Streptococcus agalactiae (n = 51), Streptococcus dysgalactiae (n = 54), Streptococcus uberis (n = 50), Staphylococcus aureus (n = 85), non-aureus staphylococci (n = 88), Escherichia coli (n = 54) and Klebsiella species (n = 52). Streptococci and staphylococci were tested against cefquinome, cefoperazone, cephapirin, penicillin, oxacillin, cloxacillin, amoxicillin/clavulanic acid and cefalexin/kanamycin. Besides cefquinome and amoxicillin/clavulanic acid, Gram-negative pathogens were examined for their susceptibility to marbofloxacin and sulfamethoxazole/trimethoprim. The examined S. dysgalactiae isolates exhibited the comparatively lowest MICs. S. uberis and S. agalactiae were inhibited at higher amoxicillin/clavulanic acid and cephapirin concentration levels, whereas S. uberis isolates additionally exhibited elevated cefquinome MICs. Most Gram-positive mastitis pathogens were inhibited at higher cloxacillin than oxacillin concentrations. The MICs of Gram-negative pathogens were higher than previously reported, whereby 7.4%, 5.6% and 11.1% of E. coli isolates had MICs above the highest concentrations tested for cefquinome, marbofloxacin and sulfamethoxazole/trimethoprim, respectively. Individual isolates showed MICs at comparatively higher concentrations, leading to the hypothesis that a certain amount of mastitis pathogens on German dairy farms might be resistant to frequently used antimicrobials.The present research study investigated the susceptibility of common mastitis pathogens—obtained from clinical mastitis cases on 58 Northern German dairy farms—to routinely used antimicrobials. The broth microdilution method was used for detecting the Minimal Inhibitory Concentration (MIC) of  Streptococcus agalactiae [The pathogens predominantly associated with bovine mastitis in Germany are substantially staphylococci, streptococci and coliforms . In genealactiae . ResultsStaphylococcus (S.) aureus (12 farms), 88 non-aureus staphylococci (NAS) (17 farms), 51 Streptococcus (S.) agalactiae (4 farms), 54 Streptococcus (S.) dysgalactiae (30 farms), 50 Streptococcus (S.) uberis (35 farms), 54 Escherichia (E.) coli (15 farms) and 52 Klebsiella species (15 farms). The number of at least 50 isolates, as well as the long period of time, enables a comprehensive statement about the susceptibilities of mastitis pathogens to commonly used antimicrobials in Northern Germany. The herd size of participating farms ranged from 55 to 2500 cows with an annual average milk yield between 7600 and 13,000 kg. At the time of milk sampling, the bulk milk somatic cell count (SCC) ranged from 88,000 to 334,000 cells/mL. According to the German Veterinary Association (DVG), an SCC of 100,000 cells/mL being present in the bulk tank milk is aimed for. An increase in SCC is due to an increased occurrence of inflammatory cells in the milk and thus reflects an important parameter for assessing the udder health status of a dairy herd. If the arithmetic mean value of the bulk tank milk sample is >300,000 cells/mL in two consecutive samples or a single sample exhibits >400,000 cells/mL, further measures are necessary for rapidly detecting animals affected by mastitis, including clinical investigations of individual animals as well as assessing secretions and cyto-microbiological analysis [The isolates included in the current study were randomly selected from the strain collection of the University of Applied Sciences and Arts . Examined mastitis pathogens were isolated from quarter foremilk samples of cows suffering from clinical mastitis. Milk sampling was performed between April 2013 and January 2018 on a total of 58 different Northern German dairy farms. Altogether, 434 isolates were included in the study, subdivided as follows: 85 2O2; Merck, Germany) as well as the performance of a clumping factor test —for the differentiation of S. aureus and NAS. S. aureus isolates were confirmed by the detection of the specific nuc gene according to Saiful et al. (2006) [S. dysgalactiae or S. agalactiae according to Lancefield group C and group B, respectively. A Rambach agar, modified as previously described by Watts et al. (1993) [S. uberis from Enterococcus species. Gram-negative rods were verified by testing the activity of cytochrome oxidase C  and the performance in an oxidative fermentative test -glucose-monohydrate, Merck, Germany). Coliform bacteria exhibiting glucose fermentation were further differentiated by Chromocult coliform agar , since E. coli appeared as blue colonies after incubation for 24 h at 37 °C. Concerning pink colonies growing on the Chromocult coliform agar, Klebsiella species could be distinguished from other coliforms by their lack of mobility during the oxidative fermentative test.The collection of milk samples as well as the microbiological identification of pathogens were performed in accordance with the guidelines of the German Veterinary Medical Association . For ide. (2006) . Serolog. (1993) , served Streptococcus species, 5% of defibrinated horse blood  was added. Gram-positive pathogens were tested against cefquinome, cefoperazone, cephapirin, penicillin, cloxacillin, oxacillin, amoxicillin/clavulanic acid (4:1) and cefalexin/kanamycin (2:1) in concentrations ranging from 0.06 µg/mL to 32 µg/mL. The used combinations referred to the ratio that is present in commercial preparations. Besides cefquinome and amoxicillin/clavulanic acid (4:1) with concentrations ranging from 0.06 µg/mL to 32 µg/mL, marbofloxacin and sulfamethoxazole/trimethoprim (19:1) were tested against coliforms with concentrations ranging from 0.015 µg/mL to 8 µg/mL and 0.03 µg/mL to 16 µg/mL, respectively. S. aureus ATCC 29213 and E. coli ATCC 25922  were used as reference strains. Inoculated microtiter plates were incubated at 37 °C for 16 to 20 h. Bacterial growth was determined due to the appearance of a visible turbidity, whereby the well without turbidity corresponded to the MIC of the respective pathogen.Antimicrobial susceptibility testing was performed using the broth microdilution method in accordance with the DIN EN ISO 20776-1:2006 protocol . The Min50 and MIC90 values are given as well. The MIC50 and MIC90 values define the lowest concentration at which proliferation of at least 50% and 90% of the tested bacteria is inhibited, respectively [The results of the MIC examination are presented in detail in the following Tables for Gram-positive  and Gramectively . S. agalactiae isolates was dense for all antimicrobials investigated, extending over less than three dilution levels. MIC50 and MIC90 values corresponded to the same concentration level for cefoperazone (0.25 µg/mL), oxacillin (0.5 µg/mL), amoxicillin/clavulanic acid (0.5 µg/mL) and cefalexin/kanamycin (4 µg/mL). Fifty-one S. agalactiae isolates were inhibited at cefalexin/kanamycin MIC of 4 µg/mL, representing the highest MIC50/90 values among the tested antimicrobial agents. MIC90 of cefquinome, cephapirin, penicillin and cloxacillin were one dilution higher than their MIC50 values. The lowest MIC50 as well as MIC90 were determined as ≤0.06 µg/mL and 0.125 µg/mL for cefquinome and penicillin, respectively , MIC90 values of cephapirin, cloxacillin, oxacillin, amoxicillin/clavulanic acid and cefalexin/kanamycin were at least one dilution higher than their MIC50. Highest MIC values were determined for the combination of cefalexin/kanamycin (MIC50/90: 0.5/1 µg/mL). One single isolate was inhibited at comparatively higher concentrations of all antimicrobials tested . Additionally, two individual isolates were inhibited at a cefalexin/kanamycin concentration of 4 µg/mL  and MIC90 (2 µg/mL) differed by three concentration levels, while oxacillin MIC50/90 was 2 µg/mL. With regard to the remaining antimicrobial agents, MIC50 and MIC90 values either differed by one  or two  concentration levels. The lowest MIC50 and MIC90 of S. uberis isolates were found for penicillin at concentrations of 0.125 µg/mL and 0.25 µg/mL, respectively. In contrast, the highest MIC90 values were investigated for cefoperazone as well as cloxacillin . Three individual S. uberis isolates showed cefalexin/kanamycin MIC values of ≥32 µg/mL, respectively, whereby two of these three isolates were also inhibited at a cloxacillin concentration of 32 µg/mL. Furthermore, one single isolate was not inhibited within tested cephapirin concentration ranges (MIC >32 µg/mL) (The widest distribution pattern of 50 2 µg/mL) . S. aureus was dense in the case of cefalexin/kanamycin, referring to three concentrations levels. In contrast, the widest distribution pattern of 85 S. aureus isolates was determined for penicillin, ranging from ≤0.06 µg/mL to 32 µg/mL with MIC50 and MIC90 values of ≤0.06 µg/mL and 0.5 µg/mL, respectively. Since at least 90% of S. aureus isolates were inhibited at a cephapirin concentration of 0.5 µg/mL, the lowest MIC90 values were detected for both penicillin and cephapirin. With the exception of penicillin, MIC90 values were either one  or two concentration levels  higher than their MIC50 values. Cefoperazone and the combination of cefalexin/kanamycin showed the highest MIC50 and MIC90 values at concentrations of 2 µg/mL and 4 µg/mL, respectively. Four S. aureus isolates were inhibited at cefoperazone concentrations of 16 µg/mL, whereby three bacterial isolates also had penicillin MICs of 16 µg/mL and one of them a penicillin MIC of 32 µg/mL  and penicillin (MIC50/90: 0.125/0.5 µg/mL). The highest MIC50 (4 µg/mL) and MIC90 (8 µg/mL) values were investigated for the third-generation cephalosporin cefoperazone. One single isolate exhibited MIC values of ≥16 µg/mL concerning all tested antimicrobial substances, except for the combination of cefalexin/kanamycin (0.5 µg/mL), while an additional one was inhibited at cefoperazone and penicillin concentrations of 16 µg/mL, respectively  and MIC90 (16 µg/mL) values of 52 Klebsiella species were determined for amoxicillin/clavulanic acid. Sulfamethoxazole/trimethoprim MIC90 values were three dilutions higher than MIC50 values, whereas MIC90 values of remaining antimicrobials were two dilutions higher than MIC50 values. One individual isolate was not inhibited at the highest concentrations of cefquinome (>32 µg/mL) and sulfamethoxazole/trimethoprim (>16 µg/mL), respectively  or four concentration levels higher (marbofloxacin) than their MIC50. The highest MIC90 value was determined for sulfamethoxazole/trimethoprim (>16 µg/mL), differing by at least six dilutions from MIC50. The lowest MIC90 was 0.5 µg/mL for cefquinome and marbofloxacin. Moreover, six (11.1%), three (5.6%) and four (7.4%) E. coli isolates exhibited MIC values above the highest concentration tested for sulfamethoxazole/trimethoprim (>16 µg/mL), marbofloxacin (>8 µg/mL) and cefquinome (>32 µg/mL), respectively ) ensures the high reproducibility of the test results. While the agar disk diffusion test represents a qualitative method, not only being inexpensive to implement and easy to use in practice, the broth microdilution method is a quantitative method considered as the “gold standard” for susceptibility testing due to its complexity and accuracy . Results90 of E. coli were at least two dilutions higher than those determined for Klebsiella species. More striking differences were apparent concerning cefoperazone MIC90 values of NAS (8 µg/mL) and S. agalactiae, as well as S. dysgalactiae , differing by five dilution levels. While S. dysgalactiae might be considered as susceptible to most tested antimicrobials due to the comparatively low MIC values, S. uberis frequently exhibited the highest MICs among examined Gram-positive pathogens. Even though oxacillin and cloxacillin are both semisynthetic β-lactams belonging to the group of isoxazolyl penicillins [Within the present study, investigated coliforms did not differ in terms of inhibition by cefquinome and amoxicillin/clavulanic acid, whereas marbofloxacin and sulfamethoxazole/trimethoprim MICicillins , most GrS. agalactiae at a penicillin concentration of ≤0.06 µg/mL [50/90 corresponded to the same concentration in trials of Minst et al. (2012) (≤0.03 µg/mL) [50/90 values (≤0.125 µg/mL) [90 values are consistent with the latest resistance monitoring [50/90: 0.25/0.5 µg/mL) and amoxicillin/clavulanic acid (MIC50/90: 0.5 µg/mL), whereas results of remaining antimicrobials were largely consistent. Previous German studies completely inhibited 06 µg/mL ,23 and c06 µg/mL . Amoxici3 µg/mL)  and the 5 µg/mL) , whereasS. dysgalactiae isolates are in agreement with former trials regarding cefquinome and penicillin, since at least 90% were inhibited at ≤0.015 µg/mL within various studies [90 of 0.25 µg/mL, whereas cephapirin MIC values were in line with our study [90 determined within a previous study was 0.06 µg/mL [50 and MIC90 determined by Tenhagen et al. (2006) was 0.125 µg/mL, respectively [50/90: ≤0.03 µg/mL) differed by at least two dilutions from our results [Our findings concerning  studies ,23,25. Iur study . Oxacill results ,23.S. uberis in the present study are in line with those in a former study [50 and MIC90 of S. uberis at concentrations of 0.03 µg/mL and 0.125 µg/mL, respectively [90 values of S. uberis that were determined as 0.125 µg/mL [90 values: 0.25 µg/mL [MIC values of penicillin and oxacillin determined for er study . Minst eectively . German 25 µg/mL  and 0.2525 µg/mL , respect25 µg/mL , 0.5 µg/25 µg/mL , respect50/90 of S. aureus agree regarding cefquinome [90 values of our trial were one dilution higher concerning oxacillin [S. aureus regarding cefquinome (MIC50/90: 1/2 µg/mL) and cephapirin (MIC50/90: 0.25/1 µg/mL) [50 (0.03 µg/mL) predominantly agree with our results (≤0.06 µg/mL), MIC90 was determined as 16 µg/mL [The MICfquinome ,26, cefofquinome , whereasxacillin ,26 and a1 µg/mL) . Penicil50/90 values of cefquinome and amoxicillin/clavulanic acid [50 and MIC90 values of 1 µg/mL and 2 µg/mL, respectively [90 (1 µg/mL) was one dilution higher [90 [50 and an MIC90 of 1 and 4 µg/mL, respectively, were previously determined [In comparison with the latest monitoring, results of NAS were in agreement with our study regarding MICnic acid . Wente eectively . Addition higher , whereasgher [90  were in termined . Klebsiella species remained at a constant level within the latest resistance monitoring programs. MIC90 of our study differed by one to four dilutions, since the BVL (Bundesamt für Verbraucherschutz und Lebensmittelsicherheit) determined MIC90 of 0.06 µg/mL (cefquinome), 4 µg/mL (amoxicillin/clavulanic acid), 0.06 µg/mL (marbofloxacin) and 0.25 µg/mL (sulfamethoxazole/trimethoprim) [Susceptibility of thoprim) ,26.90 of E. coli are in line with a former study [90 of 32 µg/mL [90 determined within the resistance monitoring also varied over the years: 0.125 µg/mL (2010), 8 µg/mL (2012), 0.125 µg/mL (2014) and 0.5 µg/mL (2016) [Whereas cefquinome MICer study , Wente a32 µg/mL . MoreoveL (2016) ,26,27,28L (2016) ,26,27,28S. agalactiae was isolated from small dairy farms only, so the results of this mastitis pathogen are not presented. The resistance monitoring achieved results from susceptibility examinations from various laboratories located in different German federal states. Minst et al. (2012) already detected higher resistance rates in districts with a high density of dairy farms and assumed that this might be due to a locally increased antimicrobial use . The cur90 values. A higher MIC50 value of 1 µg/mL was detected for isolates from larger farms concerning the following bacteria-antimicrobial-combinations: Klebsiella species (sulfamethoxazole/trimethoprim), S. aureus (cefquinome) and NAS (oxacillin). Regarding the tested NAS isolates, no additional differences were apparent between small and large farms, whereas MIC90 values of S. aureus isolates from large dairy farms were one concentration level lower  or higher  than the respective values determined for the isolates from small dairy farms. In contrast, the investigated Streptococci from small farms were frequently inhibited at elevated antimicrobial MIC90 values. In the case of the tested S. dysgalactiae isolates, this was obvious for cefoperazone, cloxacillin, oxacillin and cefalexin/kanamycin; whereas S. uberis isolates from herds >92 cows showed higher MIC90 values for all antimicrobial agents included in the study. In this respect, cloxacillin MIC90 (16 µg/mL) and cefalexin/kanamycin MIC90 (8 µg/mL) of S. uberis from small dairy farms represented the greatest difference from the isolates obtained from large farms , which differ in three concentration levels. There are also noticeable differences in the MIC90 values of the antimicrobials tested against Gram-negative bacteria. Klebsiella species isolated from large farms were therefore inhibited at MIC90 values of cefquinome (0.5 µg/mL), marbofloxacin (0.125 µg/mL) and sulfamethoxazole/trimethoprim (4 µg/mL) that were one concentration level higher than respective MIC90 from Klebsiella isolates from small herds, respectively. The amoxicillin/clavulanic acid MIC90 values of investigated Klebsiella were determined as 1 µg/mL  and 16 µg/mL (large farm). When discussing the results of the Klebsiella species, the different number of bacterial isolates from small (n = 12) and large herds (n = 40) must be taken into consideration. Due to the substantially smaller quantity of isolates from small farms, a single isolate represented 8.3% of the total and consequently has a higher impact on MIC50/90 values which refer to the total amount of tested bacteria. Regarding E. coli isolates, the MIC90 values of cefquinome (0.5 µg/mL) and amoxicillin/clavulanic (16 µg/mL) were higher on small farms, whereas a comparatively higher MIC90 was detected for isolates tested against marbofloxacin (1 µg/mL) and sulfamethoxazole/trimethoprim (32 µg/mL). The difference of five sulfamethoxazole/trimethoprim concentration levels was the highest between E. coli from small (1 µg/mL) and large (32 µg/mL) farms. This was due to four (12.1%) E. coli isolates gained from large farms, that were not inhibited within the tested concentration range and thus had MICs of >16 µg/mL. The remaining 29 isolates (87.9%) obtained from large farms as well had MICs of concentrations ranging from 0.25 µg/mL to 2 µg/mL for sulfamethoxazole/trimethoprim. Taking everything into consideration, a general statement about the occurrence of less susceptible mastitis pathogens existing on larger dairy farms cannot be confirmed on the basis of this study. Even though antimicrobial MICs were higher in isolates from larger farms, especially in coliforms, the MIC90 of streptococci were exclusively higher in large dairy herds, while NAS showed no differences, except for oxacillin MIC50. However, more investigations including a greater bacterial number are needed before an accurate statement can be made. The differences for both Gram-positive as well as Gram-negative pathogens almost exclusively refer to investigated MICS. uberis (MIC50/90: ≤0.06/0.25 µg/mL), S. dysgalactiae and S. agalactiae (MIC50/90: ≤0.06/≤0.06 µg/mL) were largely consistent with our results. Whereas penicillin MIC values of Danish NAS were ≤0.06 µg/mL (MIC50) and 0.5 µg/mL (MIC90), results of S. aureus were higher for penicillin MIC90 (2 µg/mL), while penicillin MIC50 was ≤0.06 µg/mL [90 of Danish S. aureus was previously reported at concentrations of 0.25 µg/mL, this being identical with results of S. aureus isolates from Iceland and Switzerland (MIC50/90: ≤0.06/0.25 µg/mL). Penicillin MIC values of S. aureus from England, Finland, Ireland, Sweden and the United States were in accordance with our results detected at concentrations of ≤0.06 (MIC50) and 0.5 µg/mL (MIC90) [50/90 of S. aureus investigated in our study was determined at concentrations of 0.25 µg/mL and 1 µg/mL, respectively. In comparison to results of previous surveys from England, Finland, Iceland, Ireland, Norway, Sweden, Switzerland and the United States, the oxacillin MIC90 of S. aureus was also 1 µg/mL, while MIC50 was 0.5 µg/mL. S. aureus isolates from Denmark were inhibited at lower oxacillin concentrations (MIC50/90: 0.25/0.5 µg/mL) [The susceptibility of Gram-positive mastitis pathogens obtained from dairy farms located in Northern Germany was previously described as favorable, whereas Gram-negative isolates exhibited varying degrees of resistance regarding all tested antimicrobials. Cephalosporins were considered to be effective against staphylococci and streptococci mastitis. However, penicillin represented the drug of choice for treating clinical mastitis induced by streptococci . By esta06 µg/mL . A lower (MIC90) . Oxacill5 µg/mL) . S. aureus and NAS against both oxacillin and oxacillin. Whereas NAS exhibited MIC50 values of cloxacillin and oxacillin at concentrations of 0.5 µg/mL, the MIC90 differed for cloxacillin (2 µg/mL) and oxacillin (1 µg/mL). In contrast, S. aureus was inhibited at cloxacillin MIC50 and MIC90 values of 0.25 µg/mL and 0.5 µg/mL, respectively, while MICs of oxacillin were one concentration level higher (MIC50/90: 0.5/1 µg/mL) [S. aureus did not support this hypothesis. De Jong et al. (2018) summarized the results of susceptibility testing from nine European countries: Belgium, the Czech Republic, Denmark, France, Germany, Italy, the Netherlands, Spain and the United Kingdom. Results of de Jong et al. (2018) were different to those of our study for S. aureus  as well as for NAS (cephapirin MIC50/90: 0.125/0.25 µg/mL), whereby the MICs published by de Jong et al. (2018) were one concentration level lower. The remaining MIC50/90 of amoxicillin/clavulanic acid, cephapirin and cefquinome did not differ between our results and those of de Jong et al. (2018) [S. dysgalactiae differences to our study were detected in MIC90 values only, as MIC90 values of ≤0.03 µg/mL (amoxicillin/clavulanic acid), 0.06 µg/mL (cephapirin) and 0.015 µg/mL (cefquinome) were previously described [S. uberis were lower in the case of amoxicillin/clavulanic acid (MIC50/90: 0.25/0.5 µg/mL), cephapirin (MIC90: 0.5 µg/mL) and cefquinome (MIC50/90: 0.125/0.25 µg/mL) [Klebsiella species and E. coli previously exhibited cefquinome MIC50/90 of 0.06/0.125 µg/mL and thus differed by one (MIC50) and two (MIC90) concentration levels from our trial [90 published by de Jong and coworkers (2018) was 8 µg/mL regarding both Klebsiella species and E. coli. Moreover, 90% of E. coli isolates were inhibited at a marbofloxacin concentration of 0.06 µg/mL (MIC90), representing a three concentration level lower value compared to our study. Marbofloxacin MIC50/90 against Klebsiella species were formerly detected as 0.06 µg/mL and differed concerning both values from our investigations (MIC50/90: 0.03/0.125 µg/mL) [S. dysgalactiae are mainly limited to low concentration levels and the S. uberis isolates in our study also deviates strongly from the former German and international results. The high MIC values compared to cefquinome could possibly be due to the increased use of these antimicrobials on German dairy farms. However, this is not supported in the case of other pathogens which did not show increased cefquinome MICs and the fact that MIC values of other antimicrobials  tested against S. uberis  and ceftiofur (approved in the United States). Most of the research studies only included oxacillin for detecting the in vitro efficacy of isoxazolyl penicillins against bacteria. An international study by de Jong et al. (2018) investigated the susceptibility of 1 µg/mL) . Whereas. (2018) . Concernescribed . Compare5 µg/mL) . Both Klur trial . Amoxici5 µg/mL) . As a co. uberis  were alsS. aureus in mice that were simultaneously infected with bacteria and phages [S. aureus was previously isolated from dairy cows suffering from mastitis and used to induce mastitis in mice as well. Phage therapy was able to reduce the bacterial counts as well as the clinical degree of the disease. The authors considered the use of phages for an alternative therapy option for the treatment of bovine mastitis, but also mentioned the non-comparability of mastitis in mice and cows due to anatomical and physiological conditions [p > 0.05) of the quarters infected with S. aureus achieved a bacteriological cure after a five-day treatment with an infusion of a high concentration of bacteriophage K. The authors assumed that an effective concentration of phages in the mammary gland of cattle could not be achieved in raw milk, among other things, due to an inhibition of the bacteriophages to bind to the host cell surface [The favorable results of the Danish, Norwegian and Swedish mastitis pathogens could be due to a highly restrictive use of antimicrobials in these countries. Prudent use—in order to minimize the selective pressure—should therefore be a mandatory requirement whenever the use of antimicrobial agents is considered. In the context of antimicrobial reduction in mastitis treatment, the possibility of alternative treatment methods should be considered. An alternative treatment method for mastitis cases could be the use of bacteriophages as therapeutic agents (bacteriophage therapy). Bacteriophages  are viruses that only target prokaryotic cells, mainly of one bacterial species, for inserting their DNA or RNA for propagation. After propagation, phage-coded enzymes induce the lysis of the bacterial cell (lytic propagation cycle) which leads to the release of next generation phages that are able to infect new host cells . In a mod phages . In anotnditions . The facnditions . In this surface . The invMoreover, a therapy option is the application of non-antimicrobial agents like products containing proteolytic enzymes such as chymotrypsin and trypsin  or homeoS. uberis and E. coli [S. uberis-specific vaccines were beneficial concerning the reduction in somatic cell count and bacterial count [S. aureus-induced mastitis in heifers was not successful, since neither the prevalence of intramammary infections nor the incidence of clinical mastitis was significantly approved [Another possibility for avoiding the extensive use of antimicrobials could be the application of vaccines. In several trials, the application of vaccines was able to cause a reduced severity of the clinical signs accompanied with mastitis cases caused by  E. coli ,44. Furtal count , previouapproved . The broE. coli isolates were not inhibited within tested concentration ranges  investigated in our study was considerably lower than previously described (16 µg/mL). The majority of examined Gram-positive pathogens were inhibited at lower oxacillin than cloxacillin MIC values. Except for S. agalactiae, several mastitis isolates were inhibited at comparatively higher concentrations or not inhibited at the highest tested concentration. This leads to the hypothesis that a certain number of resistant isolates are present on Northern German dairy farms. Whereas the number usually referred to individual isolates, the quantity of presumptive resistant E. coli was higher. Nonetheless, an accurate estimation of resistance is necessarily related to establishing international clinical breakpoints referring to the indication of mastitis. In conclusion, determined MIC values of Gram-negative pathogens were frequently higher than previously reported. Among Gram-positive mastitis pathogens, differences in MIC values were striking for some pathogen-antimicrobial combinations. Compared to previous German studies,


Pyroptosis is a recently identified pathway of caspase-mediated cell death in response to microbes, lipopolysaccharide, or chemotherapy in certain types of cells. However, the mechanism of how pyroptosis is regulated is not well-established.Herein, the intracellular bacteria were detected by staining and laser confocal microscopy and TEM. Live/dead cell imaging assay was used to examine macrophage death. Western blot and immunohistochemical staining were used to examine the protein changes. IFA was used to identify typical budding vesicles of pyroptosis and the STAT3 nuclear localization. SEM was used to observe the morphological characteristics of pyroptosis. ELISA was used to detect the level of inflammatory cytokines. Pyroptosis was filmed in macrophages by LSCM.S. aureus was internalized by human macrophages. Intracellular S. aureus induced macrophage death. S. aureus invasion increased the expression of NLRP3, Caspase1 (Casp-1 p20) and the accumulation of GSDMD-NT, GSDMD-NT pore structures, and the release of IL-1β and IL-18 in macrophages. Macrophages pyroptosis induced by S. aureus can be abrogated by blockage of S. aureus phagocytosis. The pyroptosic effect by S. aureus infection was promoted by either rapamycin or Stattic, a specific inhibitor for mTORC1 or STAT3. Inhibition of mTORC1 or STAT3 induced pyroptosis. mTORC1 regulated the pyroptosic gene expression through governing the nuclear localization of STAT3. mTORC1/STAT3 axis may play a regulatory role in pyroptosis within macrophages.S. aureus infection induces human macrophage pyroptosis, inhibition of mTORC1/STAT3 axis facilitates S. aureus-induced pyroptosis. mTORC1 and STAT3 are associated with pyroptosis. Our findings demonstrate a regulatory function of the mTORC1/STAT3 axis in macrophage pyroptosis, constituting a novel mechanism by which pyroptosis is regulated in macrophages.Video Abstract Macrophages were infected with S. aureus for 3 h (MOI 25:1), and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicates lysing dead cell. Pyroptosis is a lytic form of caspase-dependent cell death, which encompasses caspase-1, -4, -5, and -11. Caspase-1 is activated by various canonical inflammasomes, and caspase-4/5/11 recognizes cytosolic bacterial lipopolysaccharide directly, both of which trigger pyroptosis . GSDMD-NAs extracellular pathogens, certain types of bacteria can invade a variety of mammalian nonprofessional phagocytes and can be engulfed by professional phagocytes, such as neutrophils and monocytes, and survive in them , 9. The Several pathogens can invade cells and survive intracellularly for various periods . The sucStaphylococcus aureus, a gram-positive bacterium and human pathogen that causes a wide range of illnesses, from skin infections to severe pneumonia and sepsis, can invade cells and trigger pyroptosis in human macrophages [rophages . HoweverMammalian target of rapamycin complex 1 (mTORC1) is a central coordinator in eukaryotic cells with regard to cell growth and metabolism with environmental inputs , autophaSignal transducer and activator of transcription 3 (STAT3) is a latent transcription factor that mediates extracellular signals, such as those from cytokines and growth factors . STAT3 pS. aureus-induced pyroptosis and whether the inhibition of the mTORC1/STAT3 axis causes pyroptosis in human macrophages. Collectively, our study demonstrates that mTORC1 and STAT3 have critical functions in the regulation of S. aureus-induced pyroptosis and that inactivation of the mTORC1/STAT3 axis causes pyroptosis in human macrophages. Our results implicate a novel function of the mTORC1/STAT3 axis in regulating cell death and provide insights into the mechanism by which pyroptosis is governed in professional phagocytic cells.Pyroptosic macrophages are important in the defense against microbial infections, removing pathogens and rendering them susceptible to phagocytosis and killing by a secondary phagocyte , 32. As The anti-GSDMD (Cat# ab215191), anti-caspase-1 (Cat# ab1872), anti-S6 (Cat# ab184551), anti-4EBP1 (Cat# ab2606), anti-p-mTOR (Ser2448) (Cat# ab32028), anti-mTOR (Cat# ab10926), goat anti-rabbit (Cat# ab136817), and anti-mouse (Cat# ab205719) secondary antibodies were purchased from Abcam  The anti-p-S6 (Ser240/244) (Cat# 5346s), anti-STAT3 (Cat# 4904s) anti-p-4EBP1 (Thr37/46) (Cat# 2855s), and anti-p-STAT3 (Cat# 4113s) were purchased from Cell Signaling Technology . The anti-GSDMD (Cat# abs128820) was purchased from Absin . The goat FITC-conjugated anti-rabbit IgG (Cat# 115-095-003) and FITC-conjugated anti-mouse IgG (Cat# 115-095-146) were purchased from Jackson . The anti-β-actin (Cat# A5441) was purchased from Sigma .LPS (Cat# L2630), Stattic (Cat# S7947), CFSE (Cat# 21888), and PMA (Cat# P1585) were purchased from Sigma-Aldrich, Inc. . Rapamycin (Cat# 53123-88-9) was purchased from . IFN-γ (Cat# 121527) was purchased from PEPROTECH . β-mercaptoethanol (Cat# M8211) was obtained from Solarbio . Dil (Cat# KGMP002) was purchased from KeyGEN . DAPI (Cat# C1005) was acquired from Beyotime . Alexa Fluor® 594 Phalloidin (Cat# A12381) was purchased from Invitrogen . Cytochalasin B (Cat# HY-16928) was purchased from MCE .2. Macrophages were infected by S. aureus (ATCC25923) at a multiplicity of infection (MOI) of 25:1 (bacteria to macrophages).Human myeloid leukemia mononuclear (THP-1) cells and THP-1-derived macrophages were cultured in RPMI-1640 medium  that was supplemented with 10% fetal bovine serum , 100 U/mL penicillin G, 100 mg/mL streptomycin , and 0.1% β-mercaptoethanol . To induce the classical (M1) polarization program, the medium was replaced with fresh medium without 10% fetal bovine serum and with PMA (100 ng/mL) for 24 h and then supplemented with 2.5 ng/mL IFN-γ and 100 ng/mL LPS for 24 h. Cells were cultured at 37 °C in humidified air with 5% COS. aureus for 3 h at an MOI of 25, and the extracellular bacteria were killed and lysed by antibiotics and lysozyme for 2 h. Monolayer macrophages were lysed, and the number of intracellular bacteria was determined by spread plate method.Macrophages were infected with S. aureus) were washed with PBS and then incubated with CFSE (5(6)-carboxyfluorescein diacetate N-succinimidyl ester) at 4 °C for 15 min. The stained bacteria were centrifuged for 10 min at 3000×g at 4 °C 3 times. Macrophages were infected by the stained bacteria at an MOI of 25, washed with PBS 3 times, and then fixed with 4% paraformaldehyde for 20 min. After being treated with 1% Triton X-100 for 5 min, the macrophages were stained with Alexa Fluor® 594 Phalloidin for 1 h in the dark, washed with PBS 3 times, and counterstained with 100 μL DAPI for 3 min to assess the nuclear morphology. Finally, the slide was mounted with glycerinum for examination under a laser scanning confocal microscope (LSCM) .The macrophages were seeded onto a slide and incubated overnight. Bacteria .S. aureus) for 3 h at an MOI of 25, and the extracellular bacteria were killed and lysed by antibiotics and lysozyme for 2 h. The infected macrophages were washed with PBS 3 times centrifuged for 10 min at 3000×g at 4 °C, and fixed with 2.5% glutaraldehyde overnight. Finally, the cells were embedded in 4% AGAR and fixed with 2.5% glutaraldehyde overnight. The samples were examined by TEM  to detect intracellular bacteria. For SEM, macrophages were seeded onto a slide and incubated overnight. The macrophages were infected by bacteria or treated with rapamycin, washed with PBS, and fixed with 2.5% glutaraldehyde for 4 h. After being air-dried, the slides were examined by SEM  to detect GSDMD-NT pores on the cytomembrane.Macrophages were infected by bacteria (Macrophages were washed with cold PBS and lysed in cell lysis buffer. The lysis buffer comprised 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1% PMSF, and phosphatase inhibitors. Equal amounts (40 μg) of protein were electrophoresed on 10% (w/v) sodium dodecyl sulfate–polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and incubated with the primary antibody. Peroxidase-conjugated secondary antibody and enhanced chemiluminescence (ECL) reagent were used to detect the signals with the Western Blotting System .Cells were seeded onto slides, incubated overnight, washed with PBS, and fixed with 4% paraformaldehyde for 15 min. For immunohistochemical detection of proteins (1000× magnification), antigens were detected using UltraSensitive™ S-P Immunohistochemistry hypersensitive kits (mouse/rabbit)  with hematoxylin  for counterstaining. A biotin-streptavidin peroxidase-based method was used to detect the primary antibodies.Levels of IL-1β and IL-18 in the supernatant was quantified by enzyme linked immunosorbent assay (ELISA) using NeoBioscience ELISA kits . The amount of culture media and intracellular α-hemolysin were measured via standard Mbbiolog ELISA kit  according to the manufacturer’s guidelines. Absorbance at 450 nm and 630 nm was read on a Varioskan Flash Multimode Reader . All measurements were performed in triplicate, and the mean value of 3 independent measurements was used for statistical analysis.Mounted IFA slides were observed under an LSCM . The macrophages phenotype, IHC slides, and large bubbles on the macrophages were observed under a light microscope .CASP1, and GSDMD in macrophages in the treatment and control groups. For the bacterial infection, macrophages were infected with S. aureus for 3 h at an MOI of 25, and the extracellular bacteria were killed and lysed by antibiotics and lysozyme for 2 h. For the inhibitor treatment, macrophages were treated with 100 nM rapamycin for 6 h or 100 μM Stattic for 1 h, and the total RNA was extracted from untreated and treated cells. Total RNA was prepared with the RNAiso Plus reagent according the manufacturer's instructions . Briefly, the cells were washed with PBS and lysed in RNAiso Plus, and chloroform was added to the cell lysates for homogenization. The top aqueous layer was transferred to a new tube after centrifugation, and isopropanol was added to the supernatant and mixed well. Total RNA was precipitated by centrifugation, and the pellet was dissolved in RNase-free water. RNA quantities over 600 ng/μL and a purity of 1.90–2.0, based on the 260/280 ratio, were used to synthesize cDNA.Quantitative real-time PCR (qPCR) was performed to determine the levels of 12–18 primer using the AMV 1st Strand cDNA Synthesis Kit . ACTB was selected as the internal control gene. The primer sequences were as follows (5′–3′):CASP1: forward, CGACAAGGTCCTGAAGGAGAAGAGreverse, CGTGTGCGGCTTGACTTGTCCATTGSDMD: forward, TGTACGTGGTGACTGAGGTGCTGCreverse, CTGAGGGGATGGTGACCGTCTTCTACTB: forward, TCACCAACTGGGACGACATreverse, GCACAGCCTGGATAGCAACmRNA was reverse-transcribed with oligo (dT)−ΔΔCT values were calculated to determine expression levels, and the qPCR results were analyzed by student’s t-test to compare the expression between untreated and treated groups. Three independent experiments were performed.The KAPA SYBP® FAST qPCR Kit Optimized for LightCycler® 480  was used for the PCR according to the manufacturer’s instructions. The program comprised an initial denaturation step at 95 °C for 5 min; 40 cycles of 95 °C for 5 s, 54 °C for 30 s, and 72 °C for 20 s; and a final extension of 72 °C for 10 min. Three technical replicates were run in each experiment. 2pNA standard. The absorbance was determined at 405 nm with a 96-well plate reader and the caspase1 activity was normalized for total proteins of cell lysates.The caspase-1 activity was measured using a Caspase1 Activity Assay kit . According to the manufacturer’s instructions, about 50 μg protein from cells was mixed with synthetic tetrapeptide Ac-YVAD-pNA and incubated at 37 °C for 2 h. A standard curve was prepared using the S. aureus and incubated with 2 μM Calcein AM and 4 μM PI solution. Incubation at room temperature for 15–20 min and observe labeled cells under Live cell Imaging System It is performed using the Live & Dead® Viability/Cytotoxicity Assay Kit . Macrophages were infected with S. aureus were co-cultured at an MOI of 25 for 3 h to enable phagocytosis. Phagocytosis was stopped by placing on ice and washing the macrophages cultures twice to remove non-phagocytosed bacteria. The extracellular bacteria were killed and lysed by antibiotics and lysozyme. Monolayer macrophages were lysed, and the number of intracellular bacteria was determined by spread plate method.Macrophages were treated with 100 nM rapamycin for 6 h or 10 μg/mL Cytochalasin B for 30 min, respectively, cells and p ≤ 0.05.Statistical analyses were conducted using SPSS PASW Statistics for Windows, v18.0 . Data were analyzed using standard parametric statistics and one-way ANOVA, followed by Tukey’s method. Data are expressed as mean ± SD. The results are presented as the average of at least 3 independent experiments. Statistical significance was accepted when S. aureus-induced pyroptosis in macrophages, THP-1 cells were differentiated into macrophages in culture with 100 ng/mL PMA (phorbol myristate acetate) for 24 h. Then, the macrophages were treated in differentiation medium with 2.5 ng/mL IFN-γ and 100 ng/mL LPS for 24 h to transform them into M1-polarized macrophages   in the medium of cells that contained bacteria  was used to observe the GSDMD-NT pore structures on the plasma membrane. Pore structures with ~ 30-nm diameters were observed , NLRP3 and GSDMD-NT by western blot, immunofluorescence, and immunohistochemistry staining and the levels of IL-18 IL-1β in the cell medium by ELISA. Rapamycin upregulated the S. aureus-induced expression of caspase-1 (Casp-1 p20) and NLRP3 . Further, rapamycin promoted the formation of S. aureus-induced GSDMD-NT membrane pores . Meanwhile, using a colorimetric assay to assess caspase-1 activity during rapamycin treatment, we found that caspase-1 activity was significantly increased  and GSDMD-NT; previous study has shown that STAT3 is associated with apoptosis and autophagy and thus , we hypoCASP1 and GSDMD via STAT3, we first treated macrophages with 100 nM rapamycin for 6 h and found that repamycin reduced the phosphorylation of STAT3, indicating STAT3 activation was inhibited . These data suggest that mTORC1 regulates the expression of pyroptosic genes, likely through STAT3 in macrophages.To determine whether mTORC1 regulates the expression of ted Fig. a. Then, DMD Fig. b . In addition, Stattic treatment significantly induced caspase-1 activation  induce NLRP3-mediated signaling, triggering caspase-1 activation and pyroptosis in human and mouse monocytic cells [S. aureus phagocytosis. These data indicate that S. aureus internalized macrophages, and intracellular S. aureus induced pyroptosis in professional phagocytic cells. Furthermore, inactive mTORC1 did not affect the ability of macrophages to phagocytose bacteria, our data may reveal the biological role of pyroptosis in S. aureus invasion.Pyroptosis was initially observed in macrophages that were infected with flexneri , 47. Cytroptosis . Of noteic cells . In our S. aureus infection, mTORC1 inhibition, and STAT3 inhibition induce macrophage pyroptosis.In general, plasma membrane damage can be repaired efficiently in macrophages, for which endocytosis, membrane patching, and extracellular budding can be used . Once gaS. aureus in mouse macrophages [S. aureus-infected macrophages with rapamycin to inhibit mTORC1 and examine how S. aureus-induced pyroptosis is altered. We were surprised to find that S. aureus-induced pyroptosis was promoted by rapamycin in macrophages, for which there are 2 possibilities: rapamycin enhances S. aureus to induce pyroptosis and mTORC1 inhibition causes pyroptosis, in which case mTORC1 inactivation is an independent factor for macrophage pyroptosis. In rapamycin-treated cells, mTORC1 inactivation upregulated pyroptosic proteins, including CASP1 and GSDMD, release of IL-1β, and induced macrophage pyroptosis. Several studies in cells of mice and human reported that the negative regulation of inflammasome by rapamycin. Muhamuda et al. (2017) showed that rapamycin treatment  of lethal Ixodes ovatus ehrlichia (IOE) -infected WT-BMM attenuated production of IL-1β, which is the gold standard readout of inflammasome activity [In our previous work, mTORC1 signaling was initiated by peptidoglycan (PGN) from rophages . In the activity . In addiactivity . In contactivity  showed tactivity  reportedBECN1 and PIK3C3 [CASP1, GSDMD and triggering pyroptosis in human macrophages.As a latent transcription factor, STAT3 modulates a range of target genes, including induced and repressed target genes that mediate cellular and organismal functions . Moreoved PIK3C3 . Moreoved PIK3C3 –60. In fd PIK3C3 . In our The nuclear localization of STAT3 comprises phosphorylation, dimerization, and nuclear localization, can be blocked by Stattic , 63. In S. aureus invaded macrophages to induce pyroptosis; this process was promoted by rapamycin. mTORC1 regulates the expression of pyroptosic genes, including CASP1 and GSDMD, through STAT3. Inhibition of mTORC1 prevents the nuclear localization of STAT3. The pathogenic effects of the inhibition of the mTORC1/STAT3 axis facilitates S. aureus-induced pyroptosis. This study demonstrates a regulatory function of the mTORC1/STAT3 axis in macrophage pyroptosis, constituting a novel mechanism by which pyroptosis is regulated in macrophages.In summary, mTORC1 and STAT3 regulate human macrophage pyroptosis. The mTORC1/STAT3 axis is critical in pyroptosis in human macrophages. Additional file 1: Figure S1. PMA-induced differentiation of THP-1 to macrophages and further induction by LPS and IFN-γ into M1-polarized macrophages. THP-1 cells were treated with PMA (100 ng/mL) for 24 h to differentiate into Mϕ -polarized macrophages and then stimulated with IFN-γ (2.5 ng/mL) and LPS (100 ng/mL) to polarize them into classical M1 macrophages. (a) THP-1 and THP-1-derived macrophages and Mϕ and M1 macrophages. Scale bars represent 100 μm. (b) TFN-α and IL-1β in culture medium were determined by ELISA. M1 macrophages had more TFN-α and IL-1β than Mϕ macrophages. **p < 0.01. n = 3 independent experiments. Error bar indicates SD.Additional file 2: Table S1. Number of S. aureus in macrophages.Additional file 3: Figure S2.  (a) The amount of α-hemolysin in culture media and intracellular were evaluated by ELISA. (b) Effect of Cytochalasin B on macrophage phagocytosis. (c) Immunohistochemical analysis of pyroptosic protein GSDMD-NT in S. aureus-infected human macrophages. Scale bars represent 10 μm. (d) Large bubbles observed by light microscopy in S. aureus-infected macrophages. Large bubbles indicated by red arrows in pyroptotic cells. Scale bars represent 1 μm. *p < 0.05; **p < 0.01; NS = p > 0.05. n = 3 independent experiments. Error bar indicates SD.Additional file 4: Video 1. The Live/Dead assay was performed by simultaneously monitoring the fluorescence after macrophages were infected with S. aureus for 3 h (MOI 25:1), Green, as live cell indicator and red, as dead cell indicator.Additional file 5: Figure S3. (a) Immunohistochemical analysis of GSDMD-NT in human macrophages. Macrophages were preincubated with rapamycin for 6 h and then infected with S. aureus. Scale bars represent 10 μm. (b) Quantification of the number of CFU/ml of S. aureus in cells treated with rapamycin and without rapamycin. NS = p > 0.05. n = 3 independent experiments. Error bar indicates SD.Additional file 6: Figure S4. Rapamycin inhibits mTORC1 signaling and upregulates GSDMD-NT expression. (a) Macrophages were treated with 100 nM rapamycin for 6 h. (b) Rapamycin upregulates GSDMD-NT in macrophages. Scale bars represent 10 μm. The resolved bands were quantified using Gel-Pro Analyzer 4.0 . *p < 0.05; **p < 0.01. n = 3 independent experiments. Error bar indicates SD.Additional file 7: Video 2. Macrophages were treated with 100 nM rapamycin for 6 h, and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicates lysing dead cell.Additional file 8: Video 3. Macrophages were treated with 100 μM Stattic for 1 h, and pseudopod contracting, cell rounding, and lysing dead cells were filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicate pseudopod contracting, cell rounding, and lysing dead cells.Additional file 9: Video 4. Macrophages were treated with 100 μM Stattic for 1 h, and pyroptosis was filmed in macrophages by laser confocal microscopy. A representative field was recorded. Arrow indicate lysing dead cells.


Trichinella spiralis infection are the adult worms (AW), at the gut level, and the migrant or newborn larvae (NBL), at systemic and pulmonary levels. Most of the studies carried out in the gut mucosa have been performed on the Payer’s patches and/or the mesenteric lymph nodes but not on the lamina propria, therefore, knowledge on the gut immune response against T. spiralis remains incomplete.The main targets of the host’s immune system in T. spiralis, particularly, the events taking place between 1 and 13 dpi. For this purpose, Wistar rats were orally infected with muscle larvae of T. spiralis and the humoral and cellular parameters of the gut immunity were analysed, including the evaluation of the ADCC mechanism exerted by lamina propria cells.This study aimed at characterizing the early mucosal immune response against A marked inflammation and structural alteration of the mucosa was found. The changes involved an increase in goblet cells, eosinophils and mast cells, and B and T lymphocytes, initially displaying a Th1 profile, characterised by the secretion of IFN-γ and IL-12, followed by a polarization towards a Th2 profile, with a marked increase in IgE, IgG1, IL-4, IL-5 and IL-13 levels, which occurred once the infection was established. In addition, the helminthotoxic activity of lamina propria cells demonstrated the role of the intestine as a place of migrant larvae destruction, indicating that not all the NBLs released in the gut will be able to reach the muscles.T. spiralis infection showed that not only an effector mechanism is directed toward the AW but also towards the NBL as a cytotoxic activity was observed against NBL exerted by lamina propria cells. The characterization of the immune response triggered in the gut mucosa during  Trichinella spiralis is a nematode parasite which causes an estimated number of 10,000 cases per year with a mortality rate of about 0.2%  × 100, where NBLi and NBLf are the numbers of living NBL, judged by their motility, counted at the beginning and at the end of the reaction respectively. Results were expressed as the mean of mortality percentages of NBL ± SD obtained from five independent experiments performed in duplicate.The NBL mortality percentage (M%) was calculated according to the following formula: M% = [ = 27.10, P < 0.0001; Zvs control9 dpi  = 11.11, P < 0.001; Fig. Histamine levels in rat intestinal fluids were also quantified on 3, 6, 9 and 13 dpi and the results are shown in Fig. T. spiralis, we studied the humoral response, mainly as regards the appearance, kinetics and location of different Igs isotypes as well as the presence of different adaptive immunity cells at lamina propria. Thus, CD4+, CD8+ and the TCR+ cell counts were determined as well as the number of IgA+ and IgE+ cells present in the small intestine. The anti-T. spiralis-ESP IgE, IgA, IgG1 and IgG2a were also determined in cells from intestinal Peyer’s patches, mesenteric lymph nodes and lamina propria.During the enteral phase of the infection with + T cells increased progressively until the end of the period studied, a timepoint at which, counts reached values that were 3.5-fold higher that those observed in control animals. From 2 dpi on a significant and progressive increase in CD25+ cell counts were also observed until the end of the period studied.The cellular phenotype of cells from at lamina propria obtained by evaluating the tissue sections by IIF are presented in Table + cells increased significantly only on 4 dpi and decreased significantly to almost half of the control values on 6 dpi and returning to control values on 13 dpi. CD8β+ cells followed a similar kinetics with significant increases on 2 and 4 dpi. On 6 dpi, CD8β+ cells decreased to almost half of the control values but increasing significantly again on 13 dpi.The numbers of CD8α+ cell counts were found to be significantly elevated from 2 to 6 dpi, when they decreased drastically below control values, to increase again towards 13 dpi to control values. TCRγδ+ cell counts showed a similar kinetics, increasing significantly to reach values that were 1.5-fold higher, as compared to control animals on 2 and 4 dpi, then decreasing to almost half of the control values on 6 dpi, and finally returning to control values on 13 dpi. The lamina propria IgA+ cell counts showed statistically significant differences between 4 and 6 dpi  = 48.21, P < 0.0001; Zvs control3 dpi  = 19.43, P < 0.0001; Fig. vs 5.00 ± 0.48 IgA SCs/106 leucocytes, 3 dpi vs control, respectively, F = 47.92, P < 0.0001; Zvs control3 dpi  = 6.44, P < 0.0001; Fig. vs 1.30 ± 0.27 IgA SCs/106 leucocytes, 13 dpi vs control, respectively, F = 47.92, P < 0.0001; Zvs control13 dpi  = 20.16, P < 0.0001; Fig. To determine the sequence of secretion of anti-ML-ESP Igs occuring during the enteral phase of infection with vs 12 ± 4 μg/ml, F = 88.04, P < 0.0001; Zvs control1 dpi  = 20.77, P < 0.001 and Zvs control2 dpi  = 3.529, P < 0.05; Fig. vs 18 ± 9 ng/ml, F = 11.14, P < 0.0001; Zvs control13 dpi  = 6.25, P < 0.001; Fig. vs 503 ± 77 ng/ml, F = 7, P < 0.0001; Zvs control13 dpi  = 4.42, P < 0.001; Fig. In intestinal tissue extracts, total IgA was found to be elevated from 1 to 3 dpi and on 13 dpi. Maximum values were reached on 1 and 2 dpi, showing a 26-fold increase  = 33.85, P < 0.0001; Zvs control1 dpi  = 8.91, P < 0.001; Fig. vs 0.159 ± 0.011 OD 3 dpi and control, respectively, F = 14.54, P < 0.0001; Zvs control3 dpi  = 5.42, P < 0.001; Fig. vs 0.032 ± 0.001 OD 13 dpi and control, respectively, F = 80.73, P < 0.0001; Zvs control13 dpi  = 13.20, P < 0.001; Fig. vs 0.135 ± 0.021 OD, 1 dpi and control, respectively, F = 49.29, P < 0.0001; Zvs control1 dpi  = 12.77, P < 0.001; Fig. As for the ML stage, antibodies against ML-ESP were detected during the entire period studied: an 8-fold increase in IgA levels was detected from 1 dpi  = 10.32, P < 0.0001; Zvs control3 dpi  = 3.001, P < 0.05; Fig. vs 0.054 ± 0.013 OD 13 dpi and control, respectively, F = 21.45, P < 0.0001; Zvs control13 dpi  = 8.96, P < 0.001; Fig. vs 0.042 ± 0.007 OD 13 dpi and control, respectively, F = 43.55, P < 0.0001; Zvs control13 dpi  = 14.51, P < 0.001; Fig. vs 0.134 ± 0.009 OD 13 dpi and control, respectively, F = 19.37, P < 0.0001; Zvs control13 dpi  = 6.77, P < 0.001; Fig. Anti-AW-ESP antibodies were detected from 3 dpi until the end of the period studied. IgE was the first isotype to increase, showing a twofold increase on 3 dpi  and while IgG2a was present only in 50% of the animals ; on 13 dpi, 100% of the animals had a positive reaction for IgA, IgE and IgG1 but with higher titers in the case of IgE and IgG1 but lower for IgA . IgG2a were negative in 100% of the animals.vs 1347.0 ± 235.5 pg/ml; infected rats on 1, 2 and 3 dpi vs control rats, respectively, F = 15.61, P < 0.0001; Zvs control1 dpi  = 4.51, Zvs control2 dpi  = 5.06, Zvs control3 dpi  = 4.94, P < 0.001; Fig. Cytokines and chemokines levels were measured during the enteral phase of the infection Fig. . TNF-α lvs 472.2 ± 197.7 pg/ml, respectively, F = 49.64, P < 0.0001; Z3 dpi vs control = 4.10, P < 0.001; Fig. vs 111.8 ± 4.5 cells CCL25+/VCU, respectively; Z13 dpi vs control = 12.14, P < 0.001; Fig. With respect to the chemokines studied, CCL11 showed a significant increase from 1 dpi, with these high levels remaining until 13 dpi, and reaching the maximum value on 3 dpi, which was 12.5-fold higher than control values  = 9.85, P < 0.0001; Z6 dpi vs control = 6.87, P < 0.001; Fig. IL-4, IL-13 and IL-5 are key Th2 cytokines and their kinetics were studied in our model. IL-4 showed a statistically significant increase from 2 to reach a peak value on 6 dpi, reaching approximately a 3.5-fold increase with respect to the control  = 9.85, P < 0.0001; Zvs control2 dpi  = 3.24, P < 0.05 and Zvs control6 dpi  = 4.94, P < 0.001), decreasing on 13 dpi to control values  = 12.13, P < 0.0001; Zvs control3dpi  = 5.008 P < 0.001, respectively, Fig. vs 548.5 ± 136.5 pg/ml, F = 20.96, P < 0.0001; Zvs control2 dpi  = 2.93 P < 0.05; Fig. On the other hand, IFN-γ, a key Th1 cytokine, increased from 2 to 6 dpi to reach values that doubled those of control values  = 20.11, P < 0.0001; Zvs control13 dpi  = 12, P < 0.001 and Zvs control13 dpi  = 5.81, P < 0.05; Fig. In ADCC ies Fig. , with deies Fig. . HoweverFig. F3, 1 = 20.11Interestingly, no significant differences were observed when ADCC experiments were carried out in the presence of serial dilutions of either fresh serum or heat-inactivated serum, indicating that under our experimental conditions, complement factors do not exert cytotoxic effects (data not shown).in vitro cellular cytotoxicity assays was determined by Giemsa staining of cytospin preparations. A change in the cellular composition of these intestinal lamina propria cell suspensions occurred as the infection progressed (Table The composition of intestinal lamina propria cell suspensions used in the ed Table . IntestiT. spiralis infection. The results presented herein demonstrate that during the enteral phase of infection with T. spiralis, between 0 and 13 dpi, a marked inflammatory process occurs, which is featured by the presence of an eosinophilic and mastocytic infiltrate. These results agree with those reported the mouse model [T. spiralis caused a marked structural alteration of the mucosa characterised by early goblet hyperplasia as well as the presence of mucin in the intestinal lumen to expel the parasite. This activation of goblet cells observed from 1 dpi correlate to the early secretion of IL-13, IFN-γ and IL-4. The role of these cytokines in the induction of goblet cell hyperplasia and mucin secretion has been observed in other helminthiases [This study was aimed at characterizing the gut immune response in the rat during se model . As in tse model , 27, in nthiases , 28.T. spiralis infection [+ and CD4+ cells from 3 dpi on. The humoral response was found to be of a Th1 type profile, which shifted to a Th2 phenotype with regulatory components [The cellular and molecular assessment of the intestinal mucosa indicated that a type 1 oriented response develops from 1 dpi and that this response is characterised by the secretion of with IFN-γ and IL-12. As the infection progresses, the immune response, modulated in part by IL-10, shifts to a mixed Th1/Th2 profile and then to a Th2 profile by 6 dpi with a significant increase of IL-4, IL-5 and IL-13 levels. These results are in line with the immune response developed in the lung mucosa during the enteral phase of nfection . The gutmponents , 29.CCL25 and CCL28 are constitutively expressed by the epithelial cells of the intestine and both chemokines have a key role in directing the migration of T lymphocytes. CCL25, whose levels are regulated by the secretion of TNF-α . Not onlTogether, the results presented in this work and previous results obtained in our laboratory suggest that there is a simultaneous development of the immune response at the gut and pulmonary mucosa levels. These responses are triggered by a first antigenic stimulation taking place in the gut by the ML stage. This simultaneous mucosal activation might be attributed to cell migration through the MALT , 32 and T. spiralis infected rats. Our results showed that from 3 dpi on, there was a significant increase in histamine levels in the intestinal fluid of infected rats, although the number of mast cells were found to increase significantly from 6 dpi on. Considering these observations and the increase of anti-ESP-ML IgE levels, it could be suggested that there is an activation of the resident mast cells of the intestinal mucosa as early as on 1 dpi. Among the functions of histamine are the contraction of smooth muscle and increase vascular permeability by vasodilation [Mast cells are one of the effector cells involved in the expulsion of AW in mice, although the mechanism has not been elucidated. McDermontt et al.  have demdilation , which fdilation  who obsedilation ; in this+, CD25+, CD8β+, TCRγδ+ and IgE+ cells (Table + and CD4+ cells, together with the presence of total and specific IgE and the environment created by the rise of IL-4, IL-5 and IL-13 levels suggest that the CD4+ lymphocytes present in the gut would be of the Th2 type.In addition to the presence of mast cells and eosinophils, the inflammatory process taking place in the intestinal mucosa of infected rats from 1 dpi was also found to consist of infiltrating CD4+ cells are associated, through the secretion of IL-4, IL-5 and IL-13, with the development of a Th2 response [+ cells would be involved in the stimulation of goblet cells at the intestinal level, since they increase from 4 dpi in the gut epithelia [+ cells also promote epithelial repair [T. spiralis-damaged epithelium.In helminth infections, TCRγδresponse . In tricpithelia . Considel repair , it can T. spiralis specific IgE, total IgE levels were not increased on 1 dpi. The increase in anti-ESP-AW IgA levels during the late phase of the intestinal infection could be associated with a reduction of AW fertility and eventually expulsion [IgA is considered a crucial immunoglobulin in the GALT; however, in this model, an early strong specific IgE response was detected. The anti-ML-ESP IgE kinetics, both secretory cells and Igs present in intestinal tissue extracts, accompanying parasitic stage changes, suggests a protective role of IgE and a polarization of the immune response at the intestinal mucosa towards a Th2 phenotype. This finding is supported by the increased levels of IgG1. High total IgE levels have been reported in helminth infections; however, its biological significance is still unknown. Some authors; however, consider that the induction of the synthesis of high levels of this immunoglobulin would be an evasion mechanism since soluble IgE might bind IgE receptors to prevent specific IgE from binding to the cell surface –42. Wataxpulsion , 45. On T. spiralis demonstrated that T cells are activated on 12 dpi in the lamina propria and interact with B cells, which are drained to the mesenteric ganglia and the thoracic duct [T. spiralis begin to proliferate on 1 and 2 dpi [T. spiralis infection, the entrance of parasite in the intestinal epithelia triggers a rapid and effective immune response in the lamina propria.Previous studies conducted in rats infected with cic duct . In addind 2 dpi . In the nd 2 dpi . ConsideT. spiralis.It should be noted that the number of activated B cells does not necessarily correlate with the amount of antibody produced. This phenomenon can be evidenced by observing total IgG2a levels, which were not increased at any time, while an increase in the number of IgG2a secretory cells and an increase in anti-ML-ESP IgG2a was observed (Figs. T. spiralis NBL is known [in situ and that this process is independent of the systemic immune system. However, the role of eosinophils, present in the cellular infiltrate of the intestinal lamina propria remains unclear, as it has not been associated with the AW expulsion in a primary infection [The role of eosinophils in the systemic and pulmonary ADCC mechanism in the defence against is known , 47, 48.is known  in the lnfection , 49, 50.Regarding the ADCC mechanism in the intestine and lung mucosa, it is interesting to compare the results presented in this study with those obtained by Falduto et al. . In thatIn vitro cytotoxicity assays showed that intestinal lamina propria cell suspensions from infected rats were able to attack the NBL through an antibody-independent cytotoxicity mechanism not only on 3 dpi, when we observed an increase in the NBL mortality percentages, but also in the presence of serum anti-NBL antibodies that did not enhance the helminthotoxic capacity (Fig. It is also known that eosinophils are involved in antibody-independent cytotoxicity mechanisms during helminth infections . In vitrity Fig. . These rT. spiralis infection. We showed that not only an effector mechanism is directed toward the AW but also the NBL as a cytotoxicity activity was observed against NBL by lamina propria cell suspensions.This study provides important data characterizing the immune response triggered in the lamina propria during


The phenolic profiles, hypoglycemic activity, and molecular mechanism of the effect on type 2 diabetes mellitus (T2DM) of four highland barley varieties were investigated in the present study. The fundamental phenolics in highland barley were ferulic acid, naringin, and catechin, which mainly existed in bound form. These varieties showed favorable hypoglycemic activity via inhibition of α-glucosidase and α-amylase activities, enhancement of glucose consumption, glycogen accumulation and glycogen synthase 2 (GYS2) activity, and down-regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) activities. Specifically, ZQ320 variety exhibited the strongest hypoglycemic activity compared to the other varieties. Highland barley phenolics could inhibit gluconeogenesis and motivate glycogen synthesis via down-regulating the gene expression of G6Pase, PEPCK, and glycogen synthase kinase 3β (GSK3β), while activating the expression of insulin receptor substrate-1 (IRS-1), phosphatidylinositol 3 kinase (PI3K), serine/threonine kinase (Akt), GYS2, and glucose transporter type 4 (GLUT4). Therefore, phenolics from highland barley could be served as suitable candidates for therapeutic agent in T2DM to improve human health. Nonetheless, these drugs often have many undesirable adverse effects, including weight gain, drug resistance, and hypoglycemia [Diabetes mellitus (DM), a widespread chronic metabolic disorder with high morbidity, mortality, and healthcare expenditure, is usually characterized by persistent hyperglycemia and disorders of glucose, lipid, and protein metabolism ,2. Pursupatients . Currentglycemia . Therefoglycemia ,7,8.Hordeum vulgare L.), a cereal crop belonging to the Poaceae family, is the fourth most consumed grain worldwide, only behind wheat, rice, and maize [β-glucan (54–76 mg/L), phenolics (131–178 mg/L), flavonoids /100 g dry weight (DW)), and anthocyanins (0.06–8.98 mg C-3-G E/100 g DW) [β-glucan and phenolics [Barley  ,12,13. T00 g DW) . Concrethenolics ,16.p-coumaric acid, and protocatechuic acid were detected in highland barley [β pathway while blocking AMPK pathway. Daisy et al. [Phenolics in highland barley are present in both free and bound forms. Bound phenolics are able to survive human gastrointestinal digestion and reach the colon tract, where they exert beneficial bioactivities upon released by microbial fermentation . Phenolid barley ,12,16. Td barley ,19,20. Pd barley ,22. Pu ed barley  confirmey et al.  found thy et al. ,11. ReceNowadays, some investigations have focused on phytochemicals and bioactivities of highland barley, but little is known about the hypoglycemic effect and molecular mechanism of phenolics from different highland barley varieties, especially the cellular hypoglycemic capacity. Therefore, the present study aimed to evaluate the phenolic profiles and hypoglycemic activity of four highland barley varieties both in free and bound fractions, and explore the underlying molecular mechanism on the treatment of T2DM.The moisture content of ZQ320, ZQ2000, BQ, and HQK was 12.66% ± 0.05%, 11.95% ± 0.05%, 14.44% ± 0.03%, and 12.79% ± 0.10%, respectively . The coeThe free, bound, and total phenolic contents of highland barley varieties were depicted in p < 0.05), while the black variety had the highest free flavonoids. Moreover, bound fraction contributed at least 70.67% to total flavonoids amongst the four varieties. This phenomenon indicated the predominant contribution of bound fraction to total flavonoids, which showed a similar trend with phenolics. However, Zhu, Li, Fu, Abbasi, Zheng, and Liu [Flavonoids, belonging to the phenolics in whole grains, exhibit favorable antioxidant and anticancer activities . As show and Liu  discover and Liu  found th and Liu , but obv and Liu .p < 0.05), both belonging to the white variety. These data inferred flavonoids as the dominant phenolics in highland barley, which was in accordance with reported research [Based on a micromole basis, contributions of total flavonoids to total phenolics were ranked as follows C: ZQ2000research ,27. Closresearch . Therefop < 0.05) in contrast with the other varieties; this might be by virtue of the black color of the HQK variety. Consequently, the black and blue varieties contain more anthocyanins compared to the other two white varieties. These results were consistent with the report about the total anthocyanin content (0.06–8.98 mg C-3-G E/100 g DW) in different highland barley varieties [Anthocyanin is a subclass of flavonoids, which provides various colors to fruits, vegetables and grains. Overall, anthocyanin was detected only in the free extracts whose contents ranged between 0.42 ± 0.08 and 9.24 ± 0.79 mg C-3-G E/100 g DW . The higarieties . Howeverarieties . In the p < 0.05) amongst the four varieties, while that of ferulic acid had no obvious difference (p > 0.05). In concrete, total ferulic acid content was in a decreasing order of ZQ2000 (54.65 ± 0.77 mg/100 g DW) > BQ (51.18 ± 1.53 mg/100 g DW) > HQK (51.13 ± 2.76 mg/100 g DW) > ZQ320 (50.16 ± 1.45 mg/100 g DW). Naringin and catechin varied between 7.32 ± 0.32 (HQK) and 9.84 ± 0.23 (BQ), and between 2.47 ± 0.01 (ZQ2000) and 4.72 ± 0.01 (BQ), respectively. Amongst these phenolics, gallic acid (0.13–1.38 mg/100 g DW) was only discovered in the free extracts, quercetin (1.40–3.00 mg/100 g DW) was only observed in the bound extracts. In addition, the total contents of protocatechuic acid and chlorogenic acid ranged from 1.53 ± 0.04 (ZQ2000) to 4.17 ± 0.06 (HQK), and from 0.97 ± 0.03 (BQ) to 1.96 ± 0.02 (ZQ320), respectively. Slightly out of line with the above results, Zhu, Li, Fu, Abbasi, Zheng and Liu [Overall, seven phenolics comprising four phenolic acids and three flavonoids were detected in highland barley using HPLC-PAD . The lim and Liu  found th and Liu . Taken tα-amylase, located in the pancreas and salivary glands, can degrade dietary sugars such as starch into oligosaccharides. Subsequently, α-glucosidase secreted from small intestinal hydrolyzes oligosaccharides into monosaccharides which are absorbed into the blood circulation to increase the blood glucose levels [α-glucosidase and α-amylase activities, thus managing and treating T2DM [α-glucosidase and α-amylase, was used as a positive control in present study.Evidently, e levels . Therefoing T2DM . Acarbosα-glucosidase and α-amylase amongst four highland barley varieties were given in α-glucosidase activity was significantly suppressed by acarbose, four highland barley varieties and three phenolic compounds in a concentration-dependent manner. At the concentration of 100 mg/mL, the inhibitory rates of ZQ320 and ZQ2000 bound extracts were 97.06% ± 1.35% and 96.29% ± 1.34%, respectively, which were higher than that of acarbose (93.44% ± 0.73%). Particularly, ZQ320 bound extract showed the highest suppressive effect on α-glucosidase at each concentration point. The inhibitory efficiency of inhibitor was assessed by median inhibition dose (IC50), and thus a lower IC50 indicated a stronger inhibition effect on α-glucosidase. The IC50 of acarbose was 4.62 ± 0.68 mg/mL  indicated that the inhibitory effect of highland barley was not caused by an individual compound but by the synergistic and additive effects of phenolics. Previous researchers suggested that ferulic acid, catechin, and quercetin could be used as competitive suppressors of α-glucosidase [α-glucosidase could be inhibited by hydroxyl substitution on the B ring or the presence of 3-OH, 4-CO and 2,3-double bond. Therefore, higher ferulic acid and quercetin contents of bound extracts than free fractions may be responsible for the stronger prohibition of α-glucosidase . ZQ320 bound phenolics exhibited the strongest inhibitory effect on α-amylase at every concentration point. Generally, a lower IC50 inferred a higher inhibition effect. The IC50 (50 of ZQ320 bound phenolics (9.48 ± 1.04 mg/mL) was slightly lower than that of acarbose (3.36 ± 0.47 mg/mL). Additionally, the IC50 of ferulic acid, naringin and catechin  suggested that the inhibitory effects of highland barley may be by virtue of the synergistic and additive effects of their phenolics rather than their individual phenolic compounds. These findings revealed the potential of ZQ320 bound extract as a promising α-amylase suppressor, overcoming the adverse effects of acarbose such as diarrhea, abdominal distension, and pain. Consequently, the inhibition effect of α-amylase in bound extracts showed similar trend to α-glucosidase, this may be chiefly related to their higher phenolic and flavonoid contents, particularly ferulic acid, naringin, and catechin. These phenomenon suggested that ZQ320 could alleviate the postprandial hyperglycemia via inhibiting the activities of α-glucosidase and α-amylase [α-amylase inhibitory capacity due to the hydroxylation or the existence of 2,3-double bond and C-4 ketonic group instead of other phenolics [α-glucosidase  and α-amylase  via van der Waals and hydrogen bond to suppress their enzyme activity; in addition, they concluded that the existence of OH groups in position 3 (ring C), 7 (ring A), 4′ and 5′ (ring B) was chiefly responsible for the α-glucosidase and α-amylase inhibitory effects of polyphenols. Moreover, highland barley has confirmed to exert excellent antioxidant capacity [α-glucosidase and α-amylase inhibitory effects. Taken together, this study demonstrated that the ZQ320 bound phenolics effectively inhibited the activities of α-glucosidase and α-amylase, which could play a promising role for the treatment of the postprandial hyperglycemia.In The IC50  of sampl-amylase . Similarhenolics . Rasoulihenolics  found thcapacity . It can 50) of free and bound extracts were both over 300 mg/mL (p < 0.05), demonstrating that the IR model was induced successfully. After supplementation with highland barley free and bound extracts, the glucose utilizations were dose-dependently ascended (p < 0.01). Then they reached to the highest at 150 mg/mL, whose glucose utilizations were all higher than positive control group (8.04 ± 0.31 mmol/L). Compared to the model group, the increase of glucose consumption in free extracts at 150 mg/mL were ranked as a decreasing order of ZQ320 (4.43-fold) > HQK (3.80-fold) > BQ (3.76-fold) > ZQ2000 (3.48-fold); while the bound fractions were ZQ320 (3.90-fold) > ZQ2000 (3.85-fold) > HQK (3.81-fold) > BQ (3.60-fold). There were obvious differences between free and bound fractions in ZQ320 and ZQ2000 varieties (p < 0.05), but were inverse to that of the BQ and HQK varieties (p > 0.05). Evidently, both free and bound phenolics of ZQ320 variety exhibited the strongest glucose consumption capacity in contrast with the other varieties (p < 0.05), indicating its hypoglycemic potential for the treatment of T2DM.IR is a state of hepatic, adipose, and muscle cells/tissues with declined responsiveness to consumption and utilization of glucose, which could ultimately trigger severe metabolic syndromes such as T2DM . The liv00 mg/mL , which ip < 0.05). After the intervention of highland barley extracts, the glycogen contents were concentration-dependently ascended and reached to the maximum at 150 mg/mL extracts. In concrete, the increments of glycogen level in free extracts at 150 mg/mL were listed as ZQ320 (1.17 times) > HQK (1.01 times) > BQ (0.84 times) > ZQ2000 (0.81 times) compared with the model group; while that of bound forms were ZQ320 (1.66-fold) > HQK (1.38-fold) > ZQ2000 (1.20-fold) > BQ (1.09-fold). There were notable differences between free and bound fractions among four highland barley varieties (p < 0.01). Consequently, both free and bound phenolics of ZQ320 variety showed the highest glycogen synthesis ability compared with the other varieties (p < 0.05), which was in great compliance with that of glucose consumption . When supplemented with Met (positive control), the G6Pase and PEPCK activities of cells were observably decreased by 0.40 and 0.55-fold, respectively, and GYS2 activity were increased by 0.38-fold (p < 0.05) compared with those of model group. After the intervention of free and bound highland barley phenolics, the activities of G6Pase and PEPCK were reduced dose-dependently, whereas GYS2 activity were obviously ascended in a concentration-dependent pattern. At 150 mg/mL of bound extract, the decrease of G6Pase (0.49 times) and PEPCK activities (0.64 times) and the enhancement of GYS2 activity (0.61-fold) exceeded those of Met-treated group (p < 0.05), respectively. Interestingly, the GYS2 activity in Met-treated cells was not equivalent to its G6Pase and PEPCK activities because of the mechanism of action of Met, which was in line with the glycogen accumulation and glucose consumption assays described above. Similarly, previous researchers also confirmed that ferulic acid, naringin, and catechin could favorably reduce blood glucose via promoting glycogen synthesis and glucokinase activity in the liver [G6Pase, PEPCK, and GYS2 play crucial roles in glucose homeostasis because they were major rate-limiting enzymes of gluconeogenesis (G6Pase and PEPCK) and glycogen synthesis (GYS2) . Deficiehe liver ,24,39.p < 0.05), while those of G6Pase, PEPCK, and GSK3β were significantly augmented. However, the administration of ZQ320 caused a remarkable restoration of these gene expressions in contrast with the model group, especially in ZQ320 bound phenolics treatment group. These results were well coincided with the expressions of these markers in Met-treated group. Taken together, the transitions of gene expressions might be due to the synergetic effects of phenolics such as ferulic acid, naringin, and catechin on IRS-1/PI3K/Akt pathway as well as GLUT4 in T2DM models [Evidently, the IRS-1/PI3K/Akt signaling pathway is a major pathway involved in the regulation of glucose and insulin metabolism; it is also highly associated with IR in T2DM . GLUT4 iM models ,23,24. SM models ,42. Fromp < 0.05) after in vitro gastrointestinal digestion, and the HepG2 cell uptake contents of naringin and protocatechuic acid in digested citrus samples were 448–1684 and 127–1040 μg/100 g DW, respectively. Tagliazucchi et al. [However, the present study did not consider intestinal absorption/metabolism, which would significantly change the proportions and types of phenolic compounds HepG2 cells would be exposed to. Therefore, the known bioavailability of the free and bound phenolics identified herein were discussed below. Achour et al.  observedi et al.  found thi et al. ,47. The i et al. . These fα-glucosidase (EC 3.2.1.20) from Saccharomyces cerevisiae, α-amylase (EC 3.2.1.1) from porcine pancreas, acarbose, p-nitrophenyl-α-D-glucopyranoside (pNPG), metformin (Met), and chromatographic grade of standards were purchased from Sigma Chemical Co. . HepG2 human liver cancer cells (ATCC HB-8065) were obtained from ATCC company . Fetal bovine serum (FBS) and other cell culture reagents were purchased from Gibco Life Technologies Co. . Glucose, glycogen, and bicinchoninic acid (BCA) protein assay kits were obtained from Nanjing Jiancheng Institute of Bioengineering . Human G6Pase, PEPCK, and GYS2 assay kits were purchased from Shanghai Changjin Biotechnology Co., Ltd. .Highland barley Zangqing 320 (ZQ320), Zangqing 2000 (ZQ2000), Dulihuang (BQ), and Heiqingke (HQK) were donated by Professor Yu Liu, in Tibet Tianyu Agricultural Technology Co., Ltd. . Gallic acid, Folin–Ciocalteu reagent, catechin hydrate, n = 3).The moisture content was measured according to the oven-dry method as explained by Guo et al. . Brieflyw/v) and re-dissolved in Milli-Q-water to procure free extracts. Residues obtained were digested with 2 mol/L NaOH and acidified to pH 2 with concentrated HCl, then extracted five times by ethyl acetate. The ethyl acetate fractions were reconstituted using Milli-Q-water to yield bound extracts. Both extractions were performed in triplicates and stored at −40 °C for further analysis.Phenolics from the samples were extracted as reported before with slight modifications . In brien = 3).The phenolic content was measured by modified Folin-Ciocalteu approach  with gal18 column  maintained at 35 °C and detected at 280 nm. The gradient of binary elution-phase  was 0–2 min 8–10% B, 2–27 min 10–30% B, 27–50 min 30–90% B, 50–51 min 90–100% B, 51–56 min 100% B, and 56–60 min 8% B at a flow rate of 0.8 mL/min. Phenolic profiles were qualified by comparing retention time and peak area of the corresponding pure compounds, and were expressed as milligrams per 100 g DW .The phenolic compounds were analyzed by high performance liquid chromatography-photodiode array detector  method with little modification , using aα-glucosidase and α-amylase inhibitory activities were measured as explained by Lordan et al. [α-glucosidase inhibitory assay, appropriately diluted samples and α-glucosidase (0.1 U/mL) were kept at 37 °C for 10 min. Afterwards, pNPG (5 mM) was added and incubated at 37 °C for 20 min. Absorbance was monitored instantly at 405 nm by DU 730 Nucleic Acid/Protein Analyzer . With regards to α-amylase inhibitory assay, diluted samples and α-amylase (1 U/mL) were kept at 37 °C for 10 min, before the addition of 1% starch solution and another incubation at 37 °C for 10 min. Then, dinitrosalicylic acid reagent was added and the mixture was kept in boiling water bath for 5 min to halt the reaction. The mixture was diluted by deionized water and absorbance was measured at 540 nm . Data were both assessed by median inhibition dose (IC50) and presented as mg/mL . The α-glucosidase and α-amylase inhibitory activities were calculated as followed: The n et al.  and Obohn et al.  with min4 cells/well and incubated for 24 h. Growth medium was displaced by fresh medium containing various concentrations of extracts after incubation, then cells were counted by methylene blue assay. Results were evaluated by median cytotoxic concentration (CC50) expressed as mg/mL .HepG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 4.5 g/L glucose, 10% FBS, and 1% antibiotic antimycotic solution. The cytotoxicity of extracts were performed by modified methylene blue assay as described before . Briefly4 cells/well for 12 h, then DMEM medium supplemented with 33 mM glucose, and 1.5 μM insulin was added and incubated for 24 h to establish IR model. Subsequently, DMEM medium was displaced by fresh medium containing Met (80 mM) or diluted extracts. After administration for 24 h, the glucose concentration in the medium was determined by glucose assay kit. The glucose utilization of cells was calculated by initial glucose content minus residual glucose content and expressed as mmol/L .The glucose consumption protocol was conducted as previously reported  using Me5 cells/well in a 6-well microplate. After treatment by samples (refer to n = 3).The glycogen accumulation protocol was conducted according to Xu et al.  and Met refer to , the celn = 3).The treatment, collection, and lysis of HepG2 cells were the same as β-actin primer was used as an internal control. Sequences of primers .After administrated by samples refer to , total R primers  were desp < 0.05. Dose-effect analysis was conducted using Calcusyn software 2.0 .Data were expressed as mean ± SD for triplicates. ANOVA and Tukey’s test analysis were performed by SPSS software 21.0 . Significance was determined at α-glucosidase and α-amylase activity in vitro. Furthermore, these phenolic extracts could motivate glucose consumption and glycogen accumulation in IR-HepG2 model through IRS-1/PI3K/Akt signaling pathway and GLUT4 translocation, thus maintaining glucose homeostasis. In conclusion, this study suggested highland barley as an effective candidate for the prevention and treatment of T2DM. Therefore, the underlying mechanism of action in vivo will be sufficiently explored in our future research.Highland barley is a whole grain rich in natural phenolic compounds, and most of these phenolics partitioned in bound form. Highland barley phenolics exerted hypoglycemic effect by preventing


Moreover, the hybrid-type asymmetric supercapacitor, constructed from the flower-like β-NiS positive electrode and active carbon negative electrode, exhibits an energy density of 42.12 Wh·kg−1 at a power density of 28.8 kW·kg−1.The process of energy storage in supercapacitors via the surface reaction of the electrode can lead to a significant perfecting of the electrochemical performance of supercapacitors, for developing the morphologies of materials and increasing the specific surface areas of the electrodes. Three-dimensional nickel sulfide (NiS) superstructures with nanomorphologies, viz. coral-like, urchin-like, flake-like, and flower-like, are synthesized through simple solvothermal methods, without any template. The nanostructured flower-like β-NiS demonstrates, not only a remarkable specific capacitance of 2425.89 F·g The general applications of the renewable energy, the rapid development of the electric vehicle industry, and the constructions of smart grids paved the way for the energy storage technology to become the key link in the process of energy development ,2,3. In 3S4 electrodes were successfully synthesized, and they exhibited a specific capacitance of 1797.5 F·g−1 (0.5 A·g−1) . Ov. Ov−1 at−1, which indicated that the β-NiS//AC supercapacitor showed a favorable electrochemical performance. The GCD curves of the β-NiS//AC supercapacitor are shown in −1, at various current densities (from 0.5 to 50 A·g−1), could be deduced. −1 at the power density of 1440 W·kg−1. Even at the high power densities of 2880, 5760, 14,400, and 28,800 W·kg−1, the energy densities of β-NiS//AC could still reach the values of 30.28, 19.62, 18.88, and 12.64 Wh·kg−1, respectively. Furthermore, the electrochemical performance of the β-NiS//AC supercapacitor was relatively better than that of various transition metal sulfide//AC asymmetric supercapacitors [As a measure for evaluating the electrochemical performance of an electrode, a two-electrode asymmetric hybrid-type supercapacitor a, with tpacitors ,35,36,37−1 at 1 A·g−1. The noticeable electrochemical performance of the flower-like β-NiS benefited from its large surface area and special pore structures. The capacitance retention of the flower-like β-NiS electrode was still 99.26% after 5000 cycles at 10 A·g−1. For hybrid-type asymmetric supercapacitors, the β-NiS//AC exhibited a capacitance of 32.90 F·g−1, with a maximum energy density of 42.12 Wh·kg−1 and a power density of 28.8 kW·kg−1, which had significantly exceeded those of previously reported transition metal sulfide-based supercapacitors. Therefore, this study might provide a new way to extend the research on advancing the charge storage performance of transition metal sulfide electrodes, for highly pseudocapacitive capacitors.In summary, β-NiS with coral-like, urchin-like, flake-like, and flower-like morphologies were successfully synthesized using a solvothermal method, and without any surfactant or templating agent. The structures and compositions of the β-NiS compounds with different morphologies were mainly characterized by the XRD, SEM, TEM, and BET measurements. The flower-like β-NiS possessed a high specific capacitance value of 2424.89 F·g