Linnaeus, 1758) or the small hive beetle . Simone-Finstrom and Spivak [A possible mechanism of social immunity of colony strength has been verified that propolis directly affects the microbial load of the hive, influencing the reduction in the expression of the bee’s immunity, regardless of the level of pathogens or parasites in the hive ,9. It hawax moth  , beeswax, insoluble impurities, pollen, and spores ,19. The Another aspect to consider in the analysis of propolis is the mineral content, which contributes to improving its nutritional value. While minerals are related to botanical origin, the quantity of heavy metals depends significantly on hive management ,22. In tRegarding its biological properties, these depend on the type of flavonoid. Numerous studies on propolis show the beneficial properties of phenolic compounds, especially flavonoids ,27. PhenIn Spain, propolis is authorized as a food supplement; it is found on the market in multiple presentations, made with different types of propolis in which the main active compounds are not identified and with information on the label without any criteria regarding the doses indicated ,31. At tIn addition to its antioxidant effect, the use of propolis in the food industry is justified by various authors as a preservative ,36, thanThe aim of this study is to evaluate the total characterization of propolis collected around two harvests in the north of Spain. Knowing these products’ different capacities and properties when used in the food industry could be interesting. There is a possibility of finding an economical option for beekeepers to implement the rentability of the harvesting sector while the propolis improves its economic value.All the extraction assays used ethanol from Panreac and methanol . The equipment used for the different determinations and analyses were Soxtec System HT 1043  and Muffle Oven Mod. 10PR/300 Serial 88 , ICP-MS NexION 300D , Centrifuge 5810 R , Hewlett-Packard 1200 , Mass Spectrophotometer , SpectraMax iD3 , and Spectrophotometer DU 7400 . The preparation of the calibration graph involved the following products from different trading houses: Pinocembrin, Med Chem Express, and Galangin, Med Chem Express, both from Sweden and Quercetin, Sigma Aldrich . Different homologated and commercial kits were used: Antioxidant Activity Kit . Optical Microscope (OM) Nikon Eclipse 80i and Scanning Electronic Microscope JSM 6480 mod JEOL were used.Listeria [This study continued a previous investigation that evaluated the effect of antibacterial activity against Listeria . A totalPropolis extraction of pulverized raw propolis (10 g) comprised the addition of 600 mL of 70% hydroalcoholic solution. The extraction was carried out in two stages of 24 h each of shaking at a controlled temperature of 20 °C of the 10 g of propolis sample, first in 300 mL of 70% ethanol and after filtering a new extraction under the same conditions of shaking time and temperature [EEPs were analyzed to determine the main components responsible for their quality, as well as their bioactivity and chemical properties. The characterization of the selected propolis samples considered the proximal composition regardless of their botanical origin, including resin, water, wax, impurity, and ash content ,14,41.According to procedures described by Bankova et al. ,42, 1 g The rest of the cartridge content (propolis sample) after the procedure described in w/w [Before the wax extraction, from the extract diluted in 100 mL of ethanol 70%, 20 mL were weighed and then concentrated in a rotary evaporator to obtain a solid residue; after cooling, the concentrates were weighed again, expressing the results as % w/w .The ash content was determined using the AOAC method . A homogv/v) to which 10 ppb of Pt and Rh were also added as internal standards. The rest of the elements were analyzed in an ICPOES using the dilutions calibrated to 50 mL without dilution. The equipment was calibrated with standards of 0,1, 1, 10, and 20 ppm in nitric acid 1:5 (v/v), except for Na, which was used 1, 10, and 20 ppm. As an internal standard, 5 ppm of Y was used.For the analysis of the elements, the propolis samples were digested (0.500 g of each sample in 10 mL of 65% nitric acid and 3 mL of 37% hydrochloric acid) in a digester at atmospheric pressure with reflux programmed with the following temperature and time conditions: 20 to 45 °C in 30 min, 1 min at 45 °C, from 45 to 65 °C in 25 min, 5 min at 65 °C, from 65 to 100 °C in 15 min, and 120 min at 100 °C. The digestion tubes were allowed to cool, and the digestion tubes were volumetrically filled to 50 mL with MilliQ water. Pb, Cd, and Hg were measured on an ICP-MS. For this purpose, the digestions were diluted 1:4 in MilliQ water, and 10 ppb of Pt and Rh were added as internal standards. The ICPMS equipment was calibrated with Pb and Cd standards of 2, 10, 50, and 100 ppb and with Hg standards of 0, 2, 10, and 20 ppb in nitric acid diluted 1:20  in a 25–300 μg/mL range. A total of 0.5 mL of the 31 propolis extracts was transferred in triplicate into a 25 mL volumetric flask. Then, 7.5 mL of distilled water, 2 mL of the Folin–Ciocalteu’s regent, and 3 mL of a 20% N2CO3 solution were added. The volume was filled to 25 mL with distilled water and left at room temperature for 2 h. Absorbance measurements were performed in triplicate at 760 nm in a UV-vis spectrophotometer, expressing the results obtained as grams of pinocembrin–galangin equivalents per 100 g of raw propolis (%PGE) [Total polyphenol content (PTC) was analyzed according to Folin–Ciocalteu’s method . The cas (%PGE) ,45.3)3, and 0.10 mL of 1 M CH3CO2K were added. This was mixed well and at least 40 min/24 °C. Absorbance measurements were performed in triplicate at 425 nm in a UV-vis spectrophotometer. The results obtained are expressed as grams of galangin equivalents per 100 g of raw propolis (%GE).Total flavonoid content (TFC) was estimated according to Woisky and Salatino , using q® C18 column  thermostatic at 35 °C, using 0.1% formic acid (eluent A) and acetonitrile (eluent B) as mobile phase. A flow rate of 0.5 mL/min was set, establishing the elution gradient. The injection volume was 15 μL and spectrophotometric detection was performed by selecting 280, 330, and 360 nm as preferred wavelengths. Mass analysis was conducted using the mass spectrometer, operating in negative ionization mode at a temperature of 400 °C and recording spectra between m/z 100 and m/z 1000. Zero air was used as nebulizer gas (30 psi) and turbo gas  for eluent drying and nitrogen as curtain gas (20 psi) and medium collision gas [Previously, for HPLC analysis, 100 mg of raw propolis was pretreated to remove different impurities and substances that may interfere with the determination. Then, a similar process was used to extract phenolic compounds. In this case, the extraction was performed in triplicate, and the result was dried and dissolved in 60% methanol . Once thsion gas .The detection method employed was full scan at high sensitivity  with the following parameters: capillary voltage, −4500 V with the following potentials: declustering potential (DP) −50 V, entrance potential (EP) −6 V, and collision energy (CE) −10 V. Following this analysis, another analysis was carried out in Enhanced Product Ion (EPI) mode to obtain the characteristic fragmentation of the majority ion obtained in the first experiment. In this case, the conditions used were DP −50 V, EP −6 V, CE −25 V, and collision energy spread (CES) 0 V. The phenolic compounds were identified based on the retention time criteria observed in the chromatograms, the UV-visible spectra, and the MS and MSn data obtained in the mass spectrometer, comparing the different data with those available in the literature.max = 517 nm) stable organic nitrogen radical, is reduced by antioxidants to the colorless DPPH reduced form. Therefore, the absorbance decrease at 517 nm depends linearly on the antioxidant concentration. The synthetic antioxidant Trolox is used to standardize the sample TAC relative to Trolox . The vitamin C standard was also used to express the CEAC value [BQC DPPH TAC Assay Kit is based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. In this method, the DPPH free radical (DPPH•), a deep purple-colored . In the presence of antioxidants, the oxidized ABTS•+ radical is reduced to ABTS, resulting in a discoloration of the solution, measured by the decrease in absorbance at 734 nm. Antioxidants scavenge ABTS•+ radical cation in a concentration-dependent manner .p-value of <0.05 was set as representing statistical significance for all analyses.Continuous variables were expressed as mean values ± standard deviation (SD). The Kolmogorov–Smirnov test was applied to determine data normality. Differences between physicochemical and biological variables were analyzed using one-way ANOVA and Kruskal–Wallis tests for standard and non-normal distribution variables, respectively. Bivariate correlations (Spearman’s correlation coefficients) were used to assess associations between physicochemical variables and antioxidant parameters. Testing was conducted on several multiple linear regression models, in which differences in resin content were considered dependent variables and the rest of the variables (flavonoid content and antioxidant activity) as predictors. The software package SPSS for Windows version v.26  was used for data analysis. A As expected, the resins and waxes were the most abundant fractions of all the 31 samples studied. All the results are summarized in The amounts of the minerals studied in the 31 propolis samples are shown in Fagaceae family, where species such as Quercus rotundifolia Q. pirenaica and Castanea spp. were identified in 30 of the 31 samples. Families like Salicaceae, Ericaceae, Fabaceae, Rosaceae, and Poaceae were found in more than 2/3 samples. Conversely, others like Caprifoliacea, Boraginaceae, or Thymeleaceae appeared in only one sample. The palynological study of the propolis samples showed a rich variety of pollen grains. These included The fraction of the main bioactive compounds is shown in As expected, the chemical composition of the propolis samples showed a similar chromatographic profile (HPLC). The analyses revealed a diverse range of substances belonging to several groups. More than 50 peaks were identified in all 31 samples and are summarized in The comparison between the results obtained for the ethanolic extraction of propolis showed an extremely high antioxidant capacity compared to the scavenging method used. In both DPPH and ABTS assays, the 31 EEP showed exceedingly high antioxidant power. The results were expressed as Trolox Equivalent Antioxidant Capacity (TEAC) and Vitamin C Equivalents Antioxidant Capacity (CEAC), where units are µM of the standard. Results are detailed in p = 0.043) and flavonoid composition  and the antioxidant capacity TEAC DPPH . In contrast, the association with the wax in the propolis samples was strong although negative . The wax content also correlated negatively with the polyphenols and flavonoids. As expected, the ashes correlated positively with impurities and most mineral compounds, whereas the association with the TEACDPPH was negative. The polyphenols exhibited a moderately significant association with the antioxidant profile measured as CEAC . Lastly, a multiple regression analysis modeled the relationship between resin composition, flavonoids, and TEAC DPPH values. The significant regression equation using the resin content as a dependent variable explained 61% of the variance  = 22.629; p < 0.000; d = 2.299), thus explaining 61% of the variance in CPR global effectiveness  protocols and specifications ,41, the tandards .The levels of other proximal compounds, like waxes, were within the observed intervals of reported data from different bioclimatic conditions ,19. The Fagaceae, Salicaceae, Ericaceae, and Fabaceae families. For example, the flavonoids  are typical constituents of the exudate sprouts of Q. rotundifolia, Q. pirenaica, and Castanea spp., which are frequent spices growing in the regions where the apiaries were situated. Several phenolic acids and their esters, like caffeic, p-coumaric, ferulic, and CAPE, were abundantly present in Populus spp. Typically, from the latitude of Spain, it corresponds to the hive’s location. All of these compounds have been studied as marker compounds [The chemical characterization can also be associated with the palynological profile of propolis. Thus, the chromatographic analyses allowed the identification of more than 50 phenolic compounds related to the plant origin ,8,60. Inompounds ,25,61. Iompounds ,58.Honeybees mix the pollen grains, parts of the trees, and flowers or secretions of other plants with their fluids to produce propolis. The composition of propolis and the amount of nutrients, especially the antioxidant compounds , are influenced by the botanical family, the richness of flora diversity, the harvesting time, and/or the geographical area ,25,29,62Propolis can be considered a supplement in food because of the antioxidant role conferred mainly by phenolic compounds, such as phenolic acids and flavonoids. The amount of antioxidant compounds depends primarily on the floral source and the geographical origin ,25.Despite the geographical origin, climate, botany around the hive, year of collection, and management, among other factors, our results showed that flavonoids and flavonols were in the same range. In contrast, we observed more variations when analyzing polyphenol content . Our samSimilarly, the antioxidant activity, measured as Trolox and Vit. C Equivalents of the investigated samples were approximately 100 times higher than other studies ,38. Thesp < 0.000) and a Durbin–Watson value of 2.299, indicating the lack of autocorrelation in the sample. Consequently, our results sufficiently support the significance of the analysis to determine the validity and usefulness of the model. These findings may indicate the potential of resins as an indicator of biological activity and the need to identify the influence of phenolic content when characterizing bee products such as propolis.Previous studies attribute the propolis bioactive properties  to the compounds present in the resinous fraction. In this sense, the resin content is a feasible indicator for evaluating propolis samples’ biological activity and antioxidant capacity. Prior research has found that phenolic content is strongly linked to the antioxidant properties of propolis, even at low resin concentrations ,47,56. OAlthough we comprehensively describe the physicochemical characteristics, principal compounds, and bioactive properties of the propolis samples, this study has some limitations. For instance, quantifying specific constituents of the phenolic compounds may help establish more associations between botanical origin, the fractions with biological activity, and antioxidant effects. The knowledge of target antioxidant elements present in the propolis composition might contribute to focusing on the potential of this natural product as a relevant diet supplement with sound applications in the food industry.Understanding propolis’ intrinsic quality is essential in the search for natural products suitable in the agroalimentary field. This work shows that the biochemical characterization of propolis provides a valuable trend for predicting their antioxidant capacity. Specifically, the propolis resinous fraction indicates phenolic content and biological activity.These findings settle propolis as one of the most promising hive products, thus becoming an alternative resource for the sustainability of beekeepers and the apiculture sector. Additionally, transferring an easy-to-use tool for integrating propolis in the routine manufacturing of food products could contribute to developing healthier ingredients and additives.Therefore, propolis can provide a solid framework for addressing people’s demands while promoting appropriate consumption patterns by developing eco-friendly food supplies.


At lumbar level, tone and stiffness were higher for men at both sides , and relaxation was lower at left side (p = 0.02). The MMPs showed few correlations with sociodemographic data within women. However, within males, there were positive correlations for PF stiffness and viscoelastic parameters with age, BMI and function (0.334 < r < 0.591) and, at lumbar level, negative correlations for tone and stiffness ( − 0.385 < r < −0.590) and positive correlations for viscoelastic properties (0.564 < r < 0.719), with BMI. This indicated that between-sexes differences of lumbopelvic MMPs depend on the specific location of assessment in healthy non-obese young individuals. Women show higher tone and stiffness and lower elasticity and viscoelasticity than men, at PF level.The lumbopelvic muscle mechanical properties (MMPs) are clinically relevant, but their dependence on sex remains unknown. Therefore, this study aimed to identify if lumbopelvic MMPs depend on the sex in a young adult population. Thirty-five healthy nulliparous women and 35 healthy men were analyzed (age range: 18–50). Lumbopelvic MMPs, that is, tone, stiffness, elasticity, relaxation and creep, assessed with MyotonPRO®, and pelvic floor (PF) health questionnaires were compared between-sexes. Intra-group correlations between sociodemographic and clinical data, and MMPs were also determined. The MMPs of PF were different between healthy non-climacteric adults of both sexes, with women showing higher values of tone and stiffness and lower values of elasticity and viscoelastic properties than men (in all cases,  The pelvic floor (PF) includes a complex anatomic structure, with fascial components , muscular components (pelvic floor muscles (PFM) including levator ani muscles, muscles of the urogenital diaphragm, and superficial perineal muscles)6, and neural components 7. Despite the skeletal and visceral differences, the histomorphology8 and architecture9 of PFM are similar between female and male individuals. The most relevant anatomic difference is the genital hiatus, which is broader in women11. Furthermore, the PFM are considered an integral part of the trunk and lumbopelvic stability13. According to this, in women populations, it has been shown that pelvic floor disorders (PFD) can cause pain, disability, and instability of the spine14, and lumbopelvic interventions can improve PFD, such as stress urinary incontinence15. In men, chronic pelvic pain syndrome is associated with spasms in the quadratus lumborum and the iliopsoas and hypomobility of the thoracolumbar spine16. However, other data, such as the lumbopelvic muscle mechanical properties (MMPs), remain unknown, even when their status is relevant in both sexes18. The study of MMPs is of interest even in asymptomatic populations, a less studied population20, since the consequences of risk factors for PFD on the PFM, such as an alteration of MMPs, may not be evident until later in life21.According to the regional interdependence concept22, and muscle activity, assessed with electromyography23, both in women and men populations24. However, these methodologies do not adequately measure muscle tone or other MMPs, as they do not directly measure resistance to change in muscle length18. Moreover, there is neither a single accepted standardized way of measuring muscle tone nor normative values25, and the palpation method is not entirely validated26. Therefore, standardizing a reliable and reproducible examination is needed27. In this line, new technologies allow the evaluate MMPs, as is the case of MyotonPRO , a manual myotonometric device that does not require high levels of expertise28, contrary to other assessment tools, such as ultrasound imaging, and can be used by novices with acceptable results29, following standardized protocols30. Although manual myotonometry has been successfully applied to different clinical states and locations, including lumbar32 and PF28 regions, no study has determined whether the lumbopelvic MMPs are different between sexes.Usually, the PFM analysis focuses on strength, assessed with dynamometryThus, this study aimed to identify if lumbopelvic MMPs depend on sex in a young adult population. Secondarily, intra-sex associations between lumbopelvic MMPs and sociodemographic characteristics were analyzed.A cross-sectional case–control study with consecutive case recruitment was designed. The strengthening the reporting of observational studies in epidemiology (STROBE) method was used. The Research Ethics Committee of Córdoba  approved the project. The study was conducted in accordance with the Declaration of Helsinki. All participants signed the informed consent form.33; experience of any spinal pain in the six months before enrolment in the study34; climacteric period; any type of incontinence; body mass index (BMI) > 30 kg/m2. Participants of both sexes were paired by age (± 3 years).The inclusion criteria for women were: healthy adult women, between 18 and 50 years old; nulliparous; without any type of delivery or pregnancy. The men group inclusion criterion was: healthy adult men, between 18 and 50 years old. Exclusion criteria for both sexes were: history of surgery at lumbopelvic level; engagement in regular physical training ; moderate or heavy occupational physical activity28, an error type I of 0.05 and 0.80 of power, at least 34 women/men per group were necessary .To identify a moderate effect size (Cohen d index = 0.7) based on a minimum detectable change (MDC) of 0.86 and a pooled standard deviation of 1.23 for frequency (muscle tone), as previously reported35. A physical therapist with more than five years of experience assessed the inclusion and exclusion criteria. Two validated questionnaires for women's PF assessment were applied. The Pelvic floor distress inventory (PFDI-20) includes 20 questions divided into three scales according to the symptoms: symptoms of genital prolapse, questions 1 to 6 (POPDI-6); colorectal-anal symptoms, questions 7 to 14 (CRADI-8); and urinary symptoms, questions 15 to 20 (UDI-6). The total score is the sum of the three blocks with a maximum score of 300, where higher scores mean high distress37. The Pelvic floor impact questionnaire (PFIQ-7) includes seven questions about the impact of symptoms on activities, relationships, or feelings concerning urinary prolapse (UIQ-7), colorectal-anal conditions (CRAIQ-7), and genital conditions (POPIQ-7). Again, the total score is the sum of the three blocks with a maximum score of 300, where a higher score means a high impact37. The International Prostate Symptoms Score (IPSS), which assesses the intensity of lower urinary tract symptoms during the past 30 days, was applied to the men39. It consists of 7 questions on a Likert scale with five possible answers. The final score was interpreted as follows: mild (0–7 points), moderate (8–19 points), and severe (20–35 points)40. Sociodemographic and clinical data, such as age, BMI, and level of physical activity according to the Global Physical Activity Questionnaire (GPAQ)41, were also collected.As recommended, the patient emptied the bladder before the evaluation43. A physical therapist, previously trained in myotonometric measures , carried out the protocol with a manual tonometer . The measurements were taken with the participants in supine position, with the knees flexed and the soles of the feet on the table, to ensure that both lower limbs were symmetrical and relaxed during the measurement. The measurement site was located by visualization and palpation in the largest area of muscle bulk during contraction  on both sides of the central perineal body. This area was selected because it contains the most contractile portion of the perineal muscles. Once the muscle was relaxed, a mark was placed with a dermographic marker to ensure the measurement site18. At this moment, the men were asked to keep the sexual organs away from the perineal body, without tension or traction of the skin42. The 100 mm long probe of the MyotonPRO® was placed perpendicular to the skin's surface, on the mark location, to perform the measurements , both in PF and lumbar muscles.All recordings were performed during five seconds of apnea after exhalation45: Frequency, characterizing muscle tension or tone in resting state (Hz); biomechanical properties, such as Stiffness (N/m) and logarithmic Decrement in the amplitude of oscillation (Ø), which defines the inverse of the elasticity; and viscoelastic properties, such as Relaxation time of stress (ms), and Creep (Deborah Number -De-), that characterizes fluidity. The MMPs evaluation has been demonstrated to be valid and reliable in different body locations30, including lumbar  and PF  regions, in healthy individuals.The description of the MMPs can be summarized as28. All procedures lasted less than 30 min for each subject.At the end of the procedures, a Visual analogue scale (VAS) was applied to identify any presence of pain and its intensity during the evaluationp > 0.05) and were described by the mean, standard deviation or 95% confidence interval (95%CI), and range. Questionnaire variables were not normally distributed and were described by median and interquartile range. Since some between-sides statistical differences were identified (p < 0.05) for the MMPs when submitted to paired Student t-tests, each side was analyzed separately.Frequencies and percentages were used to describe qualitative data. Age, BMI and all outcomes showed a normal distribution of data  coefficients were calculated. Correlations were considered negligible (0.0 to 0.19), fair (0.20 to 0.39), moderate (0.40 to 0.69), strong (0.70 to 0.89), or almost perfect (0.0 to 1.00)[48.Sociodemographic and clinical data were compared between sexes with unrepeated Student t-tests, except for the level of physical activity, which was compared with Xp < 0.05 was considered significant. The IBM-SPSS version 28® software  was used for the analyses.All contrasts were bilateral, and 2, 95%CI = −3.78, −0.36). Other sociodemographic and clinical data of both groups are included in Table Thirty-five nulliparous women and 35 men were analyzed. There were no differences between groups in age, while BMI was higher in men . At lumbar level, tone and stiffness were higher for men on both sides, and relaxation was lower on the left side. No differences were identified for decrement and creep Table .Table 2MFor the women group, age showed no correlation with any lumbopelvic MMP. However, at PF level, the BMI was directly related to the viscoelastic properties at both sides in fair to moderate fashion (0.364 < r < 0.452), that is, the higher BMI, the higher relaxation and creep. Tone, stiffness and decrement of PF showed no correlations with BMI.At lumbar level, the women showed a consistent trend of correlations between BMI and MMPs. Thus, tone and stiffness were moderate and inversely related to BMI (-0.412 < r < -0.480), while viscoelastic properties were moderate and directly related to BMI (0.465 < r < 0.522). The lumbar elasticity showed no relation with BMI. Finally, considering the clinical status of PF, no correlation was identified between PFIQ-7 and PFDI-20 and any lumbopelvic MMP Table .Table 3BFor the men group, the age was correlated inversely with tone on the right side, and directly with decrement, relaxation and creep in fair to moderate intensity, at both sides of PF (|0.334|< r <|0.446|). The age was also directly related to the decrement at lumbar level (0.395 < r < 0.470). Regarding BMI, all lumbopelvic MMPs, except the PF tone, were related with the BMI in fair to strong intensity at both sides (|0.385|< r <|0.719|). In all cases, the higher BMI, the lower tone and stiffness, while the higher BMI, the higher decrement, relaxation and creep.s < 0.438|)  than between-sexes differences (> 1.5 ms). Nevertheless, it was reported that the symmetry of MMPs could depend on specific states and disorders. Thus, when asymmetrical processes are considered, such as scoliosis60 or exercise34, could be related to the MMPs of PF in nulliparous adult women. Moreover, the absence of relation between the MMPs and age found in women could also be due to the age range of our sample, exclusively composed of young adults. In fact, in healthy subjects, sarcopenia begins around the age of 50 years, which was the high range limit of the current sample61, and the lower muscular development of the female sex and the presence of adrenal androgens after menopause could make sarcopenia less apparent before the climacteric period62.Regarding the correlation analysis, on the one hand, MMPs of PF showed no relationship with age and clinical status of PF and some low but significant associations with BMI in women. On the other hand, in men, MMPs were more correlated with age and BMI, and even the clinical status of PF showed correlations with MMPs of PF. This could mean that other features than age or BMI, such as hormonal status63, and tone alterations of PFM are related to postoperative male urinary incontinence64. Similarly, stiffness, as the resistance to deformation, is also relevant for PFM assessment65 and should be measured quantitatively25. Thus, a fast, innocuous and reliable MMPs assessment of PFM can help in PF examination27. In this sense, the internal vaginal probes used for dynamometry may induce changes in PFM recruitment by the mere presence of the probes22, and the size of levator hiatus can condition the evaluations66 in women. In men, a digital rectal examination is often used to evaluate PFM, but this invasive technique might cause discomfort, resulting in a lack of cooperation and poor outcomes67. On the contrary, the external application of myotonometry and its innocuousness is described as an advantage68.The assessment of MMPs in the lumbopelvic ring is of clinical relevance, since PF hypertonicity is often associated with urological, gynecological, gastrointestinal and sexual problems as well as chronic pelvic pain in both sexes64, which could be inadequate for muscle assessment. Furthermore, the PFM assessment does not commonly consider bilateral evaluations70, as occurs with dynamometry and electromyography, while the determination of MMPs allows individualizing each side, which is considered relevant under certain conditions, such as asymmetric diseases or deliveries42 as previously commented. Moreover, the assessment of MMPs with manual tonometry has demonstrated relevance in different fields72, including lumbar 46 and PFM73.Other previous methods used to objectively fvassess perineal body tone included, in men, the Beco perineometer, which measures the introflection values of the perineal body relative to the ischiatic spines. Nevertheless, this mechanical device determines the descent of the perineum in centimeters29, increase the interest of this method in clinical setting.In summary, the differences in MMPs depending on sex suggest that the physiology of these structures could also be different in healthy states, and, consequently, specific analysis approaches could be recommended for each sex. Moreover, the external application of myotonometry, its innocuousness, speed of application, bilateral evaluation, and low need for training74. The menstrual cycle phase was not controlled in the women group, which could be relevant in clinical evaluation60. Future research, considering other ages and clinical states, is recommended.Some limitations should be recognized. Only healthy non-obese young adults who did not practice regular exercise were included, and all women were nulliparous and non-climacteric. Therefore, the external validity of the results is limited to populations with similar characteristics. All evaluations were performed in the same rest positions, which could increase the consistency of the results, but other positions and states could lead to determining different resultsBetween-sexes differences in lumbopelvic MMPs depend on the specific location of the assessment in healthy non-obese young individuals, only recreationally active. Thus, the MMPs of PF differ between healthy non-climacteric adults of both sexes, with women showing higher values of tone and stiffness and lower values of elasticity and viscoelastic properties than men. In contrast, lumbar MMPs show less and lower differences. Only MMPs of men are related to age, BMI and clinical status of PF. Lumbopelvic MMPs evaluation with external tonometry could be relevant in clinical setting, due to its innocuousness and bilateral applicability.


Aim: The purpose of this study was to assess the effectiveness of syringic acid in preventing ischemia-reperfusion injury following detorsion in a rat model of induced testicular torsion.Material and methods: In our study, a total of 24 rats, eight in each group, were used. Group 1 served as the control group. Group 2 underwent testicular torsion and detorsion. Group 3 underwent the same procedures as Group 2, but also received 100 mg/kg syringic acid immediately following ischemia. Spectrophotometric analysis was performed on blood samples, and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the values of malondialdehyde (MDA), were evaluated under direct microscopic examination of the testis to determine tissue injury. The architecture of the seminiferous tubules and spermatogenesis processes were graded using the Johnsen and Cosentino scoring systems.Results: The mean value of MDA was higher in Group 2 compared to the other groups (p < 0.001). Group 3 demonstrated a decline in the concentrations of proinflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-6, and interleukin-1 beta, as indicated by biochemical analysis of blood samples, when compared to Group 2 . In addition, the improvement in Johnsen and Cosentino scores was significantly higher in Group 3 compared to Group 2 .Conclusion: These findings suggest that syringic acid has a protective effect against testicular oxidative damage. Acute scrotum, characterized by sudden scrotal pain, redness, and swelling, is considered a urological emergency. Testicular torsion, accounting for approximately 35% to 42% of acute scrotal conditions, is the cause of this uropathy . The speThis study investigated the effects of syringic acid on ischemia-reperfusion injury in rats. This study utilized a total of 24 male Wistar albino rats, aged 12 weeks, with an average weight of 325 g. The study was conducted in accordance with institutional guidelines and followed the principles outlined in the Guide for the Care and Use of Laboratory Animals of the National Research Council. Ethical approval for the study was obtained from the Tokat Gaziosmanpaşa University Animal Studies Ethical Committee . The rats were housed in controlled conditions with a temperature range of 20-23°C and a 12-hour light/dark cycle. They were provided with standard pellets and water ad libitum to ensure their nutritional needs were met. The rats were randomly divided into three groups: Group 1 (control), Group 2 (ischemia-reperfusion), and Group 3 (treatment). Group 1 underwent only orchiectomy and no other treatments, while Group 2 underwent testicular torsion and detorsion with no medication administered. Group 3 received a single dose of 100 mg/kg of syringic acid via oral gavage immediately after ischemia with the syringic acid. Syringic acid was dissolved in dimethyl sulfoxide (DMSO) and diluted using 0.9% physiological saline solution just prior to administration. In Groups 2 and 3, ischemia-reperfusion injury was induced in the rat testes using the following method: the testis was pulled out through an inguinoscrotal incision, rotated 720 degrees in a clockwise direction, and then secured to the scrotum for a period of three hours using a 5.0 prolene suture. This procedure aimed to create a controlled model of testicular torsion to study the effects of ischemia and subsequent reperfusion. After the ischemic period, the testis was detorsioned and left in its normal anatomical position for another three hours to assess the reperfusion injury. In the final step of the procedure, orchiectomy was performed to obtain testis tissue for histopathological investigation, and blood samples were collected from the inferior vena cava for biochemical analysis. The surgical procedures were conducted under appropriate anesthesia and in sterile conditions. To achieve this, the rats were administered a combination of two anesthesia agents, i.e., xylazine hydrochloride  at a dose of 10 mg/kg via the intraperitoneal route and ketamine hydrochloride  at a dose of 50 mg/kg administered via the intraperitoneal route. Xylazine hydrochloride acts as a sedative and muscle relaxant, while ketamine hydrochloride has dissociative anesthetic effects. These agents were used to ensure the rats' comfort and minimize pain during surgical procedures.Biochemical evaluationMeasurement of Malondialdehyde LevelThe study of lipid peroxidation was performed using the Esterbauer method, which measures the levels of malondialdehyde (MDA) in the sample. The method involves a reaction between MDA and thiobarbituric acid at 90-95°C, which creates a pink-colored chromogen. The intensity of the pink color is proportional to the amount of MDA present in the sample, and this can be used to quantify the level of lipid peroxidation. After the reaction between MDA and thiobarbituric acid, the samples were rapidly cooled and then spectrophotometrically read at 532 nm after 15 minutes. The level of MDA was then expressed as nmol/mL. This method, described by Esterbauer and Cheeseman in 1990, provides a way to measure the level of lipid peroxidation in a sample by quantifying the amount of MDA produced during the oxidation of lipids .Measurement of Glutathione Peroxidase ActivityThe activity of glutathione peroxidase (GSH-Px) was assessed using the method described by Paglia et al. . This meMeasurement of Superoxide Dismutase ActivityThe activity of the superoxide dismutase (SOD) enzyme was assessed by modifying the method described by Sun et al. . This meMeasurement of Tumor Necrosis Factor Alpha, Interleukin-1 Beta, Interleukin-6 LevelsTumor necrosis factor alpha , interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6) levels were quantified using ELISA kits on the Thermo Scientific Multiskan FC Microplate device , following the manufacturer's instructions. The ELISA kits were sourced from BioAssay Technology Laboratory, and their % CV (coefficient of variation) values were below 10, indicating reliable assay reproducibility.Histopathological evaluationThe testes taken from the rats were preserved in a 10% formalin solution buffered with a buffer for a duration of two days. After being trimmed and processed, the samples were cut into 4-micrometer sections using a microtome and then stained with hematoxylin and eosin dyes. The tissue slides were examined using an upright light microscope . The pathologist was blinded to the groups. The structure of the seminiferous tubules, the processes of spermatogenesis, and the maturity of germ cells were evaluated using the Johnsen and Cosentino scoring systems. The Johnsen scoring criteria, which range from 1 to 10, were used to evaluate the samples as follows: a score of 1 indicates fibrosis, with only Leydig and peritubular myoid cells present and germ and Sertoli cells absent; 2 indicates Sertoli cells are present but do not involve germ cells; 3 indicates only spermatogonia are present; 4 indicates spermatogonia and few spermatocytes are present; 5 indicates no spermatozoa or spermatids are present, but many spermatocytes are present; 6 indicates few early spermatids are present; 7 indicates spermatogonia, spermatocytes, and early spermatids are present but no spermatozoa and late spermatids; 8 indicates only a few spermatozoa are present; 9 indicates spermatogonia, spermatocytes, spermatids, and spermatozoa are present but disorganized; 10 indicates normal spermiogenesis . The injStatistical analysisAll statistical analyses were performed using SPSS 22.0 software  for Windows. P < 0.05 was considered statistically significant in all analyses. The Shapiro-Wilk test was used to assess the distribution. For analysis, the t-test, one-way analysis of variance (ANOVA) test, and post hoc Bonferroni analysis were used for parametric variables, and the Mann-Whitney U test and Kruskal-Wallis tests were used for nonparametric variables. Data are presented as a median and interquartile range for nonparametric variables and mean ± standard deviation for parametric variables.In Group 1, the mean levels of TNF-alpha, IL-6, and median IL-1 beta were measured as 486.5 ± 94.2 ng/ml, 12.1 ± 3.3 ng/ml, and 598 (508-721) pg/ml, respectively. Group 2 exhibited significantly higher levels of pro-inflammatory cytokines, including IL-1 beta, IL-6, and TNF-alpha  when compared to Group 1. In Group 3, the mean levels of TNF-alpha and IL-6 and the median value of IL-1 beta were measured as 591.8 ± 85.8 ng/ml, 12.7 ± 2.6 ng/ml, and 598 (501-759) pg/ml, and they were found to be significantly lower than those in Group 2  . Although there was an increase in SOD and GSH-Px activities in Group 3 compared to Group 2, no statistically significant difference was found between the groups . Additionally, the levels of MDA, a marker of oxidative stress, were found to be significantly higher in Group 2 compared to Group 1 (p < 0.001). When Group 3 was analyzed, a decrease in MDA levels was noted compared to the increase in Group 2 (p = 0.001) . However, when the same scores were analyzed in Group 3, positive changes were observed compared to Group 2   level, which had initially increased. In diabetic rats, the administration of syringic acid resulted in an upregulation of renal mRNA expression of PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator) and NRF-1 (nuclear respiratory factor 1). Additionally, it increased the mitochondrial DNA (mtDNA)/nuclear DNA (nDNA) ratio, which indicates mtDNA content relative to nDNA, whereas these values were reduced in the diabetic group that did not receive the treatment. Thus, the authors concluded that syringic acid modulates renal antioxidant defense mechanisms. In another study, Sabahi et al. conducted a study to assess the protective effect of syringic acid against diabetes-induced cardiac injury in experimental rats . In theiIn our study, we found that Cosentino and Johnsen scoring systems, which assess the morphology of seminiferous tubules, germ cell morphology, and spermatogenesis processes, demonstrated a negative change in response to ischemia-reperfusion injury. However, a significant improvement was observed in Johnsen and Cosentino scores in Group 3, where we gave 100 mg/kg syringic acid following testicular ischemia, compared to Group 2, which was not treated in our study. Significant improvement in Cosentino and Johnsen's scores observed in Group 3 compared to Group 2 supports the idea that syringic acid has a protective effect against oxidative damage. Furthermore, we observed an increase in the levels of MDA, which is an end product of lipid peroxidation, in rats with induced ischemia-reperfusion injury. In our study, it was determined that the MDA level decreased significantly in Group 3 with the application of syringic acid (100 mg/kg) following testicular ischemia. Additionally, there was a decrease in the levels of SOD and GSH-Px, which are important antioxidant enzymes in organisms, indicating a decline in antioxidant enzyme capacity in our study. Although there was no statistically significant difference in our study, it was determined that there was less decrease in SOD and GSH-Px values in Group 3 compared to Group 2. This may be due to the small number of rats included in our study, but it suggests that this difference may become more significant in studies involving more rats. It has been shown that there is an increase in proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1 beta in the serum due to ischemia-reperfusion injury in several studies . ConsistOur study provided evidence that syringic acid has a protective effect against ischemia-reperfusion injury in testicular tissue. Additionally, the present study has yielded promising data regarding the use of syringic acid in routine urology practice to mitigate the effects on the reproductive system in cases of testicular torsion. However, to further validate our findings, randomized prospective clinical trials are needed. These trials will provide additional support for the efficacy of syringic acid in treating testicular torsion. Furthermore, more comprehensive studies should be conducted to determine the appropriate dosage range and optimal timing for administering syringic acid in different timeframes of testicular torsion. We believe that future studies should focus on revealing the potential side effects of syringic acid when used for its protective effect against testicular damage. Additionally, it would be important to develop appropriate algorithms to address any potential complications associated with the administration of the drug. This will contribute to a better understanding of the safety profile and facilitate the optimal use of syringic acid in clinical settings.LimitationsThe main limitation of our study is that we focused solely on the early effects of syringic acid on testicular torsion, administering only a single dose. Consequently, we were unable to investigate the long-term response of testicular tissue to this pharmacological agent. Future studies should consider assessing the prolonged effects and optimal dosing regimen of syringic acid for testicular torsion. In addition to the aforementioned limitation, it is important to acknowledge other limitations of our study. First, we were unable to demonstrate the effects of syringic acid on testicular tissue in healthy rats, which would have provided valuable comparative data. Furthermore, we did not evaluate hormone profiles, which could have provided additional insights into the mechanisms of action. Lastly, the absence of immunohistochemical analyses in our pathological examinations limited our ability to investigate specific cellular markers and molecular pathways. These limitations highlight areas for improvement and future research directions in studying the effects of syringic acid on testicular tissue.In our study, we found that the administration of syringic acid attenuated tissue damage caused by ischemia-reperfusion. Specifically, we observed an increase in the serum levels of antioxidants, such as SOD and GSH-Px, while the levels of MDA, a marker of lipid peroxidation, decreased. Furthermore, in rats treated with syringic acid, we observed improvements in seminiferous tubule morphology, spermatogenesis processes, and scores according to both the Johnsen and Cosentino scoring systems, indicating enhanced germ cell maturation. According to the findings from this experimental investigation, the utilization of syringic acid presents itself as a viable alternative therapeutic approach to alleviate the ischemia-reperfusion injury following detorsion interventions in individuals afflicted with testicular torsion. To gain a better understanding of the molecular mechanism underlying the efficacy of syringic acid, it is important to conduct prospective, randomized, and controlled clinical studies in the future. These studies will provide more robust evidence and further validate the findings obtained in our study.


Background: Melasma is a complex skin disorder characterized by brown or dark patches, primarily affecting facial areas. Despite numerous treatment options, the effective management of melasma remains challenging. This study aims to fill a gap in the literature by rigorously comparing the effectiveness of three prevalent chemical peeling agents, 15% trichloroacetic acid (TCA), 15% phenol, and 2% glycolic acid, in treating melasma.Materials and methods: A randomized controlled trial was conducted involving patients who were clinically diagnosed with melasma. Participants were divided into three groups, each receiving one of the chemical peeling treatments. The primary measure of efficacy was the Melasma Area and Severity Index (MASI) score, recorded before and after the treatment series. Side effects were also documented and analyzed.Results: Preliminary findings suggest a significant reduction in MASI scores in the group treated with 15% TCA peel. The average MASI score reduction was 8.5 points for the TCA group, 6.0 points for the phenol group, and 5.2 points for the glycolic acid group. Side effects such as redness and mild irritation were noted but were least prevalent in the TCA group.Conclusion: Our study indicates that 15% TCA peel is not only effective but also comparatively safer in treating melasma. It shows a more rapid and significant improvement in reducing melasma symptoms than 15% phenol and 2% glycolic acid peels. However, further research is warranted to validate these findings over a larger population. Melasma is a dermatological condition that affects a significant portion of the global population. Characterized by the development of brown- or dark-colored patches on the skin, this condition predominantly affects the facial region, especially sun-exposed areas like the forehead, cheeks, and upper lip . MelasmaIn this context, chemical peeling has emerged as a promising treatment modality for melasma and other benign skin conditions. Chemical peeling involves the application of a chemical solution to the affected skin area, causing the top layer of skin to exfoliate and eventually peel off. This results in the exposure of a new layer of skin that is usually smoother and less wrinkled than the old skin . While vThis research conducted a randomized controlled trial (RCT) from November 1, 2022, to January 15, 2023, involving a total of 20 patients. These patients were selected from the outpatient department of Surya Hospital and the Department of Periodontology at ITS Dental College, Ghaziabad, India. The study was divided into two primary groups as Group I, comprising 10 patients treated with 15% TCA peel, and Group II, comprising 10 patients treated with a combination peel of 15% TCA, 15% phenol, and 2% glycolic acid.The trial was designed to have several follow-up periods: at baseline and after 15 days, 30 days, 45 days, 60 days, and 75 days. Prior to the initiation of the study, written informed consent was obtained from all participating patients.Inclusion criteria were systematically healthy individuals, adults aged over 18 years, clinical diagnosis of melasma, and capacity to provide informed consent. Exclusion criteria were patients with a recent history (last 12 months) of hormone therapy, antiepileptic medications, antidepressants, or treatments for thyroid conditions, patients undergoing anticoagulant therapy or diagnosed with coagulopathy, pregnant women and women on oral contraceptive pills, and patients with a history of keloids or recurrent herpes infection.During the initial visit, demographic and clinical details were recorded for each patient. This included time of onset of the disease, duration of the disease, history related to photosensitivity, drug intake, endocrine disorders, menstrual cycles, occupation, allergic disorders, and contraception, and family history of skin conditions. Patients were informed about the treatment process, potential side effects, expected timeline for seeing results, and post-treatment care. Written informed consent was reaffirmed at this stage.Modified Melasma Area and Severity Index (mMASI)mMASI  was calcBefore the application of the peeling agent, each patient was advised to clean their face using a mild cleanser and water. Once the face was patted dry, a degreasing solution was applied to remove any residual oils from the skin. For both groups, the designated peeling agent was applied using Q-tips  or gauze in gentle strokes. After a contact period of two minutes, the agent was neutralized using a specific neutralizing solution. This procedure was repeated at various follow-up intervals: at baseline and after 15 days, 30 days, 45 days, 60 days, and 75 days. After each peeling session, patients were advised to apply a topical sunscreen with a sun protection factor (SPF) of 50. They were educated on the importance of post-peel skincare to sustain the benefits gained from the chemical peeling process. Specifically, patients were instructed to use a mild, non-detergent cleanser for facial washing, apply a deep moisturizer to prevent skin dryness and crust formation, and refrain from using cosmetics for at least one week post peel.In this study, 20 patients were evenly divided into two groups. Group I underwent treatment with 15% TCA peel, and Group II was treated with a combination of 15% TCA, 15% phenol, and 2% glycolic acid peels. Both groups had follow-up sessions and repeated treatments at baseline and after 15 days, 30 days, 45 days, 60 days, and 75 days.Table Both groups were in the range of 23-47 years. In this study, the mean age of Group I was 32.6± 4.4 years and that of Group II was 31.4 ± 6.75 years. There was no significant difference between the age distributions (p>0.05), gender distributions (p=1.000), and occupation-wise distributions (p=1.000) in both groups. In each group, out of 10 patients, only one patient was male and the remaining nine were females; eight subjects were non-working and two subjects were working.Table Pairwise comparison showed that reduction in mMASI score in group I from baseline to 15 days, baseline to 30 days, baseline to 45 days, baseline to 60 days, and baseline to 75 days was statistically significant. Also reduction in mMASI score from 15 days to 30 days, 15 days to 45 days, 15 days to 60 days and 15 days to 75 days was significant. Reduction in mMASI score from 30 days to 45 days and to 75 days was also significant. Reduction in mMASI score from 45 days to 75 days was significant. However, there was no change in mMASI scores from 45 days to 60 days and from 60 days to 75 days in Group I  and its modified version (mMASI) are commonly used clinical indicators for assessing melasma severity ,8,9. OurThe three prevalent chemical peeling agents, namely, 15% TCA, 15% phenol, and 2% glycolic acid, exhibit distinct mechanisms of action in treating melasma. TCA is a medium-depth chemical peel that works by causing controlled chemical injury to the skin. It penetrates the epidermis and reaches the upper layers of the dermis. TCA promotes skin exfoliation and stimulates collagen production. This mechanism helps to reduce pigmentation in melasma by shedding the top layers of pigmented skin cells and promoting the growth of healthier, less pigmented skin. Phenol is a deep chemical peel that penetrates deeply into the dermis. It acts as a strong exfoliating agent and also has a bleaching effect on the skin. Phenol peels can target melanin-producing cells (melanocytes) in the skin, inhibiting melanin production and reducing the appearance of melasma. Glycolic acid is an alpha-hydroxy acid (AHA) that works as a superficial chemical peel. It exfoliates the top layer of the skin (epidermis) by breaking down the bonds between dead skin cells. While it may not penetrate as deeply as TCA or phenol, it can still help improve melasma by promoting skin cell turnover and reducing the appearance of pigmentation ,13.Our study noted some complications, such as redness and post-inflammatory hyperpigmentation, in the 15% TCA group. However, no such complications were observed in the combination peel group, a finding worth noting given that other studies have reported various complications following chemical peels ,14. ChemIn the contemporary landscape of melasma treatment, phototherapy, including intense pulsed light (IPL) therapy, is widely used and effective. The study did not consider IPL therapy as one of the treatment modalities, which could be a limitation as it represents a commonly used option for melasma management.This study provides valuable insights into the potential efficacy of 15% TCA peel, 15% phenol peel, and 2% glycolic acid peel as treatment options for melasma. While 15% TCA peel showed the most promising initial results, it is essential for clinicians to consider individual patient characteristics and preferences when choosing the most appropriate treatment for melasma. Further research is necessary to refine treatment protocols, assess long-term outcomes, and evaluate safety profiles to enhance the management of this complex skin disorder.


Although Acute Kidney Injury (AKI) incidence is increasing worldwide, data investigating its cost are lacking. This population-wide study aimed to describe the characteristics and costs of hospital stays with, and without AKI, and to estimate the AKI-associated increases in costs and length of stay (LOS) in three subgroups , cardiovascular surgery with extracorporeal circulation (CVEC), and sepsis).th International Classification of Diseases and the medical acts classification. In each subgroup, the adjusted increase in cost and LOS associated with AKI was estimated using a generalized linear model with gamma distribution and a log link function.All hospital stays that occurred in France in 2018 were included. Stay and patient characteristics were collected in the French hospital discharge database and described. Medical conditions were identified using the 10vs. €735) and longer (median [IQR] 9[4–16] vs. 0[0–2] days). AKI was associated with a mean [95%CI] increase in hospitalization cost of 70% , 48% , and 68%  in the sepsis, CVEC, and MOV groups respectively, after adjustment.26,917,832 hospital stays, of which 415,067 (1.5%) with AKI, were included. AKI was associated with 83,553 (19.8%), 7,165 (17.9%), and 15,387 (9.2%) of the stays with sepsis, CVEC, and MOV, respectively. Compared to stays without AKI, stays with AKI were more expensive (median [IQR] €4,719 This study confirms the major economic burden of in-hospital AKI in a developed country. Interventions to prevent AKI are urgently needed and their cost should be balanced with AKI-related costs.The online version contains supplementary material available at 10.1186/s12882-023-03396-8. Acute Kidney Injury (AKI) may concern 1.6% to 20% of hospitalized patients, depending on the methodology used and the geographical area concerned . At the The aim of the present study was to describe in a large, recent, and unselected population, the characteristics and costs of hospital stays with AKI (AKI stays) and those without (no-AKI stays), and to estimate the increase in stays’ costs from the national insurance perspective and LOS, associated with AKI in three subgroups of interest: major open visceral surgery (MOV), cardiovascular surgery with extracorporeal circulation (CVEC), and sepsis.Programme de Médicalisation des Supports d’Information, PMSI). PMSI is hosted and controlled by the technical agency for information on hospital care . According to French law, data were collected via the platform of the secure data access center  after a declaration to the national institute of health data  through the reference methodology 006 (MR-006), n°2214295. Because all patient-level data in the PMSI database are anonymized, institutional review board, ethics approval, and informed consent at an individual patient level were not required.This study is a retrospective cross-sectional population-wide database study, using the French hospital discharge database (th revision (ICD-10) codes. The PMSI also contains all medical procedures performed during the stay, encoded according to the French medical classification of clinical procedures .The PMSI contains a discharge summary for each single stay that occurs in each public or private hospital in France . Each stay is identified by a main diagnosis  and several associated diagnoses . All diagnoses are registered in the PMSI for billing purposes, according to the International Classification of Diseases, 10According to the codes entered in the hospital stay resume (including codes for AKI), each stay is classified within a Diagnosis Related Group (DRG) of patients that shares common characteristics and have similar resources use. This DRG code is used to determine the reimbursement of the stay by the national health insurance system to the hospital. For each DRG, two tariffs coexist according to the hospital status (private or public). Supplementary fees are added to the DRG for particular procedures such as RRT, particular stays such as ICU stays and costly treatments.st, 2018 and December 31st, 2018 were included. AKI stays were identified by the presence of at least one of the following ICD-10 codes : acute kidney failure (N17*), post-procedural acute kidney failure (N990), post-partum acute kidney failure (O904), renal failure following ectopic and molar pregnancy (O084), and extrarenal uremia (R39.2). The following codes for RRT related to AKI management were also used to identify AKI patients: JVJF002, JVJF005, JVJF006, JVJF007, JVJB002. A single patient may have experienced several hospitals stays during the same year and could therefore be included more than once in the present study.All hospital stays registered in the PMSI between January 1Three subgroups of stays associated with medical procedures or conditions at risk for AKI were defined a priori. The stays with MOV and those with CVEC were identified using the corresponding CCAM codes , geographical area of residence, origin of admission  from a different hospital or emergency room (ER)), discharge destination , and LOS. The medical characteristics collected were the main diagnosis, the medical procedures performed during the stay, the treatments received during the stay , and death during the stay. Patient characteristics such as age and sex were also collected. Because they are the main comorbidities associated with AKI (susceptibilities), three medical conditions were collected using the corresponding CCAM and ICD-10 codes: cardiac failure, diabetes, chronic kidney disease . For each stay, the associated DRG  and the corresponding tariff were collected. Additional reimbursements that occurred in case of an ICU stay, medical procedures such as RRT, or costly treatments were also collected and included in the total cost of stay.The statistical unit for analysis was the hospital stay. The characteristics of AKI and no-AKI stays were described using mean and standard deviation (SD) or median and interquartile range (IQR) for continuous variables, and frequencies and percentages for categorical variables. Because the whole population was included without sampling, statistical tests were not relevant to compare populations. Missing data were not replaced.t-test or a Wilcoxon-Mann–Whitney test for continuous variables depending on the distribution and a McNemar test for categorical variables. Within matched populations, the effect of AKI on the cost of stay and LOS was estimated using a generalized linear model with a gamma distribution and a log link function. The model provided estimates for mean (95%CI) increase in cost and LOS in AKI stays, adjusted for chronic kidney disease, diabetes, and cardiac failure.To estimate the increase in cost and LOS associated with AKI in the three subgroups, a case–control analysis was performed. Control stays (no-AKI stays) were matched to AKI stays with a 1:3 ratio . Matching was considered acceptable beyond 90% of patients matched. If less than 90% of patients were matched, a lower ratio was used (1:2 then 1:1). For each AKI stay, control stays were selected randomly among the no-AKI stays sharing the same age class, sex, geographical area of residence, hospital status, and medical condition associated . Two different stays for a single patient could not be paired together. Characteristics of AKI stays and no-AKI stays were compared within each subgroup, using a Student’s The 2018 PMSI database contains 26,917,832 stays, of which 9,458,453 (35.1%) were admissions for specific procedures and health care . All of these PMSI stays were included in the main analysis and a code for AKI was present in 415,067 stays (1.54%). An explicit code for sepsis was present in 421,543 (1.6%) stays, a code for CVEC in 40,084 (0.1%) stays, and a code for MOV in 166,831 (0.6%) stays. An AKI code was associated with 83,553 (19.8%), 7,165 (17.9%), and 15,387 (9.2%) of the sepsis, CVEC, and MOV stays respectively. The study population flow-chart is reported in Fig. n = 18,212,482), and 87.7% of the AKI stays . When compared to no-AKI stays, AKI stays concerned more often male patients  and older patients (mean [SD] age 74.2[16.4] vs. 55.8[23.9]). AKI stays were mainly admitted from the ER , whereas no-AKI stays were mainly admitted from home . Discharge was more frequent to home for no-AKI stays  than for AKI stays . Death occurred in 59,338 patients with AKI during their hospital stay (14.3%) and in 237,673 patients without AKI , “respiratory tract infections, with complications”  and “other kidney and urinary tract diseases, with complications” .Public hospitals accounted for 67.7% of the total stays in 2018 . However, death occurred more frequently in the sepsis and AKI stays than in the surgery and AKI stays . The characteristics for each subgroup of stays are presented in the Supplementary Table Surgical stays (CVEC and MOV) with AKI required more often RRT than stays with sepsis and AKI (CVEC: vs €735.5 [382.8–1805.4]). Patients with AKI required more medical procedures  than those without AKI  required RRT during their stay and 17.2%  had at least one admission to the ICU whereas only 0.8%  of those without AKI were admitted to the ICU. The additional cost of an ICU stay was higher for AKI-stays compare to no-AKI stays .The total cost of stay and its main components are shown in Table .5 382.8–805.4). Pvs. 0 [0–2] days); 56%  of the AKI stays lasted > 8 days whereas 75.7%  of the no-AKI stays lasted < 2 days and 8.2%  lasted > 8 days.Stays with AKI were longer  and the LOS by 1.33 times . In the CVEC group, the presence of AKI increased the hospital cost by 1.48 times  and the LOS by 1.47 times . In the MOV group, the presence of AKI increased the hospital cost by 1.68 times  and the LOS by 1.41 times . Although a similar incidence of AKI has been previously reported in studies conducted retrospectively from large databases, it contrasts with the higher incidence of AKI reported when prospective methodologies are used . This suConcerning subpopulations, AKI incidence in surgical stays was similar to the results observed in a recent epidemiological study . For sepAmong the three observed subgroups, the highest increase in costs associated with AKI was observed in the sepsis group (+ 70%), whereas the most expensive and the longest stays were observed in the CVEC and MOV stays with AKI. Thus, although AKI in surgical patients is less frequent than AKI in patients with sepsis, the former should also be a major concern for policymakers. Bundles of nephroprotective measures have been successfully tested in surgical populations and their use should be promoted in these populations , 16.Herein, a higher incidence of AKI was observed in public hospitals compared to private hospitals and may reflect the greater complexity and severity of the case-mix in public hospital compare to the one in private hospitals.Previous studies found that up to 45% of AKI patients have not fully recovered their renal function at discharge and are at higher risk of chronic kidney disease, cardiovascular diseases, and death within five years , 28. In To the best of our knowledge, the present study is the most recent and largest economic study evaluating the financial burden of AKI in a national insurance health system. A nationwide population was included without further selection, allowing for a pragmatic analysis. In addition to the broad description of costs in the whole population, the case–control analysis provided more precise estimates of the increase in costs associated with AKI, accounting for confounding factors, in different populations. Finally, in 2018, the PMSI was filled with updated ICD-10 codes, using an improved and easier classification of AKI, probably reducing the misclassifications of AKI compared to the previously used ICD-9 codes.. found that the ICD-9 codes used for AKI had a low sensitivity of 35.4%, but a very high specificity of 97.7% to diagnose creatinine-confirmed AKI. Therefore, they suggested that the observed prevalence of AKI out of medical claims filled with ICD-9 codes may be underestimated. They also pointed out that false negatives mainly concern small changes in serum creatinine, that might be unnoticed or not perceived as significant by the medical team [plus a code for organ dysfunction, leading to an underestimation of the number of sepsis stays. Conversely, as we did not verify the presence of organ dysfunction for each sepsis-stay, we may have considered as sepsis certain cases that did not meet sepsis-3 criteria, leading to the selection of a less severe population. However, even if we had specifically sought organ dysfunctions, we would have been unable to determine whether they occurred simultaneously with the infection (defining sepsis) or not. Finally, this study is primarily an economic study, not an epidemiological one, and its aim was not to identify the incidence, determinants, or outcomes of AKI.Nevertheless, the present study has certain limitations. First, the severity of AKI and its cause cannot be determined using the PMSI. Because dates cannot be entered along with ICD-10 codes, causal relationship between diseases cannot be assessed from this database. For example, if a stay is associated with an AKI and a sepsis code, it is impossible to know what disease occurred first or if they occurred at the same time. Second, the quality (exhaustivity and exactitude) of the codes entered may be questioned. The correlation between AKI codes in databases used for billing purposes, such as the PMSI, and clinically confirmed AKI is largely debated. In a previous study, Waikar et alcal team . Recent cal team . Third, This population-wide study confirmed the major economic burden associated with in-hospital AKI episodes in a developed country and found that AKI was associated with an increase in costs ranging from 48 to 70% in three subgroups of at-risk patients. This should encourage further investigations to reduce the incidence of AKI, particularly among patients with a high risk of AKI such as those with sepsis or undergoing major surgeries. The cost of these strategies should be balanced with the high costs driven by AKI itself.Additional file 1.