The search for primary studies was performed in four databases. The sample consisted of 11 surveys. The methodological quality of the included studies was assessed using tools proposed by the Joanna Briggs Institute. Data analysis and synthesis were performed in a descriptive manner. surgical site infection rates ranged from 0.4% to 7.6%, considering the results of primary studies, in which patients underwent laparoscopic surgery. In surveys of participants undergoing surgical procedures with different approaches , infection rates ranged from 0.9% to 12%. Regarding the risk factors for the development of this type of infection, antibiotic prophylaxis, female sex, high Body Mass Index and perioperative hyperglycemia are highlighted.  conducting the integrative review generated a body of evidence that reinforces the importance of implementing effective measures for the prevention and control of surgical site infection, by health professionals, after bariatric surgery, promoting improved care and patient safety in the perioperative period. This disease has multifactorial causes linked to environmental, economic, genetic, metabolic and lifestyle aspects. Through the Body Mass Index (BMI), the World Health Organization defines the diagnosis of obesity, that is, BMI ≥ 30 kg/m-Bariatric surgery is considered a therapeutic option for the treatment of obesity, when conservative clinical treatment  has not been successful and after careful evaluation by a multidisciplinary team. In addition to the nutritional status and physical conditions of the patient, the team must assess mental health, since the success of the surgery depends on changes in lifestyle, eating habits and the search for emotional balance of the individual who will undergo the procedure. In short, bariatric surgery leads to weight loss, with improvement in body parameters, as well as a reduction in morbidity and mortality associated with obesityCurrently, in bariatric surgery, two types of procedures are the most chosen by surgeons, namely, sleeve gastrectomy and Roux-en-Y gastric bypass, which can be performed in open, laparoscopic approaches  or robotics (robot-assisted surgery)Complications after bariatric surgery can be classified as early (during the immediate postoperative period) or late . Depending on the type of surgical procedure, early complications include hemorrhage, anastomotic leakage, gastric or small bowel perforation, and deep vein thrombosis/pulmonary embolism, and major late complications are bowel obstruction, gallstone formation, and gastrointestinal hemorrhage-Surgical site infection (SSI) is also a relevant complication that can affect patients undergoing bariatric surgery, since obesity is a risk factor for the development of this type of infectionThe prevention and early treatment of complications are essential to achieve better results for the patient and, consequently, the success of the therapy. Thus, the performance of a multidisciplinary team is crucial, from the preparation for the surgery and the follow-up in the postoperative period. In this context, the nurse has a prominent role, since this professional is responsible for planning and implementing the necessary nursing care and for health education. In addition, they must have knowledge about complications after bariatric surgery, aimed at their prevention and early detection, effectively helping the patient’s well-being and the new condition of life.In view of the above, the synthesis of knowledge produced on SSI in bariatric surgery can help the multidisciplinary team, contributing to the improvement of care provided and patient safety, especially nursing care. Thus, the delimited objective of the present review was to evaluate the evidence on the risk factors for the development of surgical site infection in bariatric surgery.The method of knowledge synthesis selected for conducting this study was the integrative review of the literature (IR). The steps covered were: elaboration of the review question, literature search of primary studies, evaluation of primary studies, data analysis and presentation of the reviewOpen Science Framework (OSF). This platform is open, and the main objective is to support the conduct of research and allow collaboration between researchers, in a global context. The protocol registration took place on April 28, 2021, and the access link was: https://archive.org/details/osf-registrations-fxr6v-v1 and DOI: 10.17605/OSF.IO/FXR6.The IR protocol was registered in the The IR was carried out in the city of Ribeirão Preto, São Paulo State, Brazil.The study was carried out from March 2021 to March 2022.The review question was: “what evidence is available in the literature on risk factors for the development of surgical site infection in bariatric surgery?” To elaborate this question, the acronym PECO was adopted , where P= patient submitted to bariatric surgery; E= risk factors; C= not applicable and O= surgical site infection.Eligibility criteria for the development of IR were: primary studies whose authors investigated risk factors for the development of surgical site infection in bariatric surgery in patients aged ≥ 18 years; published in English, Portuguese, Spanish and from January 2011 to April 2021.In view of the above, editorial, response letter, secondary studies , experience report or expert opinion were excluded from the review sample. The time frame was established to ensure an adequate number of primary studies, since the inclusion of a high volume of research can make it impossible to conduct an integrative review or introduce biases in the following steps of the method.Four databases, relevant to the area of health and nursing, were selected for the search for primary studies, namely: PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Scopus and Latin American and Caribbean Literature in Health Sciences (LILACS).The three described components of the PECO acronym were used in different combinations of controlled descriptors, keywords and the Boolean operators AND and OR (search strategies for publications in the databases). In two databases (PubMed and Scopus), the controlled descriptors were delimited from the Medical Subject Headings (MeSH) and the search strategies adopted were: “Bariatric Surgery”[Mesh] OR “Bariatric Surgery” OR “Metabolic Surgery” OR “Bariatric Surgical Procedure” OR “Surgical Procedures, Bariatric” OR “Bariatric Surgeries” OR “Roux-en-Y gastric bypass” OR “Sleeve gastrectomy” OR “Weight Loss Surgery” OR “Gastric bypass” OR “Laparoscopic Adjustable Gastric Banding” OR “Duodenal Switch” AND “Surgical Wound Infection”[Mesh] OR “Surgical Wound Infections” OR “Surgical Wound Infection” OR “Surgical Site Infection” OR “Surgical Site Infections” OR “Postoperative Wound Infections” OR “Postoperative Wound Infection” OR “Wound Infections” OR “Wound Infections Surgical” AND “Risk Factors”[Mesh] OR “Risk Factors” OR “Risk Factor”.In the CINAHL and LILACS databases, the search strategies adopted were similar, but using the base vocabulary (controlled descriptors), that is, CINAHL Headings and Health Science Descriptors (DeCS), respectively. In the databases, the final search strategies for publications were implemented on May 2, 2021.The EndNote reference manager (version XII - Desktop) was used to remove duplicates of exported results (publications) from the four databasesThe Rayyan platform was used for the selection of primary studies among the reviewersThe full reading of the selected primary studies (n=36) was also performed by two reviewers independently. In the event of discrepancies, a third reviewer was consulted to resolve the queries and assist in the final selection of studies included in the IR sample.In addition to searching the databases, a reviewer manually searched for other research in the references of the primary studies included in the IR, and no new studies were included using this strategy.The search and selection of primary studies took place from May to July 2021.To collect data from the studies included in the review, a script was built with the following items: authors; study title; year of publication; journal name; objective; sample and method detail; statistical analysis; data on the occurrence/incidence of SSI; main results and conclusion. This step was carried out, from August to October 2021, by two reviewers, independently, and through meetings, differences were discussed until consensus.The identification of the type of study was according to the name given by the authors of the research included in the review. It should be noted that in two surveys, the necessary information was not found. The studies were called retrospective, since the data were collected from a database entitled Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (United States of America).The methodological quality of the primary studies was assessed using tools developed by the Joanna Briggs Institute. This international organization provides free tools for each type of study, that is, randomized clinical trial, quasi-experimental study, cohort study, cross-sectional study, among others. Such tools are composed of questions, and for each one, the reviewer answers yes, no, uncertain or not applicable. Through these questions, the internal validity and risk of bias of the study are evaluated The tool entitled JBI Critical Appraisal Checklist for Studies Reporting Prevalence Data was used to evaluate prospective or retrospective studies. For the evaluation of cohort studies, the tool adopted is called JBI Critical Appraisal Checklist for Cohort Studies and, for case-control studies, the tool is called JBI Critical Appraisal Checklist Case Control Studies.The evaluation of the methodological quality was carried out, in February 2022, by two reviewers, independently, and the divergences were discussed in meetings until consensus.Data analysis and synthesis were performed in a descriptive manner.In In In In the methodological evaluation of the primary studies, the tools proposed by the Joanna Briggs Institute were used, and none of them have a scoring system for the general evaluation of the research, but it can be affirmed that a greater quantity of “yes” answers is indicative of better results. methodological quality-In the evaluation of prospective or retrospective studies (n=6), through the JBI Critical Appraisal Checklist for Studies Reporting Prevalence Data tool, of the nine questions that make up the checklist, in three researches,,The JBI Critical Appraisal Checklist for Cohort Studies tool was used to evaluate the cohort studies (n=4). Of the 11 questions that make up the checklist, in two studies-,-To facilitate the reader’s understanding, the primary studies included in the review were grouped according to the surgical approach. Thus, in six primary studiesIn the prospective study, the authors investigated the association of comorbidities and variables (pre and postoperatively) with SSI after sleeve gastrectomy. The sample consisted of 40 patients, and SSI was diagnosed in three patients (7.5%), two cases classified as organ/space  and one superficialIn the retrospective cohort study, patients underwent the following types of bariatric surgery: Roux-en-Y gastric bypass, sleeve gastrectomy and adjustable gastric band, with a sample of 815 patients. The incidence of surgery-related infection was 4.2% in the first postoperative monthIn the retrospective study, the sample consisted of 1,400 patients undergoing Roux-en-Y gastric bypass. In this research, a surgical technique was tested , with the aim of reducing infection rates. The overall wound infection rate was 7.6% (n=106) with 9.3% (103/1109) in the primary wound closure group compared to 1.0% (3/291) in the secondary wound closure group (planned and tested) (p <0.001)In another retrospective study, the sample consisted of 1,981 patients who underwent Roux-en-Y gastric bypass or sleeve gastrectomy, 38% (n=751) had diabetes and 62%  did not have diabetes. chronic disease. Regarding SSI, in the group of patients without diabetes, the overall rate of superficial SSI was 0.7% (n=9) and organ/space SSI was 0.4% (n=5). In the group of patients with diabetes, the overall rate of superficial SSI was 2.8% (n=21) and of organ/space SSI was 0.9% (n=7)In a retrospective study, the authors developed a predictive tool (BariWound) for SSI after 30 days of surgery. Patients undergoing sleeve gastrectomy or Roux-en-Y gastric bypass  were included in the research. Of the investigated sample, 1,841 patients (0.7%) had SSI, 70.1% of which were classified as incisional SSI, 29.0% of organ/space and 0.9% a combination of both typesIn a retrospective study, the sample consisted of patients undergoing Roux-en-Y gastric bypass , who were divided into a male cohort and a female cohort with the same number . The female cohort had a higher rate of superficial incisional SSI compared to the male cohort . Regarding organ and space SSI, the infection rate was 0.41% (n=156) in the female cohort and 0.43% (n=104) in the male cohort -In view of the above, SSI rates ranged from 0.4% to 7.6%, considering the results of primary studies, in which patients underwent laparoscopic surgeryIn the literature, in a national cross-sectional study, the authors evaluated the occurrence of complications in bariatric surgery (Roux-en-Y gastric bypass). The sample consisted of 469 patients, and data were collected from medical records and records of outpatient consultations. Participants were followed up for at least one year. The occurrence of postoperative complications that required hospitalization was 24.09% (n=113), with cholecystectomy being the most frequent complication . Regarding infectious complications, one patient had a superficial abscess (0.21%) and three had a deep abscess (0.63%),,,In this grouping of primary studies included in the review, perioperative hyperglycemiaIn a prospective cohort study, with a sample of 484 patients undergoing abdominal surgery, the defined objectives were to evaluate the independent effect of perioperative hyperglycemia and the incidence of SSI. Most patients underwent cholecystectomy (50.21%), and 0.83% (n=4) underwent bariatric surgery. Of the participants, 18.39% (n=89) had diabetes and 81.61% (n=395) did not have this disease. The incidence of SSI was 20.25% (98/484), with hyperglycemia being an independent risk factor for this type of infectionIn the literature, in a cross-sectional study whose delimited objective was to identify the prevalence and factors associated with postoperative complications at the surgical site in bariatric surgery, the sample consisted of 197 patients. The results showed that females were the most prevalent  and the age group up to 45 years  was the most operated. Of the participants, 30 had postoperative complications, totaling 45 (the participant could have more than one complication). The complications identified were: seroma , incisional hernia , superficial dehiscence , deep dehiscence , hematoma , infection , fistula , hemorrhage , ischemia  and skin lesion . Of the factors associated with the outcomes, the authors highlighted the open approach (Odds ratio/OR=5.35), insertion of drains (OR=4.48) and a postoperative period of more than three days of hospitalization (OR= 5.03),-Next, we present the primary studies, whose sample was submitted to bariatric surgery through different surgical approachesIn the case-control study, patients underwent Roux-en-Y gastric bypass (n=751), with 701 laparoscopic (94%) and 46 open (6.1%) surgical procedures. The overall rate of SSI was 12% (n=91), with 71.4% of cases (n=65) being classified as superficial SSI, 19.8% (n=18) deep infections and 9.9% (n=8) of organ/spaceIn the cohort study, patients underwent gastric bypass , with 97%  laparoscopically, 2.3%  open surgeries, and 0.7%  =336) were laparoscopic procedures converted to open surgery. In this research, the authors investigated the risk of complications and mortality in relation to age. The overall deep infection/abscess rate was 9%. Superficial wound infection occurred in 1% of all patients in the cohort. The risk of developing superficial wound infection significantly increased in patients aged ≥60 years (OR=2.02) and 60-64 years (OR=2.14)In the prospective cohort study of the investigated sample , 20.9% of patients (n=334) underwent open surgery and 79.1%  underwent laparoscopic surgery. Superficial SSI occurred in 16 patients (1%) and intra-abdominal infection in 15 (0.9%). Superficial SSI rates were 3% in the open approach group and 0.5% in the laparoscopic group (p < 0.05). The results showed a correlation of superficial SSI with the investigated variables (BMI and diabetes mellitus). Thus, the increase in BMI ranges was related to a higher occurrence of infection. In the sample, 2.2% (n=9) of patients with diabetes developed infection and 0.6% (n=7) of patients without this disease (p<0.05). It is noteworthy that in this study, the authors tested a care package (bundle) aimed at reducing SSI in bariatric surgeryIn the retrospective study, the authors investigated the routine use of abdominal drain placement in bariatric surgery to assess complications associated with this practice and potential risk factors. Data were collected from the Metabolic and Bariatric Surgery Accreditation and Quality Improvement Program (MBSAQIP) database. Patients  underwent sleeve gastrectomy or Roux-en-Y gastric bypass (laparoscopic or robotic). The drain was used in 23,190 cases (15.6%) and not applied in 125,070 (84.4%). In the research, despite the SSI being an evaluated outcome, the researchers did not describe the occurrence/incidence of this complication. However, the results showed that drain placement during surgery was associated with an increased probability of superficial SSI (OR=1.57), deep incisional SSI (OR=2.04 and organ/space SSI (OR=1.8) ,-Considering the results of the mentioned primary studiesThere is evidence in the literature about SSI and the different surgical approaches. In a case-control study, the authors compared different outcomes  in bariatric surgery using robotic or laparoscopic approaches. The types of surgeries analyzed were Roux-en-Y gastric bypass  and sleeve gastrectomy . The MBSAQIP database was used for data collection in the period 2015-2016. In the robotic approach, the results showed lower occurrences of superficial SSI in patients undergoing Roux-en-Y gastric bypass (p=0.0003) and organ/space in sleeve gastrectomy (p=0.0002)2. The sample consisted of 61,493 patients . The MBSAQIP database was also used for data collection in the period 2015-2017. The group of patients undergoing robotic surgery had a longer duration of surgery  and length of hospital stay . In the multivariate analysis, the robotic approach was an independent risk factor for organ/space SSIIn contrast, in a retrospective study, the authors compared the perioperative results of sleeve gastrectomy by robotic or laparoscopic approaches in patients with a BMI≥50 kg/mIn a retrospective study with the participation of 772 patients who underwent Roux-en-Y gastric bypass or sleeve gastrectomy in a tertiary-level hospital in the United States of America, the objective was to identify risk factors for early complications in bariatric surgery. The results showed that open surgery was associated with the occurrence of superficial and organ/space SSI, in comparison with the laparoscopic approach-Antibiotic prophylaxisIn a literature review, the delimited objective was to evaluate research on the use of antibiotic prophylaxis in patients undergoing bariatric surgery to prevent SSI, with 16 studies being included in the sample . Thus, based on the results of the analyzed studies, the authors stated that cefazolin is the most effective antimicrobial, studied and used in bariatric surgery, and the administration of this drug, before anesthetic induction, should be considered as the first choice for antibiotic prophylaxis. However, dosage is still a problem, with the use of several different regimens with different reports of resultsDue to the current discussion about the use of drains in bariatric surgery, in the present review, only in one study, the device was the risk factor investigatedIn the literature, in a comparative study, the delimited objective was to describe the results achieved after the implementation of a bundle to reduce the occurrence of SSI and to identify the risk factors for this type of infection in bariatric surgery. Of the total sample , 53.6% patients underwent Roux-en-Y gastric bypass, 34.8% sleeve gastrectomy, 1.4% laparoscopic adjusted gastric band, 0.4% duodenal interruption, 7.9% were cases of revisions and 1.9% of procedures were categorized in “other category”. All surgical approaches  were included in the sample. Of the participants, 1,977 (97.8%) had no infectious complications and 45 (2.2%) developed SSIs. Before the implementation of the care package, the SSI rate was 5.1%, with a significant reduction to 1.5% (after implementation of the bundle). The predictive factors for SSI were diabetes mellitus; the placement of a drain in the intraoperative period; the number of medications for hypertension prior to surgery and the open surgical approachIn a retrospective study, the authors defined the objective of identifying common preoperative characteristics that could have led to drain placement, surgical variables associated with drain placement, and differences in postoperative complications in patients who received such a device in bariatric surgery. Data were also collected from the MBSAQIP database, in the period 2015-2017. During this period, 388,239 bariatric surgeries were performed without drains and 100,221 were performed with drains. Surgical procedures included in the study were sleeve gastrectomy, Roux-en-Y gastric bypass and revisions. The results showed that 29% of patients undergoing gastric bypass had a drain placed, but only 16.7% of patients undergoing sleeve gastrectomy. The percentage of participants with a drain dropped from 33.1% to 24.6% in the study period and from 20.3% to 13.6%, respectively. The authors concluded that despite the reduction in the use of drains in bariatric surgery, the use of this device is still very commonRegarding the limitations of the integrative review, the inclusion of primary published studies was limited, that is, the gray literature was not considered, and there were restrictions on languages and period. Data analysis and synthesis were performed in a descriptive manner. Thus, combining data from different types of studies is a challenging process that can lead to bias in the elaboration of review results.On the other hand, the search for primary studies was carried out in the main health and nursing databases. In addition, to assess the methodological quality of the research, the authors used tools developed by the Joanna Brigss Institute. This step reinforces the rigor in conducting the knowledge synthesis method.Antibiotic prophylaxis, female gender, high Body Mass Index and perioperative hyperglycemia were the main risk factors for the development of surgical site infection in bariatric surgery.Surgical site infection rates ranged from 0.4% to 7.6%, considering the results of primary studies (n=6), in which patients underwent laparoscopic surgery. In primary studies (n=5), with participants undergoing procedures with different approaches , surgical site infection rates ranged from 0.9% to 12%.Conducting the integrative review generated a body of evidence that reinforces the importance of implementing effective measures for the prevention and control of surgical site infection by health professionals after bariatric surgery, promoting improved care and patient safety in the perioperative period.


Brevibacillus laterosporus S62-9 microbial agent on the meat quality, amino acids, and volatile compounds of chicken. The experiment was carried out with 160 1-day-old Arbor Acres male broiler chickens, rearing for 42 d. The chickens were randomly divided into two groups of 8 replicates each, with 10 chickens in each group. No supplement was added to the basal diet in the control group and Brevibacillus laterosporus S62-9 microbial agent was added to the diet of the experimental group. At the end of the experiment, the meat quality, meat chemical composition, amino acid composition, and volatile compounds of chicken were determined. The results showed that pH (p < 0.05), pressing loss (p < 0.05), cooking loss (p < 0.05), and shear force (p < 0.01) were notably decreased, the percentage of breast meat (p < 0.01), protein content (p < 0.05) were visibly increased, and remarkable changes were observed in the amino acid composition (change in seven amino acids) and volatile compounds profile . In summary, it was found that Brevibacillus laterosporus S62-9 microbial agent can be used as a novel and effective feed supplement to improve the nutritional quality and flavor characteristics of broilers.Probiotics are being used in diets to improve the quality of chicken meat. The aim of the study was to investigate the effects of dietary supplementation with  Bacillus-based probiotic products [For a long time, antibiotics have been widely used in broiler farming in order to prevent disease and promote growth ,2. Howevproducts .The application of probiotics in poultry is not only limited to growth performance, gut health, and body immunity but also has a key role in improving meat quality such as water-holding capacity, tenderness, lipid oxidation stability, sensory characteristics, and microbial safety in broilers . Along wBrevibacillus laterosporus S62-9 exhibits a wide range of antimicrobial activity against fungi and bacteria. Meanwhile, good thermal tolerance, and good acid and bile salt resistance were found in Brevibacillus laterosporus S62-9 in the previous study. Thus, the Brevibacillus laterosporus S62-9 microbial agent was found to be a promising microbial agent for broiler feeding and might have favorable effects on the nutritional quality and flavor characteristics of meat. Furthermore, the effect of the probiotic, Brevibacillus laterosporus S62-9, on related meat quality in broilers has not been investigated previously. This study was designed to evaluate the effects of dietary supplementation with Brevibacillus laterosporus S62-9 on nutritional quality and flavor characteristics of chicken meat by investigating growth performance, carcass traits, meat quality, amino acids, and volatile compounds in broiler chickens.With the prevalence of broiler farming and the increased demand for meat quality, the use of probiotics instead of antibiotics to improve meat quality and maintain food safety is becoming a hot field. Antimicrobial capacity is critical in biocontrol . SimilarBrevibacillus laterosporus S62-9 was resistant to heat, acid, and bile salts in vitro. Strain S62-9 had no observable resistance to 10 types of antibiotics tested and was thus considered to be a biosafe strain. Brevibacillus laterosporus S62-9 was scraped from the agarslantculture medium of the strain stored at 4 °C to the nutrient broth medium for activation , fermenting in a specific high-yielding fermentation medium for 48 h , and by spray drying into powder to get a microbial agent with the main component Brevibacillus laterosporus S62-9 up to 109 cfu/g.In the previous study by our group, Brevibacillus laterosporus S62-9 = BS, diet containing probiotic at 106 cfu/g), each group contained 80 broilers and there were no significant differences in initial weight between the two groups. The diet composition and nutrition were according to GB/T 5916 2020 . This experiment was conducted at the Changping Experimental Base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences, which was carried out with 1-day-old Arbor Acres male broilers, divided into 2 homogeneous experimental groups  were randomly selected from each treatment for sample collection. All selected birds were weighed respectively and slaughtered by cutting the jugular vein after being stunned by carbon dioxide in a professional slaughtering room. According to a standardized procedure (NY/T 8232004) to determine the dressing percentage (%), semi-eviscerated percentage (%), eviscerated percentage (%), abdominal fat (%), breast muscle (%), and thigh muscle (%). Dressed weight was recorded after defeathering and exsanguination. The dressing percentage (%) was calculated as:After removing trachea, esophagus, crop, intestine, spleen, pancreas, gallbladder, gastric contents, and corneum membrane, the semi-eviscerated weight was recorded. The semi-eviscerated percentage (%) was calculated as:Finally, the heart, liver, proventriculus, gizzard, lung, and abdominal fat were removed, and the eviscerated weight was recorded. The eviscerated percentage (%) was calculated as:The abdominal fat (%) was calculated as:The breast muscle (%) was calculated as:The thigh muscle (%) was calculated as:After cutting the carcasses, the breast and thigh meat (10 birds/group) were selected for determination of pH, meat color, pressing loss, cooking loss, and shear force, and the remaining chicken meat was stored at −20 °C for subsequent analysis.The pH of breast and thigh muscle was measured at 45 min and 24 h (stored in air at 4 °C for 24 h) after slaughter using a pH meter  1 cm deep into the center of the meat tissue, and the right breast meat (near the bone side) and thigh meat color was measured at 24 h (stored in air at 4 °C for 24 h) after slaughter by a colorimeter . Specifically, at the time of determination, the surface color of the breast muscle close to the bony side was determined, and three positions from the top to the bottom were determined and averaged. Three values were measured in duplicate at the surface of different thicknesses of the same thigh meat, then the average value was calculated as the thigh meat color determination value; the pressing loss and cooking loss were used to assess the water-holding capacity of the chicken. Pressing loss was measured through the differences in weight before and after pressurization , and theThe operations were as follows, wrapping in gauze after weighing (volume 1.5 × 1.5 × 1.0 cm), and then wrapped in 18 layers of quantitative filter paper, placed on a YYW-2 strain-controlled lateral limitless pressure gauge and pressurized to 15 kg for 5 min, then removed and weighed; cooking loss was measured according to Liu . The cooBriefly, after samples were cut in the direction of the muscle fibers (volume 6 × 3 × 3 cm), weighed accurately, then placed in a steaming bag, and heated at 90 °C in a water bath until the central temperature of the meat sample reached 75 °C. The measurements of shear force were conducted as described by Wang  with appMoisture, protein, fat, and ash were determined with references to GB5009.3-2016, GB5009.6-2016, GB5009.5-2016, and GB5009.4-2016, respectively.The amino acid (AA) composition of the breast and thigh muscles was determined according to GB5009.124-2016. Basically, the meat samples were hydrolyzed in a hydrolysis tube at a constant temperature of 110 °C in 16 mL HCl (6 mol/L) for 22 h. The hydrolysis products were transferred and diluted in a 50 mL volumetric flask using a fixed volume of 0.02 mol/L HCl. After mixing, 1 mL of the solution was transferred to an evaporation flask and rotary evaporated to dry at 55 °C in a water bath, then dissolved with ultrapure water, evaporated with spinning, and repeated the above two steps twice. After this, the amino acids were dissolved in 2 mL of HCl (0.02 mol/L), filtered (0.22 μm), and determined using a fully automated amino acid analyzer , by comparing with standards  to determine individual amino acids.m/z. The volatile compounds were qualitatively determined based on the NIST4 mass spectrometry library, RI, and the descriptions of standard odor, and the area normalization method was adopted to the content of volatile compounds. ROAVs were calculated based on the volatile compounds content and threshold values in water.According to Ho  with sliThe evaluation of sensory was performed according to Bosque  with mint-test using SPSS 24.0 programs . Results are expressed as the mean ± standard deviation (SD). Statistical significance was indicated by p < 0.05.The analysis of data was performed with an independent sample Brevibacillus laterosporus S62-9 microbial agent to the diet affected the growth performance and carcass traits of broilers. Compared to the control group, after the addition of Brevibacillus laterosporus S62-9 to the diet, the feed conversion ratio (FCR) was reduced, accompanied by average daily gain (ADG) of 22–42 days, body weight (BW) of 42 days and breast muscle yield was increased. There were no significant changes in carcass weight, dressing percentage, semi-eviscerated percentage, eviscerated percentage, and the percentage of abdominal fat and thigh muscle in the experimental group.It was shown in Brevibacillus laterosporus S62-9 microbial agent to the diet significantly reduced brightness (L*) shear and increased redness (a*) of breast meat, while 45 min, 24 h pH, pressing loss, cooking loss, and shear force of thigh meat was decreased.As shown in Brevibacillus laterosporus S62-9 microbial agent affected the chemical composition  of broiler meat. It is worth noting that the protein content of the breast muscle and the fat content in the thigh meat of broilers in the experimental group were dramatically higher than those in the control group.Brevibacillus laterosporus S62-9 microbial agent. There was no significant difference between the experimental and the control group in the thigh muscle, but there was a tendency to increase the content of AA in the experimental group.The AA content of breast and thigh muscles is given in Brevibacillus laterosporus S62-9 microbial agent to the diet on the sensory properties of cooked meat are shown in The effects of the addition of Brevibacillus laterosporus S62-9 microbial agent in the diet on the growth performance, carcass traits, meat quality, and flavor of broilers.Antibiotic growth promotions (AGPs), as feed additives, were used to enhance and improve growth performance and carcass traits of broilers in the past ,31. HoweBrevibacillus laterosporus S62-9 microbial agent to the diet significantly reduced FCR and increased BW at 6 weeks of age but did not significantly alter ADFI. In the present study improved BW and FCR of birds might be justified by the fact that taking probiotics increases the birds’ ability to utilize nutrients. This result was in line with the findings of Wang [Bacillus laterosporus KC1 to the diet significantly increased broiler weight and decreased FCR at 42 days of age. In contrast, there were some relevant studies have shown that the addition of microorganisms had no significant effects on growth performance [Bacillus subtilis DSM 32315 to the diet, while the change in dressing percentage, semi-eviscerated percentage, eviscerated percentage, and thigh meat weight was negligible. Similar to our results, Rehman [In this study, the addition of  of Wang  that theformance . Breast formance . The notformance , the wei, Rehman  reported, Rehman . In thisBacillus to the diet was effective in improving the quality of chicken meat. The meat quality was determined mostly by its pH, color, water-holding capacity, and tenderness [Brevibacillus laterosporus S62-9 microbial agent to the diet could prevent these conditions and improve meat quality. Meat color was a useful indicator to evaluate meat quality. The lower L* was, the redder the meat color would be, which was the indicator of high meat quality perceived by consumers [Brevibacillus laterosporus S62-9 microbial had beneficial effects on the improvement of chicken meat tenderness, especially on thigh meat tenderness, and this was strongly related to the enhancement of fat content in the thigh meat [Many studies have shown that the addition of nderness . The pH nderness . Meanwhinderness . Howevernderness . Our resonsumers . The resonsumers . This imonsumers  and Zhanonsumers  reportedonsumers , the varonsumers . The amoigh meat . The criigh meat  and tendigh meat , but alsBrevibacillus laterosporus S62-9 had no effect on the content of moisture and ash in both breast and thigh muscles compared to the control group. Whereas, adding Brevibacillus laterosporus S62-9 to the diet had different effects on protein and fat in the breast and thigh. Our study was consistent with the findings of Liu et al. [The present study depicted that dietary supplementation with u et al. , in whicu et al. . In anotu et al. . Even thBrevibacillus laterosporus S62-9 significantly increased the content of seven amino acids, including three essential amino acids and four flavor amino acids. There were three important essential amino acids in chicken meat, threonine, methionine, and phenylalanine, which were higher in the experiment group, indicating that meat quality and nutritional value improved via the stimulative effects of Brevibacillus laterosporus S62-9 supplemented in the broiler diet. Flavor amino acids such as serine, alanine, glycine, and arginine can act as precursors, resulting in subtle flavors and having an important impact on meat flavor [Brevibacillus laterosporus S62-9 might improve the nutrition and flavor of chicken meat by increasing the content of essential amino acids and flavor amino acids.The content of amino acids in meat was closely associated with the nutritional value  of meat t flavor . The preBrevibacillus laterosporus S62-9 on chicken flavor, volatile compounds were determined and none of which were identified with the distinctive meat flavor. It was clear that meat flavor was the result of the interaction of many compounds. The precursors of meat aroma substances were various types of small biological molecules which originated from the water-soluble extracts of meat. The soluble components produced meat aroma under heating, which could be summarized in three pathways: the first, by the oxidation and hydrolysis of lipids to form compounds such as aldehydes, ketones, and alcohols [The concentration of volatile compounds in fresh meat also reflects the sensory quality . Volatilalcohols ; the secalcohols , it was alcohols . Most ofalcohols  or oleicalcohols . Brevibacillus laterosporus S62-9, played a role in improving flavor characteristics through the changed composition and concentration of volatile compounds in muscle. The addition of Brevibacillus laterosporus S62-9 to the diet improved the flavor of chicken by mainly affecting the content of three volatile compounds, 1-octen-3-ol, hexanal, and octanal, which contribute significantly to the flavor of chicken breast meat. However, it was the combination of volatile aromas and non-volatile taste compounds that gives meat its flavor, and further analytical studies in combination with the content of free amino acid and inosine monophosphate, and gas chromatography-sniffing were required subsequently to identify compounds that may affect meat flavor and consumer preferences. PCA analyses were performed to evaluate the difference between the control group and the experimental group (Brevibacillus laterosporus S62-9 had a significant effect on chicken quality, and this difference was more pronounced in thigh meat.Alcohols could be produced through sugar metabolism, lipid oxidation, decarboxylation, dehydrogenation of amino acids, and reduction of aldehydes ,68,69,70al group . PrincipBrevibacillus laterosporus S62-9.Combining sensory evaluation experiments with multiple perspectives such as texture and flavor, it could be more intuitive to evaluate the consumers’ preferences for chicken. A study by Horsted et al.  reportedBrevibacillus laterosporus S62-9 microbial agent contributes to improving growth performance and breast muscle yield in broilers. Furthermore, the addition of Brevibacillus laterosporus S62-9 microbial agent to the diet can improve meat quality and flavor of broilers by improving pH, meat color and tenderness, increasing water-holding capacity, protein, and fat contents, and regulating the contents of amino acid and volatile compounds. In conclusion, the current findings indicate that dietary supplementation with Brevibacillus laterosporus S62-9 could be a useful strategy for the improvement of meat quality and flavor properties of broilers. Nevertheless, further research is needed to increase knowledge regarding the mechanisms for improving meat quality and flavor.The current results suggest that the


ALP and OCN. Similarly, membranes significantly increased ECM-related and MMP8 gene expression in HGFs. In conclusion, the tested acellular porcine dermis collagen matrix membrane, mainly when it is hydrated, behaved as a suitable microenvironment for oral cells.The use of collagen membranes has remained the gold standard in GTR/GBR. In this study, the features and the biological activities of an acellular porcine dermis collagen matrix membrane applicable during dental surgery were investigated, and also by applying hydration with NaCl. Thus, two tested membranes were distinguished, the H-Membrane and Membrane, compared to the control cell culture plastic. The characterization was performed by SEM and histological analyses. In contrast, the biocompatibility was investigated on HGF and HOB cells at 3, 7, and 14 days by MTT for proliferation study; by SEM and histology for cell interaction study; and by RT-PCR for function-related genes study. In HOBs seeded on membranes, mineralization functions by ALP assay and Alizarin Red staining were also investigated. Results indicated that the tested membranes, especially when hydrated, can promote the proliferation and attachment of cells at each time. Furthermore, membranes significantly increased ALP and mineralization activities in HOBs as well as the osteoblastic-related genes  During guided tissue regeneration (GTR) and guided bone regeneration (GBR), the use of a membrane permits stabilizing the coagulum to prevent the colonization of the site by rapidly proliferating epithelium cells. It thus promotes the growth of slower-growing cells capable of forming bone and maintaining the space for regeneration. The characteristics of the membranes should be clinical manageability, space-making ability, biocompatibility, cell-occlusion properties, integration by the host tissues, and adequate mechanical and physical properties .However, porcine acellular dermal matrix membranes (PADMs) have been introduced in the market to increase the gingival thickness or to treat gingival recessions as an alternative to connective tissue grafts (CTGs) . The proIt has been shown that the combination of hard and soft tissue augmentation procedures did not influence the marginal bone level around the implants but was significant for marginal soft tissue recession .PADMs are characterized by a three-dimensional acellular network of collagen types I and III and elastin, in which cellular components and antigens have been removed to avoid any risk of tissue rejection .The advantages of using a heterologous membrane permit the avoidance of connective tissue sampling surgery, with the associated risks of bleeding, increased surgical times, increased infectious risks, increased difficulty of the clinical case, and post-operative discomfort . MoreoveSome disadvantages of PADMs are related to their malleability and degradation times. High stiffness leads to a difficult adjustment in the defect. However, if it is without any support, it will not support tissues, and malleability will be reduced. Another disadvantage of PADMs is the possibility of exposure in the oral cavity and subsequent contamination by bacteria. The presence of a high-cross-linking structure of the membranes influences their stiffness and the necessary time to fully reabsorb the material; the higher this parameter, the higher the risk of exposure in the oral cavity, contamination by bacteria, and induction of clinical failure .Many studies have shown that PADMs are able to promote the growth and proliferation of human gingival fibroblasts, osteoblasts, and endothelial cells and to facilitate the migration, proliferation, and adhesion of periodontal ligament cells and human oral fibroblasts ,9. FurthHowever, the process of production and the postprocessing treatments of PADMs have a great influence on their final three-dimensional conformation, porosity, mechanical stiffness, biodegradation process, and handling by the surgeon, also influencing their biological properties. In particular, PADMs produced by different manufacturers could differ from each other, especially in the collagen cross-links, because during production, they could be subjected to different physical treatments, such as ionizing or ultraviolet light irradiation, dehydrothermal treatment, and chemical methods involving aldehydes and isocyanates .The aim of this study was to investigate the topographical and biological properties of a novel acellular dermal matrix membrane to evaluate its use in periodontology for soft tissue augmentation. Moreover, based on the contact of the PADMs with bone tissues, the biological response of the oral osteoblasts was tested.The SEM observations permitted an evaluation of the structure of the membrane which appeared smoothed in both groups at the magnifications of 390, 1000, and 3000× . When thp < 0.001). Comparing the growth of cells on the H-Membrane and Membrane, there were similar results (p < 0.0001). In addition, the growth of HGFs cultured on the H-Membrane showed an increase compared to the Membrane.The growth of the gingival fibroblasts (HGFs) and oral osteoblasts (HOBs) was evaluated by MTT assay at 3, 7, and 14 days, as shown in  results A. Althoup < 0.0001) at 3, 7, and 14 days (p < 0.0001). The same trend was observed at 7 and 14 days.HOBs cultured on the H-Membrane showed a statistically augmented proliferation rate with respect to CTRL   was significantly enhanced when HOBs were seeded both on H-Membranes and Membranes with respect to CTRL ( 0.0001) . In detap < 0.0001). The levels of the H-Membrane and Membrane appeared comparable  at 14 days of culture . More mimparable B.COL1), fibronectin 1 (FN1), metalloprotease 8 (MMP8) in HGFs and ALP, and osteocalcin (OCN) were specifically examined. At 3, 7, and 14 days in HGFs, COL1 was statistically upregulated in the H-membrane and Membrane groups compared to CTRL. HGFs cultured on the H-Membrane and Membrane showed similar levels of expression of COL1 at 3 and 7 days, while at 14 days they were statistically higher in the H-Membrane than in the Membrane , but comparable levels were observed between the H-Membrane and Membrane. In contrast, the Membrane group showed a slightly diminished expression with respect to the H-Membrane at 7 and 14 days . At all timing points, comparable levels of MMP8 expression were observed in the H-Membrane and Membrane groups  . Thus, tp < 0.0001) was observed at 3 and 14 days, while at 7 days, the cellular metabolic activity was reduced. This observed trend may be characteristic of fibroblast cells that are usually critical components of wound healing, during which fibroblast accumulation begins 3–5 days after injury and may last up to 14 days, and it may have an up-and-down trend [p < 0.0001) increased with respect to the control in a time-dependent manner. Thereafter, the cell behavior on membranes was investigated for cell attachment, cell morphology, and cell interaction with the membrane’s surface through SEM and histological analyses. Notable differences emerged from SEM images between the two tested membranes, and both showed high biocompatibility to HGFs and HOBs that appeared strongly attached to the surface with typical morphology. One potential explanation may be that both membranes possess a dense network of collagen fibers, thereby providing a more easily attachable surface for incoming HGFs and HOBs. In addition, adhesion could be promoted through Cadherin activity [p < 0.0001), with the highest value for HOBs cultured on the H-Membrane. In a similar manner, the qualitative evaluation of calcium deposition by Alizarin Red staining at 14 days showed a more intense red color for both membranes compared to the control, indicating the presence of more mineralized nodules. Furthermore, the quantitative analysis by CPC revealed that calcium deposits resulted in the highest levels in the H-membrane. Increased mineralization rate and bone formation after surgery are fundamental, and here both membranes, especially the H-Membrane, seemed tp favor adhesion, proliferation, and mineralization activity in HOBs. Thus, the osteoblastic-related genes ALP and OCN were also investigated at the early and late stages of HOBs. ALP levels commonly indicate new bone formation and osteoblastic activity [ALP mRNA levels with respect to the control, and the increase was maximum at the late stage of cells corresponding to 14 days post-seeding, with the H-Membrane demonstrating the highest results (p < 0.001) significantly. Osteocalcin is known to be a bone tissue-specific protein, and its expression is usually correlated to the expression of ALP [OCN gene expression was similar to the trend of ALP expression and both of these bone-related genes were up-regulated more in the H-Membrane compared to the Membrane at each time. Thereafter, ECM-related genes, including collagen type I α1 (COL1) and fibronectin (FN1), in human gingival fibroblast were also evaluated by real-time PCR. It was found that both membranes provoked a significant increase in COL1 and FN1 mRNA levels when compared to the control. MMP8 (collagenase-2) belongs to the matrix metalloproteinase family of extracellular matrix-degrading enzymes, involved in tissue remodeling and wound repair. In particular, MMP8 has a protective role during the inflammatory response to various agents, despite the abnormal levels of this gene indicating chronic periodontitis [P. gingivalis infection model [MMP8 gene in HGFs seeded on tested membranes was comparable to the control at 3 days of culture. It slightly increased at 7 days and it resulted significantly (p < 0.05) higher at 14 days of culture. The up-regulation of COL1, FN1, and MMP8 may indicate that the tested membranes, especially the H-Membrane, are able to stimulate the renewal of the ECM. In addition, the level of MMP8 may be considered an input for fibroblasts involved in the wound repair process and for HOBs lined on the surface of membranes to appose bone matrix. Altogether, the results of this study suggested the acellular porcine dermal collagen matrix membrane has the capability to favor certain biological activities of the key cells in the oral cavity. The results of this study were in line with another study that evaluated the biological properties in vitro of PADMS on gingival fibroblasts and osteoblast-like cells [Given that this type of membrane can be used for soft tissue grafting in combination with GBR/GTR in this study, the biological properties of the membrane were evaluated on gingival fibroblasts and on oral osteoblasts isolated from human biopsies. The growth of HGFs and HOBs was evaluated at 3, 7, and 14 days. At each time, both HGFs and HOBs seeded on the H-Membrane had a proliferative rate higher than those seeded on the Membrane. This is in line with the study of Guarnieri and co-workers, who observed a higher proliferation rate of HPMSCs seeded on hydrated PADM compared to the non-hydrated membrane . In detawn trend . It was wn trend , whereaswn trend . In thisactivity ,18. Histactivity . In thisn of ALP . As expedontitis . Polymordontitis . MMP8 hadontitis . A previdontitis . Howeverdontitis . The paron model . Here, tke cells . Fujiokake cells  and octake cells . Howeverke cells ,31,32. PMany studies demonstrated the correlation between surface features and the behavior of cells ,34,35. Ai.CTRL: cells seeded on the plate;ii.H-Membrane: cells cultured on the matrix hydrated three times with NaCl 0.9%;iii.Membrane: cells seeded on the membrane used as manufactured.The dermal matrix was first characterized, and the biological activities of human gingival fibroblasts (HGFs) and human oral osteoblasts (HOBs) were then assessed. The following experimental conditions were used:Firstly, the adhesion and the proliferation of HGFs and HOBs cultured on the matrix were assessed at 3, 7, and 14 days. In particular, the Phosphatase Alkaline (ALP) levels of HOBs at 7 days; the mineralization of HOBs at 14 days; and the gene expression of constitutive markers of HGFs and HOBs at 3, 7, and 14 days were performed in order to evaluate the activity of cells on the matrix. The membrane , produced by Cellis Dental  was a non-pyrogenic collagen type I&III matrix obtained from porcine skin. It was supplied dry, sterilized, and did not contain any preservatives. In in vitro tests, the matrix was cut into squares of 5 mm × 5 mm under sterilized conditions.After rinsing with PBS and fixing for 1 h with 2.5% of glutaraldehyde at room temperature, the membranes were washed with PBS. Then, they were dehydrated in increasing concentrations of alcohol (from 40 to 100%) for 10 min each. The specimens were sputter coated with gold and examined using SEM . The images were taken at 390, 1000, and 3000×.The specimens were fixed in 10% buffered formalin and dehydrated in an ascending alcohol series. They were embedded in a glycol methacrylate resin  and then polymerized. Sections of about 150 μm were obtained following the long axis of the devices. The sections were subsequently ground down to about 30 μm in width and stained with fuchsin and toluidine blue. The images were taken by an optical microscope  at 40× and with polarized light.2 and 37 °C. The medium was changed every 4–5 days. Human oral osteoblasts (HOBs) were derived from bone chips of n° 12 patients undergoing the surgical removal of lower third molars at the dental clinic of the G. D’Annunzio University according to Ethical Committee reference numbers: BONEISTO N. 22-10.07.2021. The culture procedure was conducted according to Pierfelice TV et al., 2022 . Briefly2. Both cell lines, HOBs, and HGFs, were used from the 3rd and 5th passages. They were seeded on the top of the matrix at different densities and timing points depending on the test.Primary human gingival fibroblasts (HGF) were purchased from ATCC  and were cultured in DMEM low glucose (Corning), supplemented with 10% fetal bovine serum (FBS) (SIAL), 1% penicillin, and streptomycin (Corning) at 37 °C and 5% COTo assess the number of vital cells in each group, 3--2,5-diphenyl tetrazolium bromide (MTT) test was performed at 3, 7, and 14 days after the seeding of 104 cells/membrane using MTT  according to the manufacturer’s instructions. The absorbance was determined by a microplate reader  at 650 nm wavelength. The results were expressed as percentages and were calculated with respect to control (CTRL).4 cells/membrane). First, cells were fixed by 2.5% glutaraldehyde for 1 h followed by dehydration using sequential concentrations of ethanol. Then, they were sputtered with gold and observed at 390×, 1000×, and 3000× under an SEM  at 15 kV. To observe cell attachment, scanning electron microscope (SEM) images were taken 3, 7, and 14 days after cell seeding , was used to assess ALP levels in accordance with the manufacturer’s instructions and as reported by D’Amico E et al., 2022 [HOBs at the density of 5 × 10l., 2022 .4 HOBs/membrane were seeded on the membrane for 14 days. The qualitative analysis was performed using Alizarin Red Staining (ARS)  and the quantization using a 10% Cetylpyridinium Chloride (CPC) solution . These analyses were carried out in accordance with the protocol outlined by Pierfelice TV et al. 2022 [To evaluate the deposition of calcium nodules, 5 × 10al. 2022 .®2 Step RT-qPCR Kit  was used to obtain complementary DNA (cDNA) according to the manufacturer. SYBR Green  was used to perform RT-qPCR. An amount of 10 µL of mixes, composed of 1 µL of cDNA, 0.2 µL of primer mixture, and 5 µL of master mix, was plated in a 96-well plate, and gene expression was determined using Quant Studio 7 Pro Real-Time PCR System . The results were normalized to β-actin (β-ACT) for HOB and to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) using the 2 −ΔΔCt method. Primer sequences are reported in RT-PCR was performed with the objective of evaluating the gene expression of different markers in a certain stage of osteoblastic cells (ALP and OCN) and constitutive markers of fibroblast activity . Total RNA was isolated using the Trifast reagent , Italy). RNA was quantified on a Nanophotometer NP80 spectrophotometer  for analysis of RNA integrity, purity, and concentration. Afterward, the GoTaqOne-way ANOVA test followed by Tukey post hoc test was applied for the comparison of values among different groups at different time points. Statistical analysis was performed using GraphPad Prism8  with a significance level of 0.05.In conclusion, this study demonstrated that the tested PADMs, when hydrated, behaved as a suitable microenvironment for gingival fibroblasts and oral osteoblasts with significantly superior soft tissue and osteopromotive properties compared with the control. Nevertheless, these studies point to the necessity of further evaluation for both membranes in vivo.


Melissa officinalis L. extract (ME) in the rat model of myocardial ischemia/reperfusion (I/R) injury. Thirty-two Wistar rats were randomly divided into a CTRL non-treated control group with myocardial I/R injury and three experimental groups of rats treated with 50, 100, or 200 mg/kg of ME for 7 days per os. Afterward, hearts were isolated, and cardiodynamic function was assessed via the Langendorff model of global 20 min ischemia and 30 min reperfusion. Oxidative stress parameters were determined spectrophotometrically from the samples of coronary venous effluent  and heart tissue homogenate . H/E and Picrosirius red staining were used to examine cardiac architecture and cardiac collagen content. ME improved cardiodynamic parameters and achieved to preserve cardiac architecture after I/R injury and to decrease fibrosis, especially in the ME200 group compared to CTRL. ME200 and ME100 markedly decreased prooxidants TBARS, O2−, and H2O2 while increasing NO2−. Hereby, we confirmed the ME`s ability to save the heart from I/R induced damage, even after short-term preconditioning in terms of preserving cardiodynamic alterations, cardiac architecture, fibrosis, and suppressing oxidative stress, especially in dose of 200 mg/kg.We aimed to investigate the cardioprotective effects of ethanolic  During the last few decades, myocardial ischemia has been recognized as the most common form of cardiovascular disease, causing still rising morbidity and mortality worldwide. Even though the restoration of blood flow is essential for survival and better patient outcome, substantial damage to the cardiomyocytes occurs during reperfusion, consequently inducing arrhythmias, myocardial stunning, microvascular dysfunction, inflammatory response, and ultimately cardiomyocyte death. This phenomenon is called myocardial ischemia/reperfusion (I/R) injury and has received extensive scientific interest for more than three decades globally . The patMelissa officinalis L. (Lamiaceae), commonly known as lemon balm or bee balm, is a perennial herb, which is a part of the mint fragrance family of plants. It is a long-known folk medicine commonly used as a sedative or calming agent for anxiety problems, memory enhancement, and heart palpitations [itations . Today iitations . It is citations ,7,8, lutitations , apigeniitations ,11, and itations  via a nuitations ,14 but aitations , lungs [itations , and kiditations . Since MPreclinical investigations demonstrated beneficial effects of ME in the cardiovascular system using different animal models, including isoproterenol-induced infarction , left anTaking into account the abovementioned findings, this study aimed to provide new insight into the cardioprotective effects of ethanolic ME in the rat model of myocardial I/R injury by evaluating the cardiac function, morphology of the heart tissue, and the involvement of oxidative stress after short-term pre-treatment with ME in three different doses.All data regarding the chemical characterization of the investigated extract (ME), including HPLC (high-performance liquid chromatography) analyses, phenolic, and flavonoid content are provided in p < 0.05). All three doses of ME succeeded in improving dp/dt max in all three points of interest compared to CTRL, while the most prominent effect was observed in the ME200 group, which showed significantly higher values of dp/dt max compared to CTRL, ME50, ME100 in both stabs, RP1, and RP30 points, with no significant difference when compared to SHAM (Parameters of contractile function minimum and maximum rate of left ventricle pressure development (dp/dt max and dp/dt min) were characteristically impaired after ischemia in the CTRL group compared to SHAM in all three points of interest stab, RP1, and RP30. Additionally, ME treatment affected the hearts’ ability to stabilize, as shown via significantly higher dp/dt max of all treated animals  compared to CTRL and SHAM in stab point ( to SHAM A.p < 0.05).Similar changes were observed in the dp/dt min parameter, with all three experimental groups increasing this parameter values in stab, RP1, and RP30 points when compared to the CTRL group of rats. The highest dose of the applied extract ME200 induced the greatest increase in dp/dt min in all observed points of interest compared to CTRL, ME50, and ME100 groups. Additionally, only the ME200 group improved dp/dt min during the reperfusion period, similar to SHAM non-exposed to ischemia. Baseline values in stab point were affected by ME treatment, shown via increased values of dp/dt min compared to CTRL (p < 0.05).Baseline values of SLVP at stab point were significantly lower in ME50 and ME100 compared to SHAM, while ME200 stabilized the heart, similar to the healthy non-treated SHAM group. Ischemia caused a systolic dysfunction, as measured by systolic left ventricular pressure (SLVP) decrement throughout the reperfusion period in the CTRL group, especially at the end of reperfusion RP30 compared to the SHAM group, while in experimental groups, the trend was the opposite, SLVP remained more-less constant during the reperfusion period. Additionally, the ME200 group exerted the most prominent effect on SLVP, significantly increasing this parameter in all points of interest compared to both SHAM, CTRL, and lower doses of ME (ME50 and ME100) (Diastolic left ventricular pressure (DLVP) was mildly lower in the CTRL group during the post-ischemia period in the RP1 and RP30 compared to the stab. However, the treatment with ME did not cause any significant changes in this parameter compared to the CTRL or SHAM in any of the observed points of interest. Additionally, in the experimental groups, DLVP showed a constant trend during the whole reperfusion period D.p <0.05).Heart rate (HR) was significantly lowered in all three experimental groups treated with ME compared to the CTRL group prior to global heart ischemia in the stab point. Baseline values of only ME50 and ME100 were significantly lower in relation to the SHAM group in stab point. However, after 20 min ischemia, only in ME200 significantly lower HR remained constant compared to the CTRL, while at the end of reperfusion, these effects were lost. There was no significant difference in HR among all groups studied (p < 0.05), with no differences between different doses of ME and no differences compared to SHAM. However, after a period of ischemia, in the 1st min of the reperfusion period, CF was significantly higher only in the ME200 group compared to CTRL (p < 0.05), while in the 30th min of reperfusion, this difference was even more visible, with ME200 showing significantly higher CF compared to CTRL, ME50, and ME100 groups. Only ME200 succeeded in improving CF, similar to values of the non-treated SHAM group of rats who were not exposed to ischemia in RP30 point (p < 0.05).At the beginning of the experimental protocol, in the stabilization period, all three experimental groups  induced a significant elevation of coronary flow (CF) compared to the non-treated CTRL group (2−) in the stabilization point was observed in the ME200 group compared to all other groups studied (p < 0.05). These baseline values were markedly lower in treated animals  relative to CTRL and SHAM. However, in the first point of reperfusion, only ME50 significantly lowered O2− in comparison with CTRL, but at the end of reperfusion in RP1 point, all three applied doses of ME significantly lowered the level of O2− compared to CTRL, and these values were similar to SHAM (p < 0.05). At this point, ME200 seems to induce the most prominent decrease compared with the low and medium doses of ME, but this difference was not significant  compared to the CTRL group and groups of rats treated with a low and medium dose of the extract (ME50 and ME100). These differences were observed in all three points of interest  was significantly decreased in all three experimental groups  compared to the untreated control group of rats that had undergone global ischemia and SHAM group (p < 0.05). These differences were noticed in all three points of interest, during the stabilization period, first and last 30th min of the reperfusion period, with ME200 and ME100 being more efficient than the low dose ME50  was considerably increased in ME100 and ME200 compared to both SHAM, CTRL, and ME50 groups in the stabilization point (p < 0.05), while only ME200 elevated the level of NO2− in the first and last minute of reperfusion period compared to all other groups studied .The application of the highest dose of ME has mildly increased the activity of superoxide dismutase SOD B and theThe images of the heart tissues in the control group of rats were characterized by the presence of hypertrophy of individual muscle fibers with degenerative changes, stromae hypercellularity (cellular infiltrate), interstitial edema, and pycnotic nucleus. The appearance of wavy fibers was also observed A. Similap < 0.05). Additionally, treatment with the lowest dose of ME had no significant effect on collagen content compared to the CTRL. SHAM group of hearts had significantly lower collagen content than all other groups exposed to I/R injury (p < 0.05).A significantly higher amount of fibrosis and collagen deposition was observed in the CTRL group of rat hearts subjected to global ischemia , while pMyocardial I/R injury stands for a phenomenon with a complex pathology involving many pathways inducing cardiomyocytes death via necrosis and apoptosis. Despite thousands of research pieces regarding the subject, the specific mechanisms of the I/R-induced cardiomyocyte damage have not been established yet. However, the available knowledge highlights the role of phytotherapeutics, including plant extracts and their active compounds, in ameliorating myocardial I/R injury in different ways . This st+ channels or by cardiac muscarinic receptors activation [The results of our study confirmed the well-known fact that exposition of the heart to global ischemia induces changes in cardiac function, in several aspects, including depression of contractile force, but also relaxation ability at the end of the reperfusion period, which is reflected by lower dp/dt max, dp/dt min, SLVP, and DLVP in the CTRL group of rats compared to non-ischemic SHAM rats. Additionally, ME treatment also affected the hearts` ability to stabilize, as proved via dp/dt max, dp/dt min, SLVP, HR, and CF improvement at the beginning of the protocol compared to CTRL rats. Moreover, the present study demonstrated the improvement of myocardial contractile function subjected to I/R injury after the treatment with lemon balm extracts, particularly in the ME200 group, via dp/dt max and dp/dt min increment. This observation is partly in line with the previous paper, where the administration of aqueous ME induced improvement of dp/dt max after isoproterenol-induced cardiac injury. However, they highlighted the best effect of ME in lower doses of 50 and 100 mg/kg, opposite to our findings where ME200 was found to be the most effective . On the tivation ,28.2− and H2O2, after preconditioning with ME. There is evidence that lemon balm extract may lower O2− levels in different models of cardiac pathologies such as autoimmune myocarditis [2O2-induced oxidative stress in HUVECs by decreasing hydroperoxides concentration [Sedighi et al. highlighted that a two-week application of lemon balm extract may ameliorate myocardial I/R injury induced by the LAD model. One of the proposed mechanisms is oxidative stress reduction via an increase in antioxidant enzyme SOD and a decrease in malondialdehyde (MDA), a product of lipid peroxidation, which was observed for the first time 5 days post-reperfusion. In our study, 7-day application of ME200 led to a mild but nonsignificant increase in SOD and GSH in the heart tissue homogenate, possibly due to the shorter time of exposition to ME and different kinds of samples used in the previously mentioned study compared to ours . Nonethecarditis , and alsntration . Interesntration .Cardioprotective effects that ME preconditioning displayed in this study are most probably the result of synergistic effects of its active components with rosmarinic acid as the leading component, which per se has been proved to exert an ameliorating effect on myocardial I/R injury in mice . AdditioBeneficial effects of ME were also observed in vitro on cardiomyocytes involving several mechanisms, including binding and activation of NF-kB, therefore inducing decreased ROS formation ,32, prevAll experiments and analyses conducted on laboratory animals or animal material used in this study were approved by the Ethics Committee for experimental animal well-being of the Faculty of Medical Sciences, University of Kragujevac  No.01-10171. Additionally, all the experimental procedures were performed following the European Directive2010/63/EU for the welfare of laboratory animals, number and principles of Good Laboratory Practice (GLP) (86/609/EEC). The experiments were carried out according to the European Union Directive 86/609/EES for the Protection of the Vertebrate Animals used for Experimental and other Scientific Purposes and the principles of ethics.Melissa officinalis L. (Lamiaceae), was purchased from Bilje Borca, LLC, Belgrade, Serbia. The dried plant material was pulverized using a mill , stored in well-sealed paper bags, and kept at room temperature until the extract was made. Ethanolic extract was made under the reflux of the solvent (70% ethanol) at the boiling point of the solvent (78.5 °C). The extraction process lasted 2.5 h. Afterward, the obtained mixture was filtered through gauze and left at room temperature to spontaneously precipitate ballast substances. Then, the obtained liquid extract was filtered . Finally, a rotary vacuum evaporator  at 40 °C, 90 rpm, and 250 mbar vacuum was used to obtain the dry extract. The dry extract was stored in dark glass vials at + 4 ºC until administration to animals. The extract was dissolved in tap water once daily prior to administration to animals per os [The plant material used in this research, dried leaves of s per os .Wistar albino rats (body weight 300 ± 50 g) purchased from the Military Medical Academy Animal House, Belgrade. At first, animals were acclimatized for two weeks and kept in polyethylene cages (4 per cage) in quarantine under standardized, controlled environmental conditions (22 ± 2 °C and a 12-h light/dark cycle). During the whole period, animals had free access to standard food  and water (ad libitum). Animals were randomly divided into the following groups:The study involved a total of 40 eight-week-old male n = 8).SHAM—healthy rats given an equivalent amount of distilled water with no myocardial I/R injury (n = 8).CTRL—healthy rats given an equivalent amount of distilled water with myocardial I/R injury .ME100—healthy rats treated with ME in a dose of 100 mg/kg with myocardial I/R injury (n = 8).ME200—healthy rats treated with ME in a dose of 200 mg/kg with myocardial I/R injury  for 7 days. Prior to administration, the extract was dissolved in distilled water, and aqueous solution of ME was applied via gavage to rats. The volume was calculated according to the body weight of rats each day (approximately 300 μL).2O according to the Langendorff technique. Hearts were perfused with Krebs–Henseleit buffer with similar content as extracellular content , equilibrated with 95% O2 plus 5% CO2, and warmed to 37 °C (pH 7.4).After 7 days of treatment with ME, on day 8 of the experimental protocol, animals were sacrificed. Firstly, animals were anesthetized by intraperitoneal injection of ketamine (10 mg/kg) and xylazine (5 mg/kg) and premedicated with heparin as an anticoagulant and sacrificed by decapitation. Afterward, the chest was opened via midline thoracotomy, the hearts were immediately removed and immersed in cold saline, and the aortas were mounted on the cannula of the apparatus and retrogradely perfused under a constant coronary perfusion pressure (CPP) of 70 cmHThe sensor was placed  in the left ventricle, and the following parameters of myocardial function were measured: dp/dt max, dp/dt min, SLVP, DLVP, and HR. CF was measured flowmetrically. After stabilization (20–30 min) of the heart rhythm, the global ischemia was induced by stopping the perfusion through the heart, followed by 30 min reperfusion. All cardiodynamic parameters and coronary flow were measured in intervals of 5 min during the period of reperfusion (30 min) (RP1-RP7). In all of the mentioned points of interest, coronary venous effluent was collected for further analysis of oxidative stress.g for 20 min at 4 °C, and the obtained supernatant was used for the spectrophotometric determination of the following oxidative stress parameters: index of lipid peroxidation (TBARS), NO2− concentration, CAT, SOD activity and reduced GSH level . Additionally, the following oxidative stress parameters  were determined from the samples of venous coronary effluent according to our previous research [Hearts from all animals were isolated, measured, and then frozen at −80 °C. Hearts were homogenized using an electrical homogenizer in PBS on ice . Then, the samples were centrifuged at 1200× research .Immediately after the Langendorff protocol had finished, rat hearts were collected, measured, and cut into two halves so that both halves of the heart were available for further histological analysis. The hearts were fixed in 4% neutral paraformaldehyde; then, the tissues were dehydrated in increasing alcohol concentrations , cleared in xylene, immersed in paraffin, and prepared for subsequent analysis. Tissue sections (5 µm thick) were stained by the H/-E (Hematoxylin/Eosin) method in order to examine and confirm morphological changes and by the Picrosirius red staining in order to determine collagen content in the heart tissues. An Olympus BX51 light microscope  was used to capture the images of heart tissue sections.Histological analysis of H/-E-stained tissue sections was based on the following criteria: presence (+) or absence (–) of degenerative changes, dilated interstitium, and hypercellularity. The intensity of changes was evaluated by using the following score: (–) no morphological changes, (+) mild (<10% per cross-section), (++) moderate (<20% per cross-section), and (+++) severe (<30% per cross-section) .p ≥ 0.05), the following multiple comparison method was selected Bonferroni [p < 0.05 was considered significant, while p < 0.01 was considered highly significant.Statistical analyses of the obtained data were performed using IBM SPSS 20.0  for Windows. Kolmogorov–Smirnov, Shapiro–Wilk tests, histogram, and normal QQ plot tests were used to examine the normality of the distribution, while data were expressed as mean values (X) ± standard deviation (SD). The data were analyzed by one-way analysis of variance (ANOVA), followed by a post hoc Bonferroni test. This test is considered to be suitable for the determination of the statistical significance of three or more independent groups. Before applying the post hoc test, we tested the homogeneity of the variances between the groups (Levene’s test). If variances were homogeneous  and index of lipid peroxidation (TBARS). However, more mechanistic research is necessary to determine the precise signaling pathways involved. Achieved cardioprotection is assumed to be a consequence of the synergistic antioxidant and anti-inflammatory effect of ME constituents, mainly phenolic acid, rosmarinic acid, and flavonoid quercetin.Hereby, we confirmed the capability of ME, especially in a high dose of 200 mg/kg, to significantly improve heart function and salvage the heart from I/R induced damage, even after short-term preconditioning. This refers to the prevention of cardiodynamic alterations, especially the improvement of contractility (via dp/dt max and dp/dt min), but also cardiac architecture in terms of ameliorating tissue changes and collagen deposition and fibrosis caused by I/R of the heart. Assumed mechanism of the achieved cardioprotective effects certainly implies oxidative stress amelioration, as proved by significant decrement of ROS production (O