There is no standard treatment recommended at category 1 level in international guidelines for subsequent therapy after cyclin-dependent kinase 4/6 inhibitor (CDK4/6) based therapy. We aimed to evaluate which subsequent treatment oncologists prefer in patients with disease progression under CDKi. In addition, we aimed to show the effectiveness of systemic treatments after CDKi and whether there is a survival difference between hormonal treatments (monotherapy vs. mTOR-based).n:175)) after CDKi were calculated. Patients were evaluated in three groups as those who received CDKi in first-line , second-line  and ≥ 3rd-line . PFS was compared according to the use of ET and CT. In addition, ET was compared as monotherapy versus everolimus-based combination therapy.A total of 609 patients from 53 centers were included in the study. Progression-free-survivals (PFS) of subsequent treatments  or endocrine therapy . It was 6.7 (5.8–7.7) months in the ET arm of group B, and 5.7 (4.6–6.7) months in the CT arm (p = 0.311). It was 5.3 (2.5–8.0) months in the ET arm of group C and 4.0 (3.5–4.6) months in the CT arm (p = 0.434). Patients who received ET after CDKi were compared as those who received everolimus-based combination therapy versus those who received monotherapy ET: the median PFS in group A, B, and C was 11.0 vs. 5.9 (p = 0.047), 6.7 vs. 5.0 (p = 0.164), 6.7 vs. 3.9 (p = 0.763) months.The median duration of CDKi in the ET arms of Group A, B, and C was 17.0, 11.0, and 8.5 months in respectively; it was 9.0, 7.0, and 5.0 months in the CT arm. Median PFS after CDKi was 9.5 (5.0–14.0) months in the ET arm of group A, and 5.3 (3.9–6.8) months in the CT arm (Physicians preferred CT rather than ET in patients with early progression under CDKi. It has been shown that subsequent ET after CDKi can be as effective as CT. It was also observed that better PFS could be achieved with the subsequent everolimus-based treatments after first-line CDKi compared to monotherapy ET.The online version contains supplementary material available at 10.1186/s12885-023-10609-8. Approximately 70% of breast cancers are hormone receptor (HR) positive . EndocriIn some retrospective studies, it has been observed that physicians prefer chemotherapy after CDKi treatment, even if there is no visceral crisis. In these studies, there was no significant PFS difference between chemotherapy and endocrine therapy. In this multicenter study, we aimed to evaluate which subsequent treatment oncologists prefer in patients with disease progression under CDKi. In addition, we aimed to show the effectiveness of systemic treatments after CDKi and whether there is a survival difference between hormonal treatments (monotherapy vs. mTOR-based).This retrospective study was approved by local ethics committee. Fifty-three centers approved data submission for the study.Patients with breast cancer aged 18 years or older and with estrogen or progesterone receptor levels ≥ 10% (CDK 4/6 inhibitors were reimbursed for only patients whose tumors expressed ≥ 10% estrogen receptor in our country) who have progressed after CDKi-based therapy and have received at least one systemic therapy (chemotherapy or endocrine-based therapy) were included in the study (between June 2018 and March 2022). Those who received CDKi treatment in early-stage disease and those with Her2 receptor positivity were excluded. Median PFS of the subsequent treatments after CDKi was the primary endpoint. Evaluation of the PFS difference between chemotherapy and endocrine-based treatments was the secondary endpoint.Patients' age, menopausal status, date of diagnosis and date of metastasis, ECOG performance status, sites of metastasis, median duration of CDKi, treatments they received after CDKi, and dates of progression under treatment were recorded retrospectively from patient files or the hospital registry system. A total of 609 patients included in the study were evaluated in three groups: those who received CDKi on the first line , those who received it on the second line , and those who received it on the ≥ 3rd line . Groups A, B, and C were also divided into those who received endocrine therapy (ET) and those who received chemotherapy (CT). The median PFS of the ET and CT groups were compared. In addition, the median PFS of ET was compared in all groups  as monotherapy versus everolimus-based combination therapy.p < 0.05 was considered statistically significant.Continuous variables were presented as median (range or interquartile range (IQR)), and categorical variables as frequency (percent). The Mann–Whitney-U test was used to compare the continuous variables of the two groups, and the chi-square or Fisher's Exact test was used to compare the categorical variables. The time from the start of the subsequent treatment after CDKi to disease progression or death was determined as PFS. Median follow-up time and PFS were determined by the Kaplan–Meier method. The log-rank test was used to determine the median PFS difference between the groups. All statistical analyzes were performed in two ways, and The median age of patients in Groups A, B, and C was 54, 54, and 53, respectively, and the rates of patients ≥ 65 years were 21.3%, 20.3%, and 15.4%. The rates of patients with ECOG PS ≥ 2 were 6.4%, 4.0%, and 5.9% in Groups A, B, and C, respectively. The rates of bone-only metastatic patients in Groups A, B, and C were 36.1%, 37.3%, and 23.6%. The central nervous system (CNS) metastasis rate was 3.5%, 2.0%, and 5.5% in Groups A, B, and C, respectively (Table The median duration of CDKi in Group A was 10 months (range: 3–46). In group A, median CDKi was 17 months (range: 3–46 months) in the ET arm and 9 months (range: 2–39 months) in the CT arm. The median duration of CDKi in Group B was 9 months (range: 2–34). In group B, median CDKi was 11 months (range: 3–34 months) in the ET arm and 7 months (range: 2–20 months) in the CT arm. The median duration of CDKi in Group C was 5 months (range: 2–24). In group C, median CDKi was 8.5 months (range: 3–23 months) in the ET arm and 5 months (range: 2–24 months) in the CT arm. The rate of bone-only metastatic patients was 22.8%, 20.9%, and 12.6% in groups A, B, and C, respectively  patients received CT, 76 (37.6%) ET; 110 (71.9%) CT, 43 (28.1%) ET in Group B; in Group C, 198 (77.9%) received CT and 56 (22.1%) ET  in Group A, 7.5 months (95% CI: 5.7–9.5 months) in the ET arm, and 5.1 months (95% CI: 4.4–5.8 months) in the CT arm of group A. Median follow-up was 6.5 months (95% CI: 5.0–7.9 months) in Group B, 7.9 months (95% CI: 5.8–9.9 months) in the ET arm, and 5.3 months (95% CI: 4.7–5.9 months) in the CT arm of group B. Median follow-up was 7.5 months (95% CI: 6.7–8.4 months) in Group C, 7.6 months (95% CI: 6.2–8.9 months) in the ET arm, and 6.9 months (95% CI: 3.9–9.9 months) in the CT arm of group C.p = 0.073) of group A. Median PFS was 6.7 (5.8–7.7) months in the ET arm and 5.7 (4.6–6.7) months in the CT arm (p = 0.311) of group B. Median PFS was 5.3 (2.5–8.0) months in the ET arm and 4.0 (3.5–4.6) months in the CT arm (p = 0.434) of group C  months in the ET arm and 5.3 (3.9–6.8) months in the CT arm (4.0 monthp = 0.022).In Groups A, B, and C, the median duration of CDKi, median age, ECOG PS, and metastasis sites were similar in monotherapy and everolimus-based arms. The rate of denovo metastatic patients in the monotherapy arm of Group A was higher than in the everolimus-based arm  (Table p = 0.047) months, 6.7 vs. 5.0 (p = 0.164) months, and 6.7 vs. 3.9 (p = 0.763) months, respectively  was shown in Table Univariate PFS analysis of patients who received endocrine therapy after CDKi in the first line (Everolimus initiation dose was 10 mg/day. Dose reduction (to 5 mg) was performed in 19.1% of the patients. In the everolimus-based group, 42% of the patients had Grade 1 stomatitis, and 11% had Grade 2 stomatitis. There were no data on the use of primary dexamethasone prophylaxis for stomatitis. In the everolimus-based group, 15% of the patients had elevated AST or ALT, and 17% had arthralgia. In the monotherapy ET group, the most common adverse event was arthralgia, with a rate of 15%.Any grade of adverse events occurred in 93% of patients receiving chemotherapy. 84% of patients who received CT had at least one dose reduction. The most common adverse events were neutropenia (47%), anemia (38%), and fatigue (33%). There was no patient who had discontinued CT due to toxicity.A standard of care treatment recommended as subsequent therapy in patients with advanced HR + , Her2- breast cancer that has progressed under CDKi therapy has not yet been established. Results of ongoing randomized clinical trials are awaited. Therefore, real-life data of retrospective studies is crucial. In our study, the factors affecting subsequent treatment choices and the effectiveness of these treatments were evaluated. In this multicenter retrospective study, it was observed that the short duration of CDKi in patients with HR + Her2- advanced breast cancer that progressed under CDKi treatment increased physicians' preference for CT in subsequent treatment. There was no difference in PFS between the subsequent CT and ET arms. When endocrine-based treatments were compared as monotherapy vs. everolimus-based treatments among patients who received CDKi in first-line, longer PFS was found with everolimus-based treatments.In a study evaluating the factors affecting treatment choices (CT vs. ET) after CDKi, priority ET was preferred as subsequent therapy in patients who received CDKi in the first line, and priority CT was preferred in those who received CDKi in the second line . As a ren = 7) who received ET after the first line. The median duration of palbociclib in the second line was 12.8 months. In this setting, the median PFS of subsequent ET (n = 9) was 9.3 months, and CT (n = 14) was 4.7 months [n = 16) and 4.1 months in the CT arm (n = 49) [PALOMA 3, a randomized clinical trial comparing fulvestrant vs. fulvestrant + palbociclib in previously treated patients with advanced breast cancer, showed no difference in duration of treatment between subsequent CT and ET after palbociclib (5.6 vs. 4.3 months) . Similar7 months . The sub(n = 49) . SimilarIt was demonstrated in the BOLERO-2 study that the everolimus + exemestane combination achieved longer PFS than monotherapy exemestane . In thisp = 0.03) [n = 26) and CDK-received (n = 17) patients, median PFS was 4.2 vs. 3.6 months [Contradictory results were obtained from limited retrospective studies showing the efficacy of everolimus-based treatments after CDKi –15. In t = 0.03) . Another = 0.03) . However = 0.03) . In a ren = 609) was the strength of our study.Our study had some limitations. The main limitations were that the study was retrospective, and the median duration of CDKi and follow-up were short. More patients received CT in the subsequent treatment than those who received ET. In addition, the shorter median duration of CDKi in patients who received CT compared to ET suggested that this group might have a relatively poor prognosis. The difference in median duration CDKi may have caused bias in the results obtained by comparing the CT and ET groups. The short median duration of CDKi could also affect subsequent PFS. Another limitation was that the rate of patients with disease progression in the first 24 months after adjuvant ET was lower in those receiving everolimus-based therapy than those receiving monotherapy ET. Despite these limitations, the investigation of the efficacy of subsequent treatments after CDKi with a large patient population (It was observed that oncologists preferred CT rather than ET in patients whose disease progressed in a short time with CDKi. This study showed that subsequent ET could be as effective as CT in patients whose disease progressed under ET + CDKi treatment. In addition, better PFS could be obtained with the subsequent everolimus-based therapy than with monotherapy ET after first line CDKi.Additional file 1: TableS1. Chemotherapy regimens.


In this study, the antioxidant activity of 4,4'-diaponeurosporene extracted from L. plantarum subsp. plantarum KCCP11226 was examined. Maximum carotenoid content (0.74 ± 0.2 at A470) was obtained at a relatively low temperature (20°C). The DPPH radical scavenging ability of 4,4'-diaponeurosporene (1 mM) was approximately 1.7-fold higher than that of butylated hydroxytoluene (BHT), a well-known antioxidant food additive. In addition, the ABTS radical scavenging ability was shown to be 2.3- to 7.5-fold higher than that of BHT at the range of concentration from 0.25 mM to 1 mM. The FRAP analysis confirmed that 4,4'- diaponeurosporene (0.25 mM) was able to reduce Fe3+ by 8.0-fold higher than that of BHT. Meanwhile, 4,4'-diaponeurosporene has been confirmed to be highly resistant to various external stresses . In conclusion, L. plantarum subsp. plantarum KCCP11226, which produces 4,4'-diaponeurosporene as a functional antioxidant, may be a potentially useful strain for the development of functional probiotic industries.Carotenoids, which have biologically beneficial effects and occur naturally in microorganisms and plants, are pigments widely applied in the food, cosmetics and pharmaceutical industries. The compound 4,4'-diaponeurosporene is a C Carotenoids, natural pigments belonging to the isoprenoid group, exhibit yellow, orange, red, and purple colors and naturally occur in algae, yeasts, fungi, bacteria, and plants , 2. Caro40 carotenoids) such as astaxanthin, lycopene, and β-carotene. However, triterpenoids (C30 carotenoids) have been reported to be found only in several bacterial species, such as Lactobacillus plantarum, Enterococcus faecium, Staphylococcus aureus, Methylobacterium rhodinum, and some heliobacteria  × 100, where A0 and A1 represent the absorbance at 600 nm before and after mixing, respectively.Bacterial cell surface hydrophobicity was evaluated according to Ekmekci ications . BrieflyKCCP11226 was cultivated at 30°C for 24 h (100 μl) and then spread on MRS agar medium, and antibiotic discs containing ampicillin (100 μg/ml), erythromycin (25 μg/ml), gentamicin (30 μg/ml), ciprofloxacin (10 μg/ml), lincomycin (15 μg/ml), novobiocin (10 μg/ml), tetracycline (30 μg/ml), and streptomycin (50 μg/ml) were placed on the inoculated plates. All plates were incubated at 30°C for 24 h and the inhibition zones were measured.et al. (2013) /Acontrol) × 100. As a control, the same reactant that did not contain 4,4'-diaponeurosporene was used. Butylated hydroxytoluene (BHT) was also assayed as an antioxidant reference.The scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was determined according to the method described by Kim ications . The 4,4et al. (2017)[The 4,4′-diaponeurosporene was further characterized by the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical assay with a slight modification of the method described by Yang l. (2017). First, et al.(2017) [3·6H2O at 10:1:1 (v/v/v). The FRAP reagent solution was maintained at 37°C. Then, 100 μl of 4,4′-diaponeurosporene was allowed to react with 3 ml of FRAP reagent solution and 300 μl of ultrapure water for 5 min at room temperature in the dark. The absorbance values of the mixture were measured at 593 nm (A593). The FRAP values were expressed as FeSO4 (mM) equivalents using the FeSO4·7H2O standard curve.The ferric reducing antioxidant power (FRAP) of 4,4′-diaponeurosporene was estimated according to Han l.(2017) . Brieflyt-test with Welch’s correction. Differences with p values of less than 0.01 were considered significant.The results of the experiments were expressed as means ± standard deviations of three independent measurements. The significance of differences in means was evaluated using analysis of variance (ANOVA) with Tukey-Kramer multiple comparison tests, unless mentioned otherwise. The FRAP data was analyzed using an unpaired i.e., acid/bile) should be investigated using reliable in vitro/vivo experiments [L. plantarum subsp. plantarum KCCP11226 was tested. As a result, it was confirmed to have resistance to erythromycin, gentamicin, ciprofloxacin, lincomycin, tetracycline, and streptomycin, but not against ampicillin and novobiocin .To produce effective probiotic strains, the safety and functionalities such as antibiotic resistance, adhesion to intestinal mucosa, and stress tolerance , we confirmed that a significant number of cells still survived . The resistance of strain KCCP11226 to bile acid was tested at 10% and 20% oxgall. Considering that a typical bile acid resistance experiment was performed at less than 1% oxgall [2O2 at concentrations of 16 mM and 32 mM, respectively [In addition, the ability to survive after ingestion is an important characteristic of probiotic microorganisms because they are exposed to low pH and bile acids during passage through the digestive tract. As shown in % oxgall , the app% oxgall ; hence, L. plantarum subsp. plantarum KCCP11226 has considerable potential as a probiotic strain because it shows survival under very harsh acid and bile conditions as well as external stress such as heat and lysozyme.Taken together, these results indicate that 30 carotenoid production by strain KCCP11226 were investigated under static growth conditions at 30°C. The cell growth and carotenoid levels were measured every 2 h. After 12 h of incubation, cell growth reached the stationary phase and the time profile of C30 carotenoid production exhibited with similar trends to that of cell growth, showing the maximal carotenoid production of 0.46 (A470) after 24 h of cultivation (The time profiles of cell growth and Ctivation .600) at a relatively high temperature of 40°C, but little carotenoid (0.05 at A470) was produced. On the other hand, at relatively mild temperatures of 20°C and 30°C, the cells grew at 4.58 and 4.24 (OD600), respectively. However, there was a significant difference in carotenoid production. At 30°C, 0.48 (A470) of carotenoid was produced, but a much higher amount of 0.74 ± 0.2 (A470) was produced at 20°C, which is a relatively low temperature. Temperature is a significant factor affecting the carotenoid biosynthetic pathway depending on the specificity of microbial species, followed by the quantity variation of produced carotenoids [et al. (1964) investigated the increase of β-carotene production from oleaginous red yeast Rhodotorula glutinis by decrease of culture temperature [Phaffia rhodozyma was changed from torularhodin to astaxanthin by decrease of culture temperature from 30°C to 20°C [The induction of carotenoid production by strain KCCP11226 was investigated through 24 h culture at various temperatures  . As a reperature . Another to 20°C .30 carotenoid production from L. plantarum subsp. plantarum KCCP11226 by employing the oxidative and salt stresses, which resulted in aerobic condition and high concentration of sodium chloride having positive effects on the increase of carotenoid production [40 carotenoids [30 carotenoids.In fact, our previous study already investigated the enhancement of Coduction . Likewisoduction . HoweverRadical scavenging assays using DPPH and ABTS radicals are widely used to evaluate the antioxidant activity of biological samples . In thisIn addition, 4,4′-diaponeurosporene effectively scavenged ABTS radicals in a concentration-dependent manner. In the Trolox equivalent antioxidant capacity (TEAC) analysis using Trolox as a reference compound, 0.25, 0.5, and 1 mM of 4,4′-diaponeurosporene showed 2.4 ± 0.1, 4.2 ± 0.3, and 7.9 ± 1.2 μM of TEAC value, respectively . In contL. plantarum subsp. plantarum KCCP11226, which is the main Lactobacillus species producing, 4,4′-diaponeurosporene, satisfies the conditions for use as a probiotic strain by surviving under various harsh conditions. In addition, 4,4′-diaponeurosporene, a C30 carotenoid produced by strain KCCP11226, exhibits good antioxidant activity. Therefore, strain KCCP11226 may be a useful functional antioxidant producer and a strain that can also be applied in the probiotic industry.In conclusion, http://jmb.or.kr.Supplementary data for this paper are available on-line only at


Background: Ulcerative colitis (UC) is a chronic recurrent inflammatory bowel disease (IBD). The conventional drugs for UC may induce severe side effects. Herbal medicine is considered as a complementary and alternative choice for UC.Purpose: This study aims to estimate the effect of natural polyphenol gallic acid (GA) on the NLRP3 inflammasome with dextran sulfate sodium (DSS)-induced colitis in mice.Study design: The body weights and symptoms of BALB/c mice were recorded. Histological evaluation, ELISA, q-PCR, immunohistochemistry, and western blotting were carried out to observe the morphology, cytokine contents, mRNA expressions, and protein expressions, respectively. Lipopolysaccharide (LPS)-induced RAW264.7 macrophage was used to probe GA’s effect on relative protein expression.Results: GA attenuated weight loss (p < 0.05), relieved symptoms, and ameliorated colonic morphological injury (p < 0.05) in mice with colitis induced by DSS. GA also lowered the contents of TNF-α, IL-1β, IL-18, IL-33, and IFN-γ in the serum and colon of mice, which were elevated by DSS, downregulated protein, and mRNA expressions of the NLRP3 pathway in the colon tissue. Furthermore, GA downregulated the expressions of NLRP3 (p < 0.05), iNOS (p < 0.01), COX2 (p < 0.01), and P-p65 (p < 0.05), and suppressed NO release (p < 0.001) in LPS-induced RAW264.7 cells.Conclusion: GA ameliorated DSS-induced UC in mice via inhibiting the NLRP3 inflammasome. These findings furnish evidence for the anti-inflammatory effect of herbal medicines containing GA on UC. Ulcerative colitis (UC) is a chronic relapsing inflammatory bowel disease (IBD) . Long-tein vivo  in mice and its influence on the NLRP3 inflammasome.The intracellular NOD-like receptor nucleotide-binding domain-like receptor family pyrin domain-containing-3 (NLRP3) is a critical controller of intestinal homeostasis by controlling various inflammatory and autoimmune conditions . The mulin vivo, B20851 ≥ 98% for in vitro) and 5-aminosalicylic acid  were purchased from Yuanye . Lipopolysaccharides  were bought from Sigma-Aldrich (Germany).DSS (MW. 36000–50000) was bought from MP Biomedicals (USA). GA  were provided by the Experimental Animal Center, Shanghai University of TCM. All the mice were housed in a temperature- and humidity-controlled environment  with a 12-h light/dark cycle to acclimatize for one week with free access to food and water. All animal experiment protocols were approved by the Animal Research Welfare Council of Shanghai University of Traditional Chinese Medicine (Ethics No. PZ200120).via ether anesthesia, all mice were euthanized to collect blood samples and colon tissues. The blood samples of the mice were collected by removing the eyeball . GA at 40 mg/kg, 80 mg/kg, 120 mg/kg, and 100 mg/kg and 5-ASA (dissolved in 0.5% carboxy-methylcellulose sodium) were orally administered to the mice from day 7 to day 12. 5-ASA acted as the positive control. On day 13,  eyeball .The tissues were fixed in 4% paraformaldehyde for 24 h, cut into 5-µm sections after dehydration, and embedded in paraffin. HE reagents were used to stain the colonic sections. Histological scores were evaluated by assessing the indices for changes in the epithelium and for cell infiltration . The folColonic tissue samples and sera were collected from BALB/c mice. The expressions of TNF-α, IL-33, interleukin-18 (IL-18), IL-1β, and IFN-γ in serum and in the lysis buffer of the colon tissue were determined by ELISA  according to the manufacturer’s instructions.−△△CT method. The PCR primer sequences in this work are shown in The protocols and the reagents of RT-qPCR have been described in detail previously . Total RImmunohistochemistry (IHC) was performed following previously described methods . The col2 in an incubator. Cell lines were collected until the logarithmic growth phase for the experiment.RAW264.7 cell lines were bought from the Chinese Academy of Sciences . Dulbecco’s modified Eagle medium  supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum  was used for culture at 37°C with 5% CO3 cells per well and cultured overnight. Then RAW264.7 cells were incubated with serial concentrations of GA dissolved in 0.1% dimethylsulfoxide (DMSO) for 24 h. The concentrations of GA were 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM. Next, 10 μL of Cell Counting Kit-8  solution was added to each well. The culture plate was placed in the incubator for 2 h, and the absorbance of the viable cells was detected (450 nm) using a Multiskan FC microplate reader .For the toxicology experiment, RAW264.7 cell lines were seeded onto a 96-well plate at 5 × 10RAW264.7 cells were inoculated into 6-well plates for 24 h and then treated with 50 μM GA, 100 μM GA, and 200 μM GA and 1 μg/mL LPS for 24 h. The amounts of NO release were determined by a nitric oxide assay kit . In brief, 50 μL standard and cell supernatants were added to the 96-well plate and mixed with 50 μL Griess Reagent I and 50 μL Griess Reagent II at room temperature, respectively. The OD value was examined by a microplate reader at 562 nm.Total protein was extracted by cell samples and colonic tissue of different treatment groups in the lysis buffer. The cells were collected and incubated in an RIPA buffer (with the addition of phosphorylation inhibitor and protease inhibitor) for 20 min in an ice bath. After centrifugation at 15,000 rpm for 15 min at 4°C, the upper-clarified lysate was collected. For the colon tissue, 700 μL RIPA lysate was added to a 100-mg sample to extract the protein. Protein concentration was determined using a BCA kit (Beyotime Biotechnology).For western blotting, the extracted protein of each group was separated by 8%–12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane. The membrane was blocked with a 5% BSA solution. Then, the membrane was cut into different blots and probed with specific primary antibodies  overnight-test. ANOVA was performed for multiple comparisons. p < 0.05 was considered statistically significant.All experiments were performed at least three times, and the results were expressed as mean ± SD. The gray value of the protein was quantified by ImageJ software. Data were analyzed using the software SPSS version 25.0 and illustrated using Graphpad Prism 8.0. The differences between the two groups were analyzed with Methods and materials” (p < 0.001). GA treatment at 120 mg/kg attenuated DSS-induced weight loss (p < 0.05) (p < 0.001), while 120 mg/kg GA ameliorated this change (p < 0.05) (p < 0.01), while GA  restored colon damage, improved crypt architecture, relived congestion and edema (p < 0.01)  . Similar < 0.05) . HE staind edema , and red < 0.01) .The secretions of TNF-α, IL-33, IL-18, IL-1β, and IFN-γ in both the serum and colon tissues of mice in the DSS group were significantly upregulated compared with the Control group , indicatDSS-induced murine colitis showed many pathological features on UC in humans, especially type I inflammatory response. Compared with the Control group, the mRNA expression of NLRP3, ASC, Caspase-1, Caspase-4, IL-1β, and IL-18 significantly increased in the DSS group . 5-ASA avia inhibiting the activation of the NLRP3 inflammasome.We have demonstrated that GA reduced the levels of TNF-α, IL-33, IL-18, IL-1β, and IFN-γ in the serum and colon tissues, which provides the possibility of suppressing the activation of NLRP3 inflammasomes. We also detected a relative downstream protein expression of NLRP3. IHC results illustrated that the expressions of NLRP3 were hardly found in the Control group but were significantly elevated in the DSS group, and after treatment of GA, the expression of NLRP3 was reduced . The resp < 0.001) and was suppressed after GA treatment (p < 0.05) and dose-dependently downregulated LPS-induced iNOS and COX2 expressions (p < 0.01). Further investigation demonstrated that GA significantly reduced the phosphorylation of p65-NF-κB (p < 0.05) and ERK (p < 0.01) in the RAW264.7 cell primed by LPS (The CCK-8 assay indicated that the concentrations of GA applied in the subsequent experiments exhibited no evident influence on RAW264.7 cell viability . NO relereatment . Similarreatment . GA tread by LPS .UC is an inflammatory disease of the intestine, highly susceptible to relapse and progression into cancer . AlthougInflammasomes play a critical role in the progression of UC . The infvia inhibiting the NF-κB pathway (In our previous study, we found that QCS ameliorated DSS-induced colitis by inhibiting the expression of Caspase-1 and IL-1β during inflammasome activation . GA, the pathway . Consist pathway . Further pathway , and dow pathway , 4, demo pathway . Accordi pathway . Moreove pathway , and IL- pathway . Our fin pathway .in vitro results verified our speculation that GA not only inhibited NLRP3 inflammasome activation but also inhibited the endogenous NLRP3 expression subsequently with a suppression of p65-NF-κB phosphorylation (It is generally believed that NLRP3 must be primed before activation and formation of the inflammasome complex. An NF-κB-activating stimulus such as LPS priming first induces elevated expressions of NLRP3 and IL-1β . Murine rylation .via inhibiting the NLRP3 inflammasome. Herbs and preparations containing GA may have an anti-inflammatory effect on UC, but the deeper mechanisms deserve further investigation.In conclusion, this study shows that GA ameliorated DSS-induced UC in mice


Inflammatory cytokines, cell adhesion molecules, and toll-like receptors (TLRs) play an important role in atherosclerosis. The aim of this study was to further evaluate the role of inflammatory cytokines, cell adhesion molecules, and toll-like receptors in atherosclerosis. Forty local breed domestic male rabbits were divided randomly into 4 groups, 10 rabbits each. Group I was the control group, group II received a high cholesterol diet, group III received the drug solvent dimethyl sulfoxide (DMSO), and group IV received Atorvastatin (3.5 mg/kg/day). Blood samples were collected at 0 times, 5 weeks, and at the end of 10 weeks. TLRs expression on monocyte was measured by flow cytometry, IL-10, IL-17, IL-1β, intracellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) were measured by ELISA. In group II, a high cholesterol diet led to a statistically significant elevation of lipids profile  at both 5 weeks and 10 weeks compared to the control. The expression of TLRs was also increased compared to the control (13.53±2.5 to 25.79±6.5). The intimal thickness increased from 103.46±13.85 to 248.43±11.11. IL-17 increased significantly from 3.4±0.4 to 7.7±1.00, and IL-1β increased from 1.04±0.19 to 9.66±1.4 (P 0.05) at 10 weeks. ICAM and VCAM increased from 1.7±0.16 to 8.2±0.74 and from 0.89±0.07 to 5.2±0.45, respectively. Atorvastatin significantly reduced TLRs at 10 weeks to 21.98±3.4 and intimal thickness to 191.6±15.59. IL-17, IL-1β, ICAM, and VCAM were significantly reduced by Atorvastatin. Cytokines, cellular adhesion molecules, and probably TLRs have a role in the pathogenesis of hyperlipidemia and atherosclerosis. Atherosclerosis is an inflammatory disease and a complex condition in which arteries become hard due to the formation of plaques. It is a multifocal and immune-inflammatory disease of large and medium-sized arteries . It is cThe objective of our study was to evaluate further the role of TLRs as:Cytokines , intracellular and vascular;Adhesion molecules (ICAM and VCAM) in the pathogenesis of atherosclerosis and to explore the modulator effect of Atorvastatin on these parameters.ad libitum. After one week of adaption, the animals were randomized into four groups, ten rabbits each: normal control group (Ι) were kept on a standard chew diet and tap water, group (ΙΙ): atherogenic high cholesterol diet, were kept on an atherogenic diet  [@). Atorvastatin was dispersed in DMSO since it is insoluble in water. About 3 ml of blood was collected from the central ear vein of each rabbit following overnight fasting. Blood samples were collected at zero time, at the end of 5 weeks, and the end of 10 weeks of drug treatment for each group of rabbits. Sera were collected to measure serum parameters. In the 10th week, the animals were sacrificed under ketamine and xylazine (5 to 10 mg/kg) anesthesia. A segment of the aorta was dissected to estimate intimal thickness and histopathological examination.Forty local domestic male rabbits were used in this study. Their weight ranged from 1–1.5 kg, and their age was between 6–12 months. These were housed in the same experimental conditions with constant humidity, and they were allowed to drink tap water and pellet diet  (wt/wt)  and tap  (wt/wt)  by oral Plasma cholesterol, low-density lipoprotein (LDL), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were determined enzymatically by standard methods.Measurements of TLRs were done by flow cytometry.An enzyme-linked immunosorbent assay (ELISA) kit was used for IL-10, IL-17, IL-1ß, ICAM, and VCAM serum levels measurement.Measurements of intimal thickness were carried out by an ocular microscope.Aorta segments were fixed in 10% formaldehyde at room temperature. The sections were stained with hematoxylin-eosin.Data are expressed as mean±SD. Paired t-tests were used to compare differences between the mean values within each group at different times. The Chi-square test was also used to compare histopathological findings in different groups. Statistical significance was considered as P<0.05. Analyses were performed using SPSS software update version 21.vs. 12.66±3.8 and 25.79±79 vs. 13.53±2.5), and the intimal thickness at 10 weeks significantly increased (P<0.05) from 103.46±13.85 in the control group to 248.43±11.11 in the atherogenic diet group. Atorvastatin treatment in group (IV) produced a statistically significant (P<0.05) reduction in lipid profile at both 5 and 10 weeks following treatment, with the largest effect at 10 weeks as compared to the high cholesterol diet group (group II), reduction of total cholesterol from 1301±443 to 290±23 and TG from 256.0±24.0 to 101.0±28 and LDL from 929±251 to 296±38 at 10 weeks. Atorvastatin also reduced serum level of IL-1β1 from 9.66±1.4 group (II) to 2.44±0.49 at 10 weeks, with similar changes in IL-17 and effect on IL-10. Atorvastatin also reduced both ICAM from 8.2±0.74 in group II to 2.7±0.13 and VCAM from 5.2±0.45 to 1.6±0.14 TLRs decrees from 25.79±6.5 to 21.98±3.4. This reduction was not statistically significant (P>0.05). Intimal thickness significantly decreased in the Atorvastatin group at 10 weeks from 248.43±11.11 to 191.6±10.59. In this study, all rabbits (100%) fed with high cholesterol diet developed hypercholesterolemia and atherosclerosis lesions with different stages in the branches of the aorta artery, including fatty streaks, atheroma, and fibrous cap formation. Treatment of rabbits with Atorvastatin resulted in a significant reduction in the severity of atherosclerotic lesions compared with the untreated group (Atherogenic diet (group II) led to a clear and statistically significant (P<0.05) elevation of all the parameters of lipid profile  . The effThe high cholesterol diet in this study produced a clear and significant (P<0.05) increase in all the lipid profile parameters in rabbits. This is in agreement with many other studies . It is kThis study found that 30% of rabbits treated with Atorvastatin for 10 weeks developed initial lesions, 50% developed intermediate lesions, and 20% developed advanced lesions. It was found that Atorvastatin can reduce inflammatory cell accumulations and smooth muscle cell proliferation and migrations, and plaque formation . There iIn conclusion, a high cholesterol diet induced elevation of all serum lipid parameters. It also led to increased serum levels of IL-17 and IL-1β and increased adhesion molecules ICAM and VCAM. In addition to its lipid-lowering effect, Atorvastatin can also modulate cytokines and adhesion molecules, which could explain some of its pleiotropic effects. The role of TLR in the mediation of statins action requires further exploration.


Bacillus cereus sensu lato (B. cereus sl) is important spoilage bacteria causing milk structure and flavor changes and is ubiquitous in the environment. This study addresses the biodiversity, toxicity, and proteolytic activity of B. cereus sl from 82 environmental samples and 18 raw bovine milk samples from a dairy farm in the region of Tianjin. In sum, 47 B. cereus sl isolates were characterized through biochemical tests, 16S rRNA gene sequencing, and panC gene analysis. Fourteen sequence types (STs) of B. cereus sl were found in raw bovine milk samples, and five new STs  were identified in this study. ST1150 was the dominant ST, associated with fecal, air, drinking water, teat skin, teat cup, and teat dip cup. The results of toxin gene analyses showed that 12.77% and 8.51% of isolates carried hblACD and nheABC operons, respectively. In addition, the detection rate of emetic cesB gene was 21.28%. B. cereus sl demonstrated high spoilage potentials even at 7°C, which has the proteolytic activity of 14.32 ± 1.96 μmol of glycine equivalents per ml. Proteolytic activities were significantly (p < 0.05) decreased after the heat treatment. The residual activity of protease produced at 7°C was significantly higher than that produced at 25°C and 37°C after treatment at 121°C for 10 s and 135°C for 5 s (p < 0.01). Together, the results provide insights into the characteristics of B. cereus sl from farm environment and raw bovine milk and revealed that B. cereus sl contamination should also be monitored in raw milk for ultra-high temperature (UHT) products. This knowledge illustrates that strict cleaning management should be implemented to control B. cereus sl and assure high-quality milk products. Bacillus cereus sensu lato (B. cereus sl) is a bacterial group that can be commonly detected in environment samples such as soil , B. thuringiensis, and B. weihenstephanensis  and European Centre for Disease Prevention and Control (ECDPC), B. cereus sl could induce two types of food poisoning: emetic and diarrheal  milk  isolates would help to evaluate the population dynamics in China's dairy farm.A total of 100 samples from a dairy farm in Tianjin, China were collected from September to November 2019. They included 82 environmental samples  and 18 raw milk samples. All the samples were collected according to Du et al. . Then, aB. cereus sl isolation, all the samples were diluted and first chosen according to the National Food Safety Standard Method for Food Microbiological Examination (GB 4789.40-2010)  thermal cycler  via 16S rRNA and panC gene. DNA of B. cereus isolates was extracted with the InstaGene Matrix DNA Extraction Kit (Bio-Rad) following the instructions of Owusu-Kwarteng et al. , and conditions are shown in B. cereus ATCC 11778; China Center of Industrial Culture Collection, Beijing, China) controls.Identification of g et al. . The panhttp://www.ncbi.nlm.nih.gov/BLAST/). The degree of homology for the universal 16S rRNA gene and the panC gene was higher than 99%.The PCR products were sequenced by SinoGenoMax Co. Ltd. Then, the sequencing results were BLASTed in the GenBank database  were chosen according to the B. cereus MLST database (http://pubmlst.org/bcereus/) as described by Zahner et al.  were selected as specific reference strains in this study.After the seven housekeeping gene sequences were spliced in order, the molecular evolutionary genetics analysis  software was used for the phylogenetic relationship analysis  and an emetic gene (cesB). All the primers  and PCR conditions are also shown in Eight virulence genes have been detected, including seven enterotoxigenic genes (Bacillus cereus sl isolates were streaked on nutrient agar (Beijing Land Bridge Technology Co. Ltd.) supplemented with UHT milk   and cultured at 37°C to produce protease. After 7 days, all incubated samples were immediately centrifuged at 12,000 × g for 20 min at 4°C. About 1-ml supernatant was added to 9-ml skim UHT milk (Granarolo S.p.A.) followed by the addition of bronopol  and sodium azide  at final concentrations of 0.025% and 0.01%, respectively, to prevent the growth of bacteria. Finally, the samples were then incubated at 7°C, 25°C, and 37°C for another 14 days, respectively.The e et al. . The isoB. cereus sl isolates were calculated according to a standard curve and expressed as a concentration of glycine.In order to quantify proteolytic activity, the trinitrobenzenesulfonic acid (TNBS) method was performed according to Meng et al.  to trackAbout 2 ml of protease supernatant was added to a glass tube and heated at 55°C for 5 min, 95°C for 5 min, 121°C for 10 s, or 135°C for 5 s. All the samples were immediately cooled with ice. Then, 1 ml of heated protease supernatant was diluted with 9 ml of skim UHT milk (Granarolo S.p.A.) and incubated at 7°C, 25°C, and 37°C for 14 days, respectively.via GraphPad Prism 9.0  through the one-way analysis of variance (ANOVA). The results were presented as the mean ± standard error of mean (SEM).All data were analyzed B. cereus sl from 43 samples  were identified according to the biochemical experiments , followed by raw milk samples (11 isolates), and post-dip cup samples (5 isolates).In total, 47 strains of eriments  and PCR B. cereus sl STs based on the allelic coding of the seven housekeeping genes and the MLST database reference (http://pubmlst.org/bcereus/) , followed by ST205 , ST1419 , ST744 , ST127 , ST281 , ST1013 , ST1943 , ST2749 , ST2750 , ST2751 , ST2752 , ST2753 , and ST142 . Moreover, ST1150 was isolated from ten different types of samples, including milk, fecal, air, drinking water, teat skin, teat cup, pre-dip solution, post-dip solution, pre-dip cup, and post-dip cup. The new STs were numbered from 2749 to 2753, which were from fecal (ST2749 and ST2750), milk (ST2751), drinking water (ST2752), and post-dip solution (ST2753), as shown in B. cereus sl, a neighbor-joining phylogenetic tree was constructed. All the isolates in this research and the 13 selected reference strains showed a clear phylogenetic relationship as seen in In view of the linkage sequences of the seven housekeeping genes of 47 B. cereus sl STs with a close phylogenetic relationship differed only by a few bases. The novel ST, designated ST2749, ST2751, ST2752, and ST2753, had a closer phylogenetic relationship with B. cereus strains; however, ST2750 was close to B. paranthracis.According to the MLST database, it was found that the gene sequences of these hblACD operon), nonhemolytic enterotoxin complex genes (nheABC operon), cytotoxin K (cytK-2), and cereulide (cesB)  and hblC (57.45%). However, only 12.77% (n = 6) and 8.51% (n = 4) isolates carried hblACD and nheABC gene clusters, respectively. No virulence genes were detected in one isolate (2.13%). Furthermore, ten strains (21.28%) of B. cereus sl carried the virulence gene cesB together with hemolytic and/or nonhemolytic enterotoxin complex genes.The virulence genes are divided into hemolytic enterotoxin complex genes (B. cereus sl isolates (27.66%) carried hblA, hblC, nheB, and nheC, and ten isolates (21.28%) carried only nheB and nheC. Isolates carrying only hblA/C/D and nheA/B/C were detected in 31 isolates (65.96%), which suggests that diarrheal strains were more common among isolates from raw bovine milk and dairy farm environmental samples.In total, 14 different virulence patterns were detected in this study. Thirteen n = 1; ST205, n = 1; ST1150, n = 2; and ST1419, n = 1), no cesB gene was detected.The proteolytic activity of the 47 isolates was evaluated at 7°C, 25°C, and 37°C. The appearance of a clear zone or halo in milk agar was used to determine the phenotypic results. Halos were detected in 42 isolates (89.36%) at 25°C and 37°C. No halos were found on the milk agar at 7°C . MoreoveR2 value was 0.998 (data not shown). Isolates were identified as proteolytically active when the value was above 2 μmol of glycine equivalents per ml.The quantified proteolytic activity was performed in skim UHT milk . First, B. cereus sl isolates. At 37°C, the proteolytic activity of these isolates  was significantly higher than at 25°C and 7°C (p < 0.001). At 25°C, the proteolytic activity was 17.25 ± 3.56 μmol of glycine equivalents per ml, and it was 14.32 ± 1.96 μmol of glycine equivalents per ml at 7°C.Differences in proteolytic activity were found in proteases produced by 42 B. cereus sl isolates at 7°C, 25°C, and 37°C were then heated to investigate the heat resistance at 55°C for 5 min, 95°C for 5 min, 121°C for 10 s, and 135°C for 5 s, respectively. In sum, all the proteases were resistant to 55°C for 5 min and 95°C for 5 min. However, after heating at 121°C for 10 s, proteolytic activity of 9, 9, and 10 isolates produced at 7°C, 25°C, and 37°C, respectively, was below 2 μmol of glycine equivalents per ml. Moreover, after heating at 135°C for 10 s, protease of 22, 21, and 21 isolates produced at 7°C, 25°C, and 37°C, respectively, was considered as no proteolytic activity . The percentage of residual protease activity produced at 7°C was 13.40 ± 0.98%, 20.35 ± 12.74%, 56.65 ± 14.07%, and 70.36 ± 5.81% after 55°C for 5 min, 95°C for 5 min, 121°C for 10 s, and 135°C for 5 s, respectively. These values were 5.70 ± 5.93%, 19.45 ± 11.52%, 62.08 ± 8.64%, and 83.56 ± 7.80% for protease produced at 25°C and 5.09 ± 5.20%, 19.35 ± 12.80%, 62.19 ± 9.02%, and 79.18 ± 7.61% for protease produced at 37°C. Moreover, when the protease was heated at 55°C for 5 min, the percentage of residual proteolytic activity produced in the 25°C and 37°C groups was significantly higher than that for 7°C (p < 0.001).All the proteases produced by 42 activity . The perB. cereus foodborne outbreaks usually occurred in dairy-based products  to identify B. cereus from other Bacillus species. In this way, B cereus was distinguished from strains that were highly similar in both phenotype and 16S rRNA sequence , followed by raw milk samples (61.11%). BioNumerics software analysis has illustrated that B. cereus sl isolates in raw milk might be from fecal, teat skin, teat cups, and dip cups (data not shown). The incident was similar to B. cereus prevalence on an Australian dairy farm, where B. cereus group was positive for 41% of 120 samples. Moreover, a high occurrence of B. cereus group was found in soil , feces , and raw milk  , followed by bedding , feces , feed , and raw milk . Fei et al. (B. cereus strains from 50 raw bovine milk samples and 54 isolates (18%) from 300 environmental samples. In that study, bedding  and excrement  had the highest prevalence. B. cereus was most widely distributed at the Bacillus genus level from Korean dairy farms  in Egypt . The occurrence of B. cereus sl in this study has a similarity to other studies, which support the speculation that Bacillus spp. in raw milk is contaminated from the upper layer of soil in pastureland or from feces ], which is similar to the presence (12.5%) in milk and milk products in Ghana  were present in 51% B. cereus sl isolates from Norwegian bovine milk products  from 306 milk and environmental samples, 94.4% (51/54) from 500 dairy products , emetic gene cesB was only detected in 21.28% of the strains. Furthermore, proteases of B. cereus sl produced at low temperatures had higher resistance to UHT treatment. Therefore, from this study, an understanding of the hygienic conditions and control of B. cereus sl is needed to maintain milk quality and safety.In this study, 47 The original contributions presented in the study are included in the article/LM designed experiments and wrote the manuscript. RZ carried out experiments. LD and HH helped in the experiments. HL, JC, and NZ gave advice. JW gave opinions on the research design and supported the findings. All authors contributed to the article and approved the submitted version.This work was supported by the Project of Risk Assessment on Raw Milk (GJFP20220304), the Agricultural Science and Technology Innovation Program (ASTIP-IAS12), and the China Agriculture Research System of MOF and MARA (CARS36).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In conclusion, the enteric-coated Cologrit tablets demonstrated good small intestine-specific phytochemical delivery capability, and decreased UC-like inflammation, and oxidative stress through the regulation of TNF-α/NFκB/IL6 signaling axis.Ulcerative colitis (UC) is an inflammatory bowel disease that affects the patients’ colorectal area culminating in an inflamed ‘leaky gut.’ The majority of UC treatments only provide temporary respite leading to its relapse. Therefore, this study investigated the efficacy of the enteric-coated ‘Cologrit’ (EC) tablet in alleviating UC-like inflammation. Cologrit is formulated using polyherbal extracts that have anti-inflammatory qualities according to ancient Ayurveda scriptures. Phytochemical profiling revealed the presence of gallic acid, rutin, ellagic acid, and imperatorin in Cologrit formulation. Cologrit treatment decreased inflammation in LPS-induced transformed THP-1 macrophages, and TNF-α-stimulated human colorectal (HT-29) cells through the modulation of NFκB activity, IL-6 production, and NFκB, IL-1β, IL-8, and CXCL5 mRNA expression levels. Cologrit also lessened human monocytic (U937) cell adhesion to HT29 cells. Methacrylic acid-ethylacrylate copolymer-coating of the enteric Cologrit tablets (EC) supported their dissolution, and the release of phytochemicals in the small intestine pH 7.0 environment in a simulated gastrointestinal digestion model. Small intestine EC digestae effectively abridged dextran sodium sulfate (2.5%  Ulcerative colitis (UC) is an idiopathic chronic inflammatory bowel disease of the colon and rectum. The disease’s aetiology is mostly unknown, but it is frequently linked to genetic, dietary, lifestyle, microbial infection, and environmental factors, alone or in combination ,2. AdultAegle marmelos (L.) Corrêa, Holarrhena antidysenterica (L.) Wall. ex A. DC, Cuminum cyminum L., Trachyspermum ammi (L.) Sprague, Foeniculum vulgare Mill., Rosa indica L., and Cinnamomum camphora (L.) J. Presl (see Aegle marmelos (L.) Corrêa fruit extract were found to modulate inflammation-associated responses in inflammatory bowel syndrome (IBD)  extract of Cinnamomum camphora (L.) J. Presl leaf was also applied. All components were combined and blended for 15 min at 25 rpm in an octagonal blender into Cologrit formulation.Cologrit powder (Batch no. PRFT/CHIN/0422/0196) was created by combining different quantities of the plant extracts listed in Cologrit tablets (Batch No. CHIH/COLA/0222/2320) were prepared by blending Cologrit powder with microcrystalline cellulose in an octagonal blender for 15 min at 25 rpm . The mixture was then mixed with excipients at the concentrations listed in w/w) EC, appropriate weight of MMA was dissolved in isopropyl alcohol (IPA) for 10 min while stirring at 200 rpm. Dichloromethane (DCM) was added to the prepared solution (Ratio of IPA: DCM set at 60:40), and stirred for another 30 min at 300 rpm [For 6%  was used to determine the average weight of 20 tablets. Average friability of 20 tablets at 25 rpm for 4 min was measured using test apparatus . A hardness tester  was used to determine the average hardness of ten tablets. A Vernier Calliper  was used to measure the thickness and diameter of the tablets.4NO3 (80 mM), KH2PO4 (9 mM), KCl (5 mM), K3C6H5O7 2 mM, C5H4N4NaO3 0.2 mM, Urea 0.6 mM, Sodium DL Lactate (2.6 mM), mucin (3.0 mg/mL) and amylase (150 unit/mL), and vigorously shaken for 15 s.EC Cologrit tablets (11 × 500 mg) were suspended into 20 mL of MilliQ water for the study. As a negative control, only water without the Cologrit tablet was used. Simulated digestion of the Cologrit tablets was done following the protocol mentioned by Deloid et al. . In the 2 (10 mM), bile salt (5.0 mg), and lipase  for 2 h at 37 °C with constant stirring. The pH of the solution was kept at 7.0 by adding 10 mL of 0.1 N NaOH solution. The dissolved Cologrit concentration in the final digestae was 11.06 mg/mL. All the digestae were stored at −80 °C and used for biochemical analysis.In the stomach phase, 20 mL of mouth phase bolus were mixed with 20 mL of simulated gastric fluid (chyme) containing NaCl (684 mM), HCl (72 mM) and pepsin (3.2 mg/mL), adjusted to pH 2.5, and incubated at 37 °C for 2 h on a shaker (100 rpm). 30 mL of stomach phase chyme with pH adjusted to 7.0 was mixed with small intestinal fluid (NaCl (150 mM) and CaClIndividually, Gallic acid, Rutin, Ellagic acid, and Imperatorin standards were dissolved in methanol to make 1000 ppm standard stock solutions. Imperatorin was further diluted for preparing a 100 ppm working standard solution. All the other standards were diluted to a working standard solution of 25 ppm. 250 mg of Cologrit was sonicated for 30 min in 10 mL of water: methanol (20:80) mixture. The mixture was centrifuged at 10,000 rpm and filtered through a 0.45 µm nylon filter. HPLC analysis was performed on the standards’ filtrate, and Cologrit tablet containing chyme and digestae samples collected under sub-HPLC was performed using Prominence-XR HPLC system  equipped with Quaternary pump (Nexera XR LC-20AD XR), DAD detector (SPD-M20 A), Auto-sampler (Nexera XR SIL-20 AC XR), Degassing unit (DGU-20A 5R) and Column oven (CTO-10 AS VP). Separation was achieved using a Shodex C18-4E  column subjected to binary gradient elution. The two solvents used for the analysis were water containing 0.1% acetic acid in water (solvent A) and acetonitrile (solvent B) set in a gradient program (Time (T) = 0: A% = 100%, B = 0%; T = 5: A%= 95%, B = 5%; T = 10: A% = 90%, B = 10%; T = 50: A% = 70%, B = 30%; T =60: A% = 45%, B = 55%; T =70: A% = 20%, B = 80%; T =71: A% = 100%, B = 0%; T =75: A% = 100%, B = 0%). Column temperature was kept 30 °C and flow was set 1.0 mL/min during the analysis. 10 µL of standard and test solutions were injected. Wavelength was set 253 nm  and 270 nm .During the digestion phase, chyme and digestae samples were collected every 20 min, and analyzed for Cologrit tablet dissolution using UV-1800 spectrometer . A full spectrum UV scan (200–1000 nm) was performed using the samples and Cologrit specific absorbance spectra λmax were determined at 339 nm, with no optical interferences in the presence of biomolecules. Spectragryph software  was used to spectra and data processing.Caco-2, HT-29, THP-1 and U937 cells were obtained from National Centre for Cell Science, Maharashtra, India . THP-1-Blue NFκB reporter cell was purchased from InvivoGen . HT-29, and Caco-2 cells were grown in high-glucose DMEM media supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), 10 mM HEPES buffer, 100 IU/mL Penicillin, 100 µg/mL Streptomycin and 1% non-essential amino acids. U937, THP-1 and THP-1-Blue NFκB reporter cells were grown in normal RPMI-1640 media supplemented with 10% FBS, 10 mM HEPES buffer, 100 IU/mL Penicillin, 100 µg/mL Streptomycin and 1% non-essential amino acids.For the cell-based studies, fresh digestae containing 11.06 mg/mL of enzyme digested Cologrit tablet were prepared using the protocol described in sub-4 cells/well in 96 well plates and incubated overnight. Next day, the cells were treated with different concentrations of Cologrit for 24 h. Caco-2 cells were plated at the concentration of 1 × 104 cells/well in 96 well plates and pre-incubated overnight. Digestae w/wo Cologrit tablets were dissolved in serum-free DMEM medium at concentrations of 1, 3, 10, 30, and 100 μg/mL. In parallel, Caco-2 cells following pre-incubation were also treated for 24 h with 0.65 percent, 1.15 percent, 2.5 percent, and 5 percent (w/v) Dextran Sodium Sulfate (DSS) colitis grade  solution prepared in serum-free medium. At end of treatment time, media was removed and the cells were washed with sterile PBS. Cells were incubated for 3 h with a 10 μg/mL concentration of Alamar blue™, and fluorescence was measured at Ex. 560 nm/Em. 580 nm using Envision multimode plate reader .Cologrit was suspended in 2% FBS containing DMEM at the concentrations of 1, 3, 10, 30, and 100 μg/mL. Following pre-incubation, HT-29 cells were plated at the concentration of 2 × 105 THP-1 cells/mL were differentiated using 20 ng/mL PMA overnight. The next day, the PMA-containing media was replaced with fresh complete cell culture media, and the differentiated cells rested for 4 days with intermittent media change. Pre-treatment of Cologrit was given to the differentiated cell at the concentrations of 3, 10, 30 and 100 µg/mL for 24 h. Following day, the cells were co-treated with LPS (100 ng/mL) and varying concentrations of Cologrit for 6 h. Cell soup was collected and used for the quantification of cytokines according to manufacturer instructions.For the IL-6 and TNF-α release estimation using ELISA assay, 1 × 105/mL and pre-incubated overnight. Cells were subsequently co-treated for 24 h with 10 ng/mL TNF-α and Cologrit at 10, 30, and 100 µg/mL concentrations. The expression of NFκB was measured as Secreted Embryonic Alkaline Phosphatase (SEAP) and quantified using the QUANTI-Blue assay  according to the manufacturer’s instructions. The optical density at 630 nm readout was determined using an Envision multimode plate reader .THP-1-Blue NFκB reporter cells were plated in 96-well plates at a seeding density of 5 × 105/well in 12 well plates and incubated overnight. Cells were pre-treated with/without Cologrit at the varying concentrations of 10, 30 and 100 µg/mL for 24 h. Cells were subsequently co-treated for 24 h with 10 ng/mL TNF-α and Cologrit at 10, 30, and 100 µg/mL concentrations. Qiagen’s RNeasy mini kit was used to extract total RNA from the treated cells. 1 µg of RNA was reverse-transcribed into cDNA using the Verso cDNA synthesis kit according to the manufacturer’s instructions. The mRNA expression was analyzed using human primer sequences of IL-8: Fw- 5′ GCTTGAAGTTTCACTGGCATCT 3′; Rv- 5′ AGTTTTTGAAGAGGGCTGAGAAT 3′; CXCL5: Fw- 5′ AGCTGCGTTGCGTTTGTTTAC 3′; Rv- 5′ TGGCGAACACTTGCAGATTAC 3′; NFκB: Fw- 5′ GTGGTGCCTCACTGCTAACT 3′; Rv- 5′ GGATGCACTTCAGCTTCTGT 3′, and IL-1β: Fw- 5′ GTCGGAGATTCGTAGCTGGA 3′; Rv- 5′ ATGATGGCTTATTACAGTGGCAA 3′; and β-actin (housekeeping gene): Fw- 5′ CATGTACGTTGCTATCCAGGC 3′; Rv-5′ CTCCTTAATGTCACGCACGAT 3′. For RT-PCR analysis, a mixture containing 5 µL of PowerUp SYBR Green Master Mix, 5 ng of cDNA template, and 0.5 µL (200 nM) forward and reverse primers was prepared. qRT-PCR was performed using qTOWER3G  instrument set at conditions- 95 °C for 5 min, 95 °C for 30 s, and 55 °C for 30 s for 40 cycles, with a melt curve obtained for each sample. The obtained mRNA expression values were normalized to β-actin, and relative mRNA expression was calculated using the 2−Ct technique. Obtained expression values were normalized against β-actin and relative mRNA expression was quantified using the 2−∆∆Ct method [HT-29 cells were seeded at a density of 2 × 10t method .4 cells/well in 96-well plates. Pre-treatment of the HT-29 cells was given with Cologrit at the concentrations of 3, 10, 30, and 100 µg/mL for 24 h. Next day, cells were co-treated with TNF-α (10 ng/mL) and varying concentrations of Cologrit for 24 h. U937 cells were pre-labelled with Calcein AM dye (0.5 µM) for 30 min at 37 °C. Subsequently, HT-29 cells were co-incubated with the pre-labelled U937 cells (1 × 105 cells/well) for 2 h. Non-adherent cells were washed with 1 × PBS, and fluorescence was measured with using Infinite 200Pro multimode plate reader  at Ex. 485 nm and Em. 530 nm.HT-29 cells were plated at a density of 5 × 10TM for 3 h. Fluorescence was measured at Ex. 560 nm and Em. 580 nm using Envision multimode plate reader .Caco-2 cells were pre-incubated with Cologrit digestae doses of 1, 3, 10, 30, and 100 µg/mL prepared in incomplete DMEM medium for 24 h. The cells were subsequently co-treated with 2.5% percent DSS and varying concentrations Cologrit digestae prepared in serum-free cell culture media. As negative and positive controls, water-based digestae w/wo 2.5 percent DSS were used. The treated cells were incubated for 24 h and then washed with PBS. The cells were treated with a 10 µg/mL of Alamar blueCaco-2 cells were pre-treated with the Cologrit digestae concentrations of 1, 3, 10, 30, and 100 μg/mL prepared in serum-free DMEM media for 24 h. Next day, cells were co-treated with 2.5% DSS and varying concentrations of Cologrit digestae prepared in serum-free media for 24 h. Water based digestae w/wo 2.5% DSS were treated as negative and positive controls, respectively. At the end of incubation time, cells were washed with 10 µg/mL (100 μL) of 2′, 7′-dichlorofluorescein diacetate dye and incubated in dark for 45 min at 37 °C. Fluorescence was measured at Ex. 490 nm and Em. 520 nm using the Envision multimode plate reader .p-value of <0.05, the results were deemed statistically significant.GraphPad Prism 7 was used for statistical analysis . The data were presented as mean ± standard deviation (SD). Significance between distinct treatment groups was determined using one-way ANOVA, followed by Dunnett’s posthoc analysis. At a


Aims: Carbapenem-resistant K. pneumoniae (CRKP) is the most common carbapenem-resistant Enterobacteriaceae with high mortality. Ceftazidime-avibactam (CAZ-AVI) has exhibited excellent in vitro activity in vivo against CRKP. However, the efficacy of CAZ-AVI in KPC-producing CRKP-infected patients with different kidney statuses varies, such as renal insufficiency, normal renal function, and augmented renal clearance (ARC). We explored the use of therapeutic drug monitoring (TDM) to evaluate the concentration and efficacy of CAZ-AVI in CRKP-infected patients with different kidney statuses.Methods: Serum concentrations for CAZ and AVI were determined by the high-performance liquid chromatography method. Bacterial identification, routine susceptibility testing, renal function index, and others were performed in standard protocols in the hospital’s clinical laboratories.Results: In the two patients with ARC, in case 1, CAZ-AVI 2.5g q6h was used with good efficacy, and the concentrations were up to the pharmacokinetics/pharmacodynamics targets. In Case 2, 2.5 g q8h was used with invalid effectiveness, and AVI Cmin was only 0.797 mg/l, which is lower than the PK/PD target. Case 3 was renal insufficiency using CAZ-AVI 1.25 q8h, and case 4 was normal renal function using 2.5 g q8h. Their concentrations were both up to the PK/PD targets.Conclusion: TDM results demonstrated that CAZ-AVI steady-state plasma concentration varies among patients with different kidney statuses, providing evidence for the utility of TDM of CAZ-AVI in individualized drug dose adjustment. ARC patients may need more CAZ-AVI daily doses than the standard dose. K. pneumoniae (CRKP) is also the most common CRE with high mortality  as the “critical” group of bacteria that pose the greatest threat to human health . Especiaortality .Enterobacteriales derives from two main mechanisms, including the acquisition of carbapenemase genes that encode enzymes capable of hydrolyzing carbapenems and a decrease in the uptake of antibiotics by a qualitative or/and quantitative deficiency of porin expression in association with overexpression of β-lactamases that possess a very weak affinity for carbapenems  profiles and protein binding . TherefoWe report a case series about KPC-producing CRKP-infected patients with different kidney statuses treated with CAZ-AVI. Therapeutic drug monitoring (TDM) evaluated the adequacy of therapy and guided the dose selection and dose optimization for CAZ-AVI.−1 potassium dihydrogen phosphate solution, mobile phase B of the mixed organic phase (acetonitrile/methanol/water = 7∶2∶1), A/B , a flow rate of 1.0 ML min−1, and a wavelength is 254 nm.Serum samples were planned at the following intervals 3 days after CAZ-AVI treatment: 0.5 h immediately before the next dose and 5 min after intravenous drip. Blood samples were collected and were then immediately sent to the central laboratory for centrifuging and serum separation. Serum samples were stored in transport tubes at −80°C until they were assayed. The high-performance liquid chromatography (HPLC) method determined serum concentrations for CAZ and AVI referring to relevant literature studies . Plasma Enterobacteriaceae phenotypically . The MICs of CZA for each strain were evaluated using the trace broth double dilution method. A fixed avibactam concentration of 4 μg/ml in conjunction with two-fold dilutions of ceftazidime was used in these tests according to Clinical and Laboratory Standards Institute (CLSI) guidelines . Resultsypically .2. On the third day in the hospital, bilateral blood cultures showed ESBL (-), carbapenem-sensitive pneumonia. The sputum culture showed stenotrophomonas maltophilia and hence SMZco was added. On day 11, bilateral blood cultures turn negative. While on day 15, the urine culture revealed the CRKP (800 cfu/ml) urinary tract infection, only sensitive to polymyxin B (PMB) (MIC ≤ 1) and CAZ-AVI(MIC = 2/4). On day 21, CRKP strains were isolated from sputum and bronchoalveolar fluid (BALF) cultures, only sensitive to PMB (MIC ≤ 1) and CAZ-AVI (MIC = 2/4). Then, the antibiotic regimens were changed to meropenem 2g q8h plus fosfomycin 4g q8h. Subsequently, CRKP was repeatedly detected in sputum, alveolar lavage fluid, and urine. While the clinical efficacy was not satisfactory, considering that the CrCl was 194.98 ml/min/1.73 m2, CAZ-AVI 2.5g q6h was substituted for fosfomycin on day 25. Three days later, TDM of the concentration of CAZ-AVI showed the following: CAZ Cmax 149.21 mg/L and Cmin 14.33 mg/L; AVI Cmax 37.76 mg/L, and Cmin 3.16 mg/L. After 10 days of CAZ-AVI treatment, the sputum and urine cultures turned negative. Five more days later, the patient’s bacterial infection condition improved, and the antibiotic regimens were discontinued. On day 58, the patient’s intracranial condition was stable and he was transferred to a local hospital for rehabilitation.A 56-year-old man was admitted to the Neurological Intensive Care Unit (NICU) due to fever for 3 weeks, and abnormal mental behavior for 2 weeks. The patient was admitted with a temperature of 39.3°, accompanied by chills, fatigue, and head pain. Head MRI showed abnormal signals in the left hippocampus, and the CT scan of the lungs showed lung infection. Considering the possibility of viral meningoencephalitis and pneumonia, meropenem 1g q8h, acyclovir, and foscarnet sodium were used for antiviral and antibacterial purposes. The patient was lethargic. Peripherally inserted central catheter and ventilator were used in the patient. The creatinine clearance rate (CrCl) was 145.32 ml/min/1.73 m9/L, a neutrophil ratio of 91.60%, the C-reactive protein of 278.00 mg/L, the procalcitonin of 0.321 ng/ml, and t interleukin-6 of 6137.00 pg/ml. The initial anti-infection regimen was meropenem (2g q8h) combined with linezolid (600mg q12h). On day 16, CRKP was isolated from the cerebrospinal fluid and sputum, both sensitive to PMB (MIC ≤ 1) and CAZ-AVI (MIC = 2/4). PMB was administered intravenously (75mg q12h) with intrathecal administration (3.3mg qd). Twelve days later, CRKP strains were still found positive in sputum and BALF culture but clean in cerebrospinal fluid. On day 35, the regiments for CRKP were changed to CAZ-AVI  and meropenem (1g q6h). The patient’s CrCl was 295.49 ml/min/1.73 m2 on day 35. Three days later, the TDM of CAZ-AVI results showed the following: CAZ Cmax 57.13 mg/L and Cmin 4.96 mg/L, and AVI Cmax 21.267 mg/L and Cmin 0.797 mg/L. On day 42, the BALF culture results showed that CRAB strains instead of CRKP strains were found after 7 days of CAZ-AVI treatment. So, tigecycline (TGC) (100mg q12h) combined with meropenem was used. However, CRKP was detected in BALF several times later. The patient’s infection failed to improve and he was transferred to a rehabilitation hospital for further treatment.A 32-year-old man with cerebellar infarction and bodyweight of 80 kg was transferred to NICU after the venous embolus, posterior fossa decompression, dilated dural repair, and pulmonary infection on May 18, 2021. Upon admission, the patient had a temperature of 39°C, a pulse of 140 beats/min, and was delirious, with a white blood cell count of 16.25 × 102. CAZ-AVI  combined with meropenem was used to anti-CRKP. TDM showed that CAZ Cmax 77.22 mg/L, Cmin 37.83 mg/L; AVI Cmax 20.24 mg/L, Cmin 8.67 mg/L. After 12 days of treatment with CAZ-AVI, the CRKP strains were clean in sputum and BALF culture, while the CRAB strains were isolated. The CAZ-AVI was replaced by PMB (75 mg loading dose with 50mg q12h maintenance dose) on day 40. On day 44, the patient died of gastrointestinal bleeding and lowered blood pressure.A 77-year-old man was hospitalized in NICU with progressive limb weakness and a temperature of 38.6°C on day 1. After admission, systemic examinations revealed pulmonary infection, hypertension, and chronic renal insufficiency. The initial anti-infective therapy was piperacillin/tazobactam. After 2 days, the patient was delirious, unresponsive, had low oxygen saturation, and was intubated. The inflammatory markers were high , and the antibacterial regiment was switched to meropenem 1g q8h. On day 23, the sputum and BALF culture results showed CRKP infection, which was just sensitive to CAZ-AVI (MIC = 4/4) and PMB (MIC≤1). The patient had renal insufficiency, and the CrCl was 39.78 ml/min/1.73 m2 and so TGC was replaced with CAZ-AVI 2.5g q8h. Three days later, the patient’s clinical infection symptoms and inflammatory indicators did not improve; considering ARC, CAZ-AVI was changed to q6h. Two days later, the TDM of CAZ-AVI results showed the following: CAZ Cmax 111.18 mg/L and Cmin 28.81 mg/L; and AVI Cmax 32.95 mg/L and Cmin 7.73 mg/L, while the CrCl decreased to 103.06 ml/min/1.73 m2. PMB was discontinued on day 17. On day 29, the patient’s PCT value decreased to 0.415 ng/ml and the CrCl was 100.29 ml/min/1.73 m2, while CRKP was still isolated from sputum. CAZ-AVI was given with 2.5g q8h to continue until day 39. Although the CRKP strains were still isolated from sputum on days 45 and 47, the patient had no fever with a slight cough and no sputum, and inflammatory markers tended to be normal. After 50 days of treatment, the patient was discharged.A 35-year-old man had a poor appetite, fatigued for more than a month, and in coma for 20 days. On April 15, 2021, this patient was diagnosed with chronic and acute liver failure and hepatic encephalopathy. The patient was admitted to the department of liver transplantation on May 10, 2021. Under general anesthesia, the patient underwent orthotopic classical liver transplantation plus intestinal adhesiolysis. After surgery, the inflammatory markers were high , and the patient was given the anti-infection treatment of meropenem (1g q8h), PMB (75mg q12h), teicoplanin (400mg qd), and caspofungin (50mg qd). On the 4th day of admission, CRKP  and CRAB were isolated from the sputum culture. Meropenem and teicoplanin were discontinued, and TGC (100mg q12h) combined with PMB was replaced to treat negative bacterial infection. Later, CRKP  was isolated from wound secretion, blood, and stool cultures. However, multiple sputum cultures, blood, and abdominal drainage fluid cultures indicated CRKP. On day 11 (2021.05.21), the CrCl of the patient was 170.44 ml/min/1.73 mThe basic information of the four cases is shown in All the CRKP strains produced KPC enzymes and were isolated from urine, sputum, BALF, cerebrospinal fluid, wound secretion, blood, and stool . All str2. The AVI Cmin was 3.16 mg/L when Case 1 was given CAZ-AVI 2.5g q6h. Moreover, in Case 2, after CAZ-AVI 2.5g q6h was given on the first day and 2.5q8h was given subsequently, the AVI Cmin was only 0.797 mg/L, and the CAZ Cmin was only 4.96 mg/L. Case 3 had renal insufficiency with CRCL 39.78 ml/min/1.73 m2, and TDM showed that the Cmin of CAZ and AVI were higher than the MIC value after CAZ-AVI 2.5g q8h was given on the first day and 1.25 q8h was given subsequently. In case 4, the renal function was normal, and the plasma concentration of CAZ-AVI was higher than the MIC value after CAZ-AVI 2.5g q8h was given for 3 days and then 2.5g q6h for 2 days (Cases 1 and 2 were ARC patients with CRCL 194.98 and 295.49 ml/min/1.73 mr 2 days .K. pneumoniae carbapenemase (KPC)-producing CRKP-infected patients with different kidney statuses, such as augmented renal clearance (ARC), normal renal function, and renal insufficiency. TDM results demonstrated that CAZ-AVI steady-state plasma concentration varied among patients with different kidney statuses, providing evidence for the utility of TDM of CAZ-AVI in individualized drug dose adjustment.This report described the therapeutic drug monitoring of CAZ-AVI concentrations in CAZ and AVI have similar pharmacokinetics and both are eliminated mainly through kidneys . Therefo2 in males and ≥120 in females coupled with a normal serum creatinine value (0.6–1.4 mg/dl) (T of 1 mg/L) were therefore deemed appropriate for the probability of target attainment (PTA) analyses to select ceftazidime-avibactam dosage (Enterobacteriaceae members and P. aeruginosa (min was 3.16 mg/L. Case 2 used 2.5q8h (first day 2.5g q6h), and AVI Cmin was only 0.797 mg/L. It implies that ARC patients may need more CAZ-AVI daily doses than the standard dose.Augmented renal clearance (ARC) is a pathophysiological condition defined as the occurrence of a measured CrCl ≥130 ml/min/1.73 m4 mg/dl) . ARC occ4 mg/dl) . Real-wo4 mg/dl) . For mul4 mg/dl) . Further4 mg/dl) . Previoum dosage . A studyruginosa . In the 2. The dose of CAZ-AVI given was standard on the first day and was adjusted to 1.25g q8h later. TDM results showed that the Cmin values of CAZ and AVI were higher than the target concentration, and the PK/PD target rate was 100%.Due to the predominantly renal excretion of both CAZ and AVI, the concentrations of both drugs increase with increasing severity of renal impairment . CAZ-AVIK. pneumoniae. The primary mechanism is the production of a carbapenemase classified into the Ambler class A , B , and D   . A previrn China . The codn Tn4401 . NDM-1 Kvibactam . To datein China .We explored the use of TDM to evaluate the concentration and efficacy of CAZ-AVI in CRKP-infected patients with different kidney statuses. The limited clinical samples limit the results of this study. Large-scale clinical sample experiments are needed to confirm the formulation of an individualized administration scheme of CAZ-AVI based on different renal functions.In conclusion, our study demonstrated that CAZ-AVI steady-state plasma concentration varies among patients with different kidney statuses, including ARC, normal renal function, and renal insufficiency, providing evidence for the utility of TDM of CAZ-AVI in individualized drug dose adjustment. ARC patients may need more CAZ-AVI daily doses than the standard dose.


Moreover, we observed a significant improvement in arterial stiffness parameters and in endothelial function indices. Our study demonstrated that 6 months treatment with Sacubitril/Valsartan, in patients with HFrEF, improves endothelial dysfunction and arterial stiffness, by reducing oxidative stress, platelet activation and inflammation circulating biomarkers, without adverse effects.Heart failure (HF) is associated to endothelial dysfunction that promotes the increase of arterial stiffness thus augmenting myocardial damage. Sacubitril/Valsartan is used in the treatment of HF reduced ejection fraction (HFrEF) and has been proven effective in reducing cardiovascular disease (CVD) progression and all-cause mortality. The aim of this study was to evaluate the effect of Sacubitril/Valsartan on endothelial dysfunction, arterial stiffness, oxidative stress levels and platelets activation in patients with HFrEF, at baseline and after 6 months of treatment. We enrolled 100 Caucasian patients. Endothelial function was evaluated by the reactive hyperemia index (RHI) and arterial stiffness (AS) by the measurement of carotid-femoral pulse wave velocity (PWV), augmentation pressure (AP) and augmentation index (AI). At baseline, among enrolled outpatients, 43% showed a NYHA class II and 57% a NYHA class III. At 6 months, there was a significant improvement of several hemodynamic, clinical and metabolic parameters with a significant reduction in oxidative stress indices such as 8-isoprostane ( Heart failure (HF) is a widespread health problem and represents a major cause of cardiac morbidity and mortality representing the second cause of hospitalization in subjects >65 years old, despite optimal treatment . Among aNotably, endothelial dysfunction is a frequent condition in patients affected by HF and in this setting it predicts major cardiovascular (CV) events and mortality. Endothelial dysfunction is characterized by reduced bioavailability of nitric oxide (NO), in response to increased reactive oxygen species (ROS) production, with an imbalance between vasoconstrictor and vasodilator factors, predisposing to a pro-atherogenic and pro-trombotic phenotype, characterized by leukocyte adhesion, mitogenesis, platelet activation and vascular inflammation . EndotheIt is known that inflammation and oxidative stress, characterized by increased 8,12-isoprostane F (2α) concentration, low endothelium-bound extracellular superoxide dismutase activity and increased endothelium-bound xanthine oxidase (XO) activity and cytokines production, play a central role in pathogenesis of both endothelial dysfunction and HF . MoreoveArterial stiffness, resulting from reduced elasticity of the arterial wall, increases with age and different cardiometabolic abnormalities promoting endothelial dysfunction and representing an independent predictor of CV events and HF . AccordiIt is known that, arterial stiffness and endothelial dysfunction are strongly interconnected recognizing similar pathophysiological mechanisms. In particular, arterial stiff-ness is correlated with increased activity of angiotensin II, which activates matrix metalloproteinases (MMP) and pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Inflammatory cytokines promote the production of C reactive protein (CRP) by vascular smooth muscle cells, which increases vascular inflammation and reduces endothelium-dependent vasodilation . The incSacubitril/Valsartan is the first angiotensin receptor-neprilysin inhibitor (ARNI) acting by reducing the degradation of natriuretic peptides and blocking selective AT1-receptor with positive effects on CV system . In HFrEMoreover, a post hoc analysis from PARADIGM-HF demonstrated that subjects with type 2 diabetes mellitus (T2DM) in treatment with Sacubitril/Valsartan presented an improved glycaemic profile, compared to patients in treatment with enalapril, after a follow-up of 12 months . In addiTaken together, all these findings could suggest a possible direct protective effect of Sacubitril/Valsartan on CV system by modulating inflammation and oxidative stress. According with this, a recent study conducted by Qing By the way, in literature no study investigated the possible effect of treatment with Sacubitril/Valsartan on endothelial dysfunction and arterial stiffness in patients with HF.With the aim to better understand pathophysiological mechanisms underlying cardiovascular protective effects of Sacubitril/Valsartan, and in agreement with evidence al-ready in the literature, the aim of the present study was to evaluate the effect of Sacubitril/Valsartan on endothelial dysfunction and arterial stiffness in patients with HFrEF evaluated at baseline and after 6 months of treatment. Moreover, we evaluated the effects of Sacubitril/Valsartan on oxidative stress levels and platelets activation and the correlation with arterial stiffness and endothelial dysfunction.2], angioedema or side effects due to ACE-I or ARB. Moreover none had systolic blood pressure (SBP) < 100 mmHg or potassium levels >5.4 mmol/L. Women were exclude if pregnant or breastfeeding. In addition to previous treatments, eligible patients for Sacubitril/Valsartan discontinued ACE-I (at least 36 h before) or ARB, and received initial dosages of 24/26 mg or 49/51 mg bid according to clinical conditions. Moreover, Sacubitril/Valsartan dosage was increased every 2–4 weeks up to the maximum tolerated dose, as recommended.We performed a prospective, one-center study, analysing all clinical, laboratory, and vascular parameters of 100 consecutive HFrEF Caucasian subjects  followed at the Catanzaro University Hospital, Geriatric Department. Patients’ inclusion criteria were age >18 years, left ventricular ejection fraction (LVEF) < 35%, NYHA class II or III, symptomatic patients despite optimized treatment with stable doses of angiotensin- converting enzyme inhibitor (ACE-I) or angiotensin receptor blocker (ARB) for at least four weeks. All enrolled patients underwent to Sacubitril/Valsartan treatment according to International Guidelines recommendations . No patiIn all patients a complete medical history and careful physical examination were performed.Ethics Committee Regione Calabria “Area Centro” approved the protocol (code protocol number 2012.63). All participants gave their informed consent for personal data treatment and all investigations were made according to Helsinki Declaration.All clinical evaluations and laboratory tests were performed at baseline (time T0) and after 6all patients a complete medicalmonths of treatment (time T6).After 5 min of rest, clinical blood pressure (BP) measurement were obtained with an aneroid sphygmomanometer in the left arm of supine patients. The diagnosis of arterial hypertension was carried out in all subjects with SBP >140 mmHg and/or diastolic BP (DBP) > 90 mmHg according to current guidelines . The difAfter at least 12 h fasting, the laboratory measurements were performed. The glucose oxidation method  was utilized to measure plasma glucose, and a chemiluminescence-based assay (Roche Diagnostics) for plasma insulin determination. An enzymatic method  was used to detect total, low and high-density lipoprotein  cholesterol and triglyceride concentrations. Serum creatinine was determined using a Roche Creatinine Plus assay  on a clinical chemistry analyser . Renal function was evaluated by calculating the estimate glomerular filtration rate (e-GFR) using the CDK-EPI equation . An enzyBlood samples were taken in tubes with separator gel and centrifuged at 4,000 rpm for 15 min to obtain serum samples. In order to prevent oxidation samples were stored at −80°C in the presence of 0.005% butylated-hydroxy-toluene (BHT) (10 µL of 5 mg/ml solution in ethanol per 1 ml sample). Specific ELISA kits were employed to detected serum levels of 8-isoprostane ; NAPDH Oxidase 2 (Nox-2), human glycoprotein VI (GPVI) and Sp-selectin . Values of 8-isoprostane were expressed as pg/mL; the lower detection limit of the assay was 0.8 pg/ml and interassay coefficient of variation (CV) was <9.6%. Values of Nox-2 were expressed as nmol/L, the lower detection limit of the assay was 0.25 nmol/L, the intra-assay CV was <9%, the interassay CV was <11%.Values of GPVI were expressed in pg/ml, the lower detection limit of the assay was 46.88 pg/ml, the intra-assay CV was <8% and the interassay CV was <10%. Finally, Sp-selectin concentrations were expressed in ng/ml, the kit had a lower detection limit of 15 ng/ml, the intra-assay was CV <10% and the interassay CV was <15%.The Biochip Array (Randox Labs) was used for the assay of serum cytokines  , throughWe used high-fidelity applanation tonometry (Millar) and pressure wave analysis computer software (Sphygmocor™), which is a validated system, to measure arterial stiffness. Automatic, non-invasive recording of supine brachial blood pressure of the dominant arm after a 30-min rest was used for pressure calibration . Within 10 min, 5 BP measurements were taken and the average of the last 3 was used for calibration. The radial artery of the dominant arm with the wrist slightly hyperextended was used to record pres-sure waves representing the average of individual pressure waves recorded consecutively for 8 s. Only pressure waves with peak and background pressure variation of individual waves <5% were used. Using an integrated generalised transfer function, the central pressure wave was automatically derived from the radial pressures. Pressure wave measurements were also obtained at the level of the right carotid artery, resulting in a more precise central augmentation index (AI) . In addiA semi-plethysmographic method with digital pulse volume amplitude (PVA) measurement was used to assess endothelial function with patients in a supine position in a quiet, temperature-controlled environment set at 22°C. PVA was measured using the peripheral arterial tonometer in the fingertip of the index finger  both at rest and during reactive hyperemia (RH). The peripheral arterial tonometer consists of a finger-mounted probe that encloses the fingertip with an inflatable, pressurised air cushion that is electronically controlled and confined in a rigid outer shell. Pressure changes within the probe accompanying PVA changes in the fingertip were trans-mitted to a personal computer where the signal was band-filtered (0.3–30 Hz), displayed, amplified and stored. From the release of an inflated upper arm BP cuff above the SBP for 5 min, the RH was obtained. The ratio of the mean PVA over a 1-min time interval from 1 min after cuff deflation (RH) to the mean PVA assessed for 1 min before cuff inflation (baseline) defines the digital PVA-RH. Any deviations in signal magnitude due to systemic factors were assessed by constant evaluation of the PVA of the index finger of the other non-ischemic hand (which was not subjected to RH) throughout the study .p < 0.05. All comparisons were performed using SPSS 20.0 statistical software for Windows .Continuous variables were expressed as mean ± standard deviation (SD)  or as median and interquartile range (IQR) . Categorical data were expressed ass percentage. For all continuous variables, comparisons between baseline (T0) and post-treatment values (T6) were performed using paired Student’s t test for normally distributed data or Wilcoxon’s test for non-normally distributed data. For categorical data comparison was conducted by chi-square test. All variables which deviate from the normal distribution were log-transformed (ln) before correlational analysis. A linear regression analysis was performed to assess the relation-ship between variation in arterial stiffness (PWV) and endothelial function (RHI) indices, expressed as Δ of variation between baseline and follow-up (ΔT0–6) and the variation of metabolic, inflammatory, oxidative stress and platelets activation covariates that significantly changed after the treatment (expressed as ΔT0–6). Variables reaching statistical significance were inserted in a stepwise multivariate linear regression model to assess the magnitude of their individual effect on ΔPWV and ΔRHI. Moreover, a linear regression analysis was performed to assess the relationship between variation in oxidative stress indices (expressed as ΔT0–6) and different covariates (expressed as ΔT0–6) and to evaluate the relationship between variation in platelets activation and oxidative stress bi omarkers (expressed as ΔT0–6). Differences were assumed to be significant at Of the 100 outpatients evaluated, 82% were males, 13% active smokers, 43% had NYHA class II and 57% had NYHA class III. p = 0.032). At baseline, all patients with T2DM were in treatment with oral antidiabetic drugs (OADs), at 6 months follow-up 87% were still taking OADs. In addition, at baseline 38% of diabetic patients were treated with insulin whereas at 6 months of follow-up 23% continued insulin treatment (p = 0.038). At baseline 35% diabetic patients were treated with OADs and insulin concomitantly, while at 6 months only 23%. Moreover, among diabetic patients, only 20% were in treatment with glucagon-like peptide 1 receptor agonist (GLP-1 RA) at baseline. On the other hand, regarding the intake of beta-blockers, there was no change in their use. All 100 enrolled patients completed the follow-up.The main aetiologies for HF were ischemic heart disease in 76% of the study group, valvulopathies in 30% and arterial hypertension in 84%. Considering the associated comorbidities, atrial fibrillation was present in 32% of the study population, 66% of the patients presented type 2 diabetes mellitus (T2DM), 35% showed chronic obstructive pulmonary disease (COPD) and 87% had dyslipidaemia. The different drug classes taken by the study patients are assumed in At baseline, 71% of the population started the lowest dose of Sacubitril/Valsartan (24/26) and 29% of the patients the intermediate dose (49/51). After 6 months of follow-up, there was a significant improvement of the functional status, thus 41% of patients shifted to NYHA class I, 51% of patients showed a NYHA class II and 8% remained in NYHA class III. After 6 months of follow-up, 16% of patients were taking the lowest dose of Sacubitril/Valsartan (24/26), 61% of the patients the intermediate dose (49/51) and 23% the highest dose (97/103).p < 0.0001), respiratory rate (RR) , SBP , DBP , NT-ProBNP  to 2,845  pg/ml, p < 0.0001), sodium , creatinine ; on the contrary, e-GFR , potassium , haemoglobin (Hb)  and insulin-like growth factor-1 (IGF-1)  significantly increased. Moreover there was a statistically significant improvement in the glycometabolic profile as shown in p < 0.0001), IL-6 (7.5 (7.0–9.5) vs. 5.6 (5.0–7.5); p < 0.0001), TNF-α (5.0 (4.1–5.7) vs. 4.0 (3.2–4.8); p < 0.0001), indicating an improvement in the inflammatory state, after 6 months treatment with Sacubitril/Valsartan.The main anthropometric and biochemical parameters of the study population at baseline and after the 6-month treatment period are reported in p < 0.0001) (p < 0.0001) (p < 0.0001) (p < 0.0001)   and 8-is 0.0001)  and plat 0.0001)  and sP-s 0.0001)  significp < 0.0001) and central DBP (c-DBP) . Similarly, we observed a significant reduction in arterial stiff-ness parameters such as PWV , AI (%) , AP  and a significant improvement in endothelial function as demonstrated by RHI increase . In addition, we performed a sub-analysis in the three groups of patients taking the different dosages of Sacubitril/Valsartan to assess changes in PWV and RHI (p < 0.0001) as shown in p = 0.001 and RHI from 1.8 ± 0.6 to 3.1 ± 0.9, p < 0.0001. Finally, in the 23 patients taking the highest dosage (97/103 mg bid), PVW decreased from 7.9 ± 3.7 to 6.6 ± 4.5 m/s, p = 0.024; while RHI increased from 1.8 ± 0.7 to 3.2 ± 0.9, p < 0.0001. Finally, we performed a sub-analysis according to the different comorbidities to assess changes in PWV and RHI. In particular, in patients with T2DM (66 pts) we observed changes in PWV from 8.1 ± 3.9 to 6.5 ± 4.5 m/s, p = 0.018 and in RHI from 1.8 ± 0.6 to 3.1 ± 0.9, p < 0.0001; in patients without T2DM (34 pts): PWV from 8.7 ± 4.6 to 6.6 ± 4.8 m/s, p = 0.005 and of the RHI from 1.7 ± 0.7 to 3.1 ± 0.9, p < 0.0001; in AF pa-tients (32 pts) we observed changes in PWV from 7.6 ± 3.4 to 6.3 ± 4.7 m/s, p = 0.030; and of the RHI from 1.8 ± 0.6 to 3.0 ± 0.8, p < 0.0001; in patients without AF (68 pts): PWV from 8.8 ± 4.5 to 6.7 ± 4.6 m/s, p = 0.007; and of RHI from 1.8 ± 0.6 to 3.1 ± 1.0, p < 0.0001. In dyslipidaemic patients (92 pts), PWV varied from 8.4 ± 4.1 to 6.3 ± 4.2 m/s, p < 0.0001; and RHI from 1.8 ± 0.6 to 3.1 ± 0.9, p < 0.0001; in non-dyslipidaemic (8 pts) PWV from 9.5 ± 7.7 to 6.7 ± 4.2 m/s, p = 0.02; and RHI from 1.6 ± 0.7 to 2.6 ± 1.0, p < 0.033. In patients with CKD (27 pts), PWV varied from 8.9 ± 5.1 to 7.6 ± 5.5 m/s, p < 0.0001; and RHI from 1.6 ± 0.6 to 3.0 ± 1.0, p < 0.0001; in non-CKD patients (73 pts) PWV from 8.0 ± 3.7 to 6.2 ± 4.2 m/s, p < 0.0001; and RHI from 1.8 ± 0.6 to 3.1 ± 0.9, p < 0.0001.Peripheral and central hemodynamic parameters at baseline and 6 months follow-up are reported in  and RHI . In partp = 0.013), ΔlnIL-6 , and Δhs-CRP ; furthermore ΔNox-2 resulted directly correlated with ΔHOMA  and Δhs-CRP  (p = 0.002), ΔNox-2 , ΔSp-selectin , ΔGPVI , Δhs-CRP , ΔLogIL-6 , ΔLogTNF-α , ΔHOMA . ΔRHI was inversely correlated with Δ8-isoprostane , ΔNox-2 , ΔSp-selectin , ΔGP6 , Δhs-CRP , ΔLogTNF-α , ΔLogIL-6 , ΔHOMA .A linear correlation analysis was performed to test the correlation between Δ values of oxidative stress markers  and different covariates express as Δ variation between baseline and follow-up. Δ8-isoprostane was directly correlated with ΔHOMA  . Moreove= 0.011) . ΔPWV wap < 0.0001), Δhs-CRP and ΔHOMA added respectively another 6.9% (p < 0.0001) and 5.6% (p < 0.0001) of its variation. Furthermore, Δ8-isoprostane was the major predictor of ΔRHI explaining 20.4% of its variation, Δhs-CRP added another 8.4% (p < 0.0001) . ΔLogIl-6 was the stronger predictor of ΔPWV, justifying 13.6% ( 0.0001) .To the best of our knowledge, this is the first study to demonstrate the possible effect of Sacubitril/Valsartan, a combination of Ang II receptor blocker and neprilysin inhibitor, on endothelial function and arterial stiffness in HF patients.The endothelium is a monolayer of cells covering the inner surface of blood vessels and acts as structural and functional barrier between vessels wall and blood. It is known that the endothelium is involved in the control of thrombosis and thrombolysis, leukocytes adhesion and platelets aggregation, and the regulation of vascular tone by balancing vasoactive substances production  El-Batt El-Batt.In particular, endothelium-derived NO is released in response to physical stimuli, hormones, and platelet-derived substances and it induces vascular relaxation and platelet function inhibition.Endothelial dysfunction, characterized by a shift in the properties of the endothelium toward impaired vasodilation promoting a pro-inflammatory and pro-thrombic state, is associated with reduced NO bioavailability . It has Notably, endothelium-dependent vasodilation is impaired in patients with both reduced and preserved HF . In partOf interest, endothelial dysfunction can be found not only in patients with ischemic but also non-ischemic HF, affecting non-coronary vascular beds . MoreoveThe endothelial dysfunction observed in HF patients is amply justified by increased formation of superoxide radicals and other oxidant substances in the vascular beds. The increase in oxidative stress promotes a direct inactivation of NO thus favouring a reduced endothelium dependent vasodilation . Furtherp = 0.663). Therefore, the entire population of our study can be considered older.Present data obtained from this longitudinal, one-center study, demonstrated that six months treatment with Sacubitril/Valsartan had positive effects on the improvement of endothelial dysfunction and arterial stiffness in a consistent cohort HFrEF symptomatic patients, as well as a significant improvement of clinical, biochemical and haemodynamic parameters, confirming previous evidence . In accoThe novelty of our study is the significant betterment of inflammatory profile, as evidenced by the decreased values of IL-6, TNF-α, hs-CRP, and a reduction of oxidative stress and platelets activation biomarkers, after 6 months of treatment with Sacubitril/Valsartan; all these mechanisms may account for the improvement in vascular function.via excessive production of ROS . The decrease in oxidative stress levels could be due to the ability of Sacubitril/Valsartan to block angiotensin II receptor, as well as the reduction of inflammatory indices. In fact, it is known that, in patients suffering from HF, Angiotensin II, pro-inflammatory cytokines and other pathological stimuli such as hypercholesterolemia and hyperglycaemia, significantly stimulate the expression and activity of NAD(P)H oxidase . In partn of ROS .in vitro study conducted by Furthermore, previous studies demonstrated that oxidative stress might lead to platelets hyperactivity; in fact, ROS over production is relevant for increase in important markers of platelets activation including sP-selectin, glycoprotein VI (GPVI) and, thromboxane B2 (TXB2) . PlateleIn addition, our results demonstrated a significant improvement in metabolically in-dices such as FPG, FPI, HbA1c and HOMA, and these data are in agreement with our previous evidence . Many stThe increased levels of IGF-1 observed in our cohort of patients, at six months follow-up, confirm the amelioration of metabolic profile; according with this lower IGF-1 levels have previously been shown to correlate with endothelial dysfunction , so the In conclusion, the present study strengthens scientific evidence supporting the use of Sacubitril/Valsartan in HFrEF patients and confirms its beneficial effects on hemodynamic, clinical, biochemical and metabolic profile. Moreover, the novelty of our study is that six months treatment with Sacubitril/Valsartan is able to improve endothelial dysfunction and arterial stiffness in HFrEF patients, likely through the reduction of oxidative stress, platelet activation and inflammation biomarkers circulating levels with the improvement of cardio-metabolic burden.