Objective: Yiqi Huazhuo Decoction (YD) reduces blood glucose, glycated hemoglobin, body weight, and insulin resistance in patients with type 2 diabetes mellitus (T2DM), but its exact mechanisms are unknown. This study investigated the therapeutic effects and mechanisms of YD on impaired insulin secretion in T2DM rats.Methods: T2DM rats were randomized to the model, YD-lo , YD-hi , positive drug (TAK-875), and healthy control groups. The rats underwent an oral glucose tolerance test (OGTT), glucose-stimulated insulin secretion (GSIS) test, and serum lipid measurements. High-fat and high-glucose-injured RIN-m5f cells were treated with YD (30 or 150 mg/mL) for 48 h. GPR40 and IP3R-1 expression levels were determined by immunofluorescence, qRT-PCR, and western blot.Results: Compared with the model group, the OGTT area under the curve (AUC) in the YD-hi group was decreased by 26.7%, the insulin release test (IRT) AUC in the YD-hi group was increased by 45.9%, and the GSIS AUC was increased by 33.9% (p < 0.05). Compared with the model cells, the insulin secretion after glucose stimulation in the YD-hi group was increased by 24.5%, similar to the TAK-875 group (23.1%) (p > 0.05). GPR40 and IP3R-1 mRNA in the model cells were decreased by 49.5% and 51.2% compared with the control cells (p < 0.05). In the YD-hi group, GPR40 and IP3R-1 mRNA levels were increased by 58.1% and 39.3% (p < 0.05), similar to the TAK-875 group. The changes in protein expression were similar to mRNA.Conclusion: YD promotes insulin secretion from pancreatic islet β-cell in T2DM rats by regulating the GPR40-IP3R-1 pathway, thereby reducing blood glucose. Diabetes mellitus (DM) is a chronic disease with a high incidence. According to the International Diabetes Federation (IDF) Diabetes Atlas (10th Edition), as of 2021, 537 million adults aged 20-79 (10.5%) have diabetes, and China has the largest number of diabetics in the world. By the end of 2017, the number of adults with DM had reached 116 million in China, accounting for 11.2% of the national population . The potType 2 diabetes mellitus (T2DM) is characterized by variable degrees of insulin resistance and deficiency, resulting in hyperglycemia . T2DM ac2+ from the endoplasmic reticulum (ER) through the phospholipase C (PLC) pathway under the stimulation of free fatty acids (FFAs) ) belongs to the G protein-coupled receptor family and is a natural receptor for medium- and long-chain fatty acids. GPR40 is mainly distributed in the cell membrane of islet β-cells in the human pancreas and is a transmembrane protein that is characteristically highly expressed in β-cells . GPR40 cs (FFAs) . Neverths (FFAs) . In addis (FFAs) . GPR40 cs (FFAs) . GPR40 is (FFAs) . The acts (FFAs) . Therefos (FFAs) .For centuries, herbal drugs have been widely used as complementary and alternative medicine (CAM) to treat chronic diseases such as diabetes. In recent decades, more and more diabetes patients (72.8%) have used herbs, dietary supplements, and other CAM therapies . CurrentAstragalus mongholicus Bunge (Fabaceae)), Atractylodes Rhizoma (Atractylodes lancea DC), Salviae Miltiorrhizae Radix et Rhizoma , and Radix Puerariae Lobatae (Pueraria montana var. lobata (Willd.) Maesen & S.M.Almeida ex Sanjappa & Predeep). The plant names have been checked with https://wfoplantlist.org/plant-list. YD has been used for a long time in treating T2DM with mildly impaired pancreatic islet function (in vitro that YD could improve insulin secretion by decreasing the expression of miR-124-3p and decreasing the inhibition of the PLC-B1/IP3R-1 pathway in INS-1 cells (Yiqi Huazhuo Decoction (YD) is a traditional Chinese medicine (TCM) preparation consisting of four herbs: Radix Astragali ; all were inspected based on the Pharmacopoeia 2015 edition and the enterprise standard TS-ZL-03. YD comprises Radix Astragali 30 g, Atractylodes Rhizoma 30 g, Radix Puerariae Lobatae 30 g, and Salviae Miltiorrhizae Radix et Rhizoma 20 g. After the medicines were crushed, 550 g of water was added for decoction for 1 h. The filtrate was adjusted to pH 7.0 using sodium citrate and sodium carbonate. The obtained YD had a crude drug content of 3 g/mL, determined by a colorimetric method. The YD liquid was added to pure water, placed in a freeze-drying box, pre-frozen at −50°C for 4 h in a medicinal vacuum freeze dryer , dried, sublimated at −20°C for 12 h, and dried at 32°C for 8 h. The YD freeze-dried powder was obtained, which was refrigerated at 4°C for later use. Before use, the powder was dissolved in the corresponding medium to the required concentration, centrifuged at 4000 ×g for 30 min to remove the precipitate, and the supernatant was used.Radix Astragali (lot no. 20108) from Gansu Province of China, Atractylodes Rhizoma (lot no. 20115) from Liaoning Province of China, Radix Puerariae Lobatae (lot no. 20117) from Jiangxi Province of China, and Salviae Miltiorrhizae Radix et Rhizoma (lot no. 20201) from Zhejiang Province were purchased from Zhejiang University of Traditional Chinese Pharmaceuticals Co., Ltd.  flow rate: 35 arbitrary units; auxiliary gas (N2) flow rate: 13 arbitrary units; purge gas flow rate: 0 arbitrary units; spray voltage: 2.8 kV (negative) and 3.2 kV (positive); S lens RF level: 50 V; auxiliary gas heater temperature, 300°C; scan mode: full MS; scan range: 80–1,200 m/z. The maximum injection time (IT) was 200 ms. The scan resolution was 70,000 FWHM (m/z/s). The automatic gain control (AGC) target was 1.0e6. The typical chromatographic fingerprint of the YD extract is shown in The chemical analysis of YD was carried out by ultra-high performance liquid chromatography-Q precision hybrid quadrupole orbitrap high-resolution mass spectrometry . UHPLC (Thermo Scientific Dionex Ultimate 3000) was controlled with the Chromeleon 7.2 software. The autosampler was set to 10°C and protected from light. The column was set to 40°C. A Waters ACQUITY UPLC BEH C18 Column  was used. The mobile phase consisted of A (0.1% formic acid) and B (methanol) at a flow rate of 0.3 mL/min, gradient elution: 0–4 min (4% B), 4–10 min (4%–12% B), 10–30 min (12%–70% B), 30–35 min (70% B), 35–38 min (70%–95% B), 38–42 min (95% B), 42–45 min (95% B) −4% B). The injection volume was 2 μL. The mass spectrometer Q-Orbitrap system was connected to the UHPLC system https://www.aaalac.org/) and the Institutional Animal Care and Use Committee (IACUC) guidelines.Thirty-two specific pathogen-free (SPF) 8-week-old male ZDF (fa/fa) rats, weighing 200–220 g, and eight SPF 8-week-old male ZL (fa/+) rats, weighing 200–220 g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. . The animals were housed in the SPF experimental rearing room at the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine. All animal studies were approved by the Institutional Animal Care Committee of Zhejiang University of Traditional Chinese Medicine (#2017A610167). All experiments were performed with humane care according to the 3R principles  and in aAll rats were acclimatized for 1 week. The ZDF rats were fed a high-glucose and high-fat diet  for 4 weeks, while the control ZL rats were fed a normal diet for 4 weeks. A fasting blood glucose ≥11.1 mmol/L was used as the success criterion for establishing T2DM rats. After successful modeling, the ZDF rats were randomly divided into four groups, with eight rats in each group. Referring to a previous study , the dosBefore the end of the experiment, the rats in each group were fasted for 12 h, euthanized with pentobarbital, and blood was collected by cardiac puncture. The abdominal cavity was quickly cut open, and the pancreas was taken out. Part of the pancreatic tissue was taken and fixed in a 10% formalin solution to prepare sections.After successful modeling, each rat was weighed weekly, blood samples were collected from the tail vein once a week, and the fasting blood glucose level was measured using glucose test strips.The OGTT test and IRT were conducted every 2 weeks. For the OGTT, the rats were fasted for 12 h, and the blood was collected from the tail vein. The glucose oxidase method  was used to test the plasma fasting blood glucose levels. At the same time, 0.2 mL of blood was collected, the serum was collected after centrifugation, and the fasting insulin levels were determined by ELISA. Each group was given the corresponding drugs or distilled water for 30 min to measure the blood glucose and insulin levels, then the rats were given 20% glucose by gavage at 10 mL/kg body weight, and the glucose dose was 2 g/kg. Blood glucose and insulin were measured at 30, 60, 120, and 180 min after glucose administration. The area under the curve (AUC) of blood glucose and insulin was observed at each time point after glucose administration in each group. The formula was AUC = (0 min + 30 min × 0.25 + (30 min + 60 min) × 0.25 + (60 min + 90 min) × 0.5 + (90 min + 120 min) × 0.5.For the GSIS, the rats were fasted for 12 h and anesthetized by an intraperitoneal injection of 3% pentobarbital (45 mg/kg) . The ratThe rats were sacrificed after fasting for 12 h. Blood was collected from the heart, and serum total cholesterol (CHOL) and triglyceride (TG) levels were detected using a U2700 automatic biochemical analyzer  using the manufacturer’s reagents and instructions.The rat pancreatic tissues were fixed in 10% formalin for 5 days. The specimens were dehydrated with ethanol, treated with xylene for 30 min, and placed in paraffin for 5 h. The paraffin blocks were sectioned at 4 μm thickness. The paraffin sections were dewaxed with xylene, rehydrated with ethanol at 100%, 95%, 85%, and 70%, stained with hematoxylin, treated with 0.2% ammonia, and stained with 0.5% eosin. After ethanol dehydration and xylene transparency, the sections were sealed with neutral gum. The histopathological changes in the pancreas were observed under light microscopy. The nuclei were blue, the cytoplasm was pink, and the erythrocytes were orange-red. The Schmidt pathology scale was used to perform the pathological score of pancreas . The scoThe paraffin sections were dewaxed and rehydrated with xylene I for 20 min, xylene II for 20 min, 100% ethanol I for 5 min, 100% ethanol II for 5 min, 95% ethanol for 5 min, 80% ethanol for 5 min, and PBS three times, 3 min/time. The sections were placed in 0.01 M citrate buffer (pH 6.0) for antigen retrieval in a microwave, cooled to room temperature naturally, and washed with PBS three times, 3 min/time. The primary anti-rabbit polyclonal GPR40 antibody  was incubated overnight at 4°C in a refrigerator, transferred to room temperature to equilibrate for 30 min, and washed with PBS three times, 5 min/time. Fluorescent secondary antibody donkey anti-rabbit antibody  was incubated at 37°C for 60 min, rinsed with PBS three times, 5 min/time. The nuclei were stained with DAPI  at room temperature for 10 min. After sealing with glycerol PBS sealer , the sections were observed using a laser confocal microscope .2 at 37°C with saturated humidity. The modeling was performed as previously described . The RINm5f cell line was inoculated in a 5.6 mmol/L glucose low-glucose DMEM medium , 10% FBS  with 1% penicillin/streptomycin and incubated in 5% COescribed . The mod7/L and inoculated in a 96-well plate. After the cells adhered, the serum-free medium was replaced, and the culture was continued for 12 h. The cells were treated as above for 48 h. Then, 10 μL of CCK8 detection solution  was added to each well and incubated at 37°C for 2 h in the dark. The optical density was measured at 450 nm with a microplate reader. Cell viability (%) = OD value of experimental group/OD value of non-drug group×100%. The experiment was repeated three times independently.The effect of different concentrations of YD on cell proliferation was determined by the CCK8 assay. YD freeze-dried powder was added in a gradient of crude drug content of 0.01%, 0.1%, 1%, 5%, 10%, and 100%. The optimum concentration and time  were screened for the subsequent experiments. The RIN-m5f cells in the logarithmic growth phase were adjusted to 2.5 × 10The cell supernatant after drug intervention was discarded, and the cells were incubated in a 5.6 mmol/L glucose medium for 1 h. Then, the supernatant was taken, and the basal insulin secretion (BIS) levels were measured by ELISA using an insulin ELISA kit . The cells were incubated in a 30 mmol/L glucose medium for 1 h, and the supernatant was taken. GSIS levels were determined by ELISA. The total protein of each well was extracted and expressed as unit mass insulin (insulin content per well/corresponding protein content).−ΔΔCt) represented the relative amount between groups.Total cell RNA was extracted using a TRIZOL RNA extraction kit , followed by RT-PCR amplification after reverse transcription. The primers were designed with Primer 5 software and synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. . The sequences are shown in ×g, 4°C). The protein concentration was measured using a BCA kit . The proteins were separated by SDS-PAGE and transferred to PVDF membranes . The membrane was blocked with 5% nonfat milk powder for 1 h at room temperature. Primary anti-rabbit monoclonal anti-GPR40 , anti-IP3R  and anti-GAPDH  were incubated overnight at 4°C. The membranes were incubated with HRP-conjugated secondary antibody goat anti-rabbit  for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence in the VersaDoc 4000 MP system . Optical density values were analyzed using the Image-Pro Plus 6.0 software  to determine the relative expression levels of GPR40 and IP3R proteins  and centrifuged for 20 min  or Dunnett’s-T3 post hoc test (uneven variance). p < 0.05 was considered statistically significant. SPSS 23.0  was used for statistical analysis. All graphs were drawn using Prism 6.02  and Illustrator CS6 .All data were shown as mean ± standard deviation. One-way ANOVA was used for comparison among multiple groups, with the least significant difference (LSD) Nine compounds were analyzed to obtain the characteristic spectra: salvianic acid A , protocatechualdehyde , chlorogenic acid (characteristic component of Atractylodis Rhizoma), puerarin (characteristic component of Puerariae Lobatae Radix), calycosin-7-O-β-D-glucopyranoside , salvianolic acid B , atractylenolide I (characteristic component of Atractylodis Rhizoma), astragaloside IV  and astragaloside II  were used for quantitative and qualitative analysis.The contents of salvianolic acid A, protocatechuic aldehyde, chlorogenic acid, puerarin, verbascoside-7-O-β-D-glucopyranoside, salvianolic acid B, atractylodes I, astragaloside IV, astragaloside IV in YD were 0.32 mg/g, 0.35 mg/g, 0.10 mg/g, 0.34 mg/g, 0.14 mg/g, 0.35 mg/g, 0.11 mg/g, 0.10 mg/g and 0.11 mg/g .p < 0.05). The YD-lo and TAK-875 groups (positive control) were similar to the model group (p > 0.05). Hence, YD appeared to prevent weight gain in the YD-hi group (p < 0.05). The YD-lo group was similar to the model group. These results indicated that YD-hi and TAK-875 could reduce FBG in rats , while the blood glucose levels in the YD-hi and TAK-875 groups were decreased more than in YD-lo group, and these two groups were similar (p > 0.05)  of the YD-lo, YD-hi, and TAK-875 groups were all decreased compared with the model group after 8 weeks of treatment ( > 0.05) ; Table 5p < 0.05), while the insulin release levels in the YD-hi and TAK-875 groups were higher than in YD-lo group, and these two groups were similar (p > 0.05)  in the YD-lo, YD-hi, and TAK-875 groups were increased compared with the model group after 8 weeks of treatment ( > 0.05) ; Table 6p < 0.05); the YD-lo group was 106.4% of the model group (p < 0.05), while those in the YD-lo group were lower than in the YD-hi and TAK-875 groups. The insulin secretion levels in the YD-hi and TAK-875 groups were close (p > 0.05) (p < 0.05), while YD decreased glucose and increased insulin levels . YD-hi had similar effects to TAK-875 (p > 0.05).The GSIS AUC values in the YD-hi and TAK-875 groups were 133.9% and 134.2% of the model group, respectively, which were significantly higher than in the model group (el group . The ins > 0.05) ; Table 7p < 0.05). The TG levels in the YD-lo and TAK-875 groups were 88.6% and 96.7% of the model group, respectively, higher than in the YD-hi group (p < 0.05). The CHOL levels in the YD-lo and TAK-875 groups were 93.4% and 98.8% of the model group, respectively, which were also higher than in the YD-hi group (p < 0.05)  .p < 0.05). A small amount of fat deposition was observed in the YD-hi group, and no acinar cell hyperplasia or telangiectasia was found. Partial fat deposition was seen in the YD-lo and TAK-875 groups, and a small amount of acinar cell dysplasia and telangiectasia were found. The pathological scores of YD-lo, YD-hi and TAK-875 groups were lower than those of the model group (p < 0.05), while those of YD-hi group were lower than those of TAK-875 group (p < 0.05)  .p < 0.05). The YD-lo and model groups showed the weakest GPR40 expression, accounting for 53.8% and 42.6% of the control group, respectively (p < 0.05). There were no significant differences between the YD-lo and YD-hi groups. Therefore, the increased insulin levels observed in the YD-hi group might be due to a higher expression of GPR40.The GPR40 protein was observed using immunofluorescence . The flup < 0.05), but YD at 0.1% and 0.01% had no effect (p > 0.05)  . Because > 0.05) . Therefop < 0.05), and were 123.1% and 172.6% of the model group in the TAK-875 group, respectively (p < 0.05), without significant differences between the YD-hi and TAK-875 groups. The levels of BIS and GSIS in the YD-lo (30 mg/mL YD) group were not significantly different from the model group (p > 0.05) (The BIS and GSIS levels of the YD-hi group (150 mg/mL YD) were 124.5% and 178.3% of the model group, respectively ( > 0.05) .p < 0.05), the YD-lo group was close to the model group (p > 0.05), and the TAK-875 group was higher than the YD-hi group (p < 0.05) (p > 0.05) (p < 0.05). There were no differences between the YD-lo and model groups. There were no differences in mRNA expression between the YD-hi and TAK-875 groups (p < 0.05) (The relative expression levels of the GPR40 mRNA in the YD-hi and TAK-875 groups were higher than in the model group ( < 0.05) . The pro > 0.05) . The rel5 groups . The tre < 0.05) .YD reduces blood glucose, glycated hemoglobin, body weight, and insulin resistance in patients with T2DM , but thein vitro.In a previous study, YD was found to significantly improve blood glucose, blood lipids, and body weight in patients with T2DM . YD alsoThe effects of YD, a natural plant compound decoction, were investigated on blood glucose, blood lipids, insulin secretion, and pancreatic GPR40 expression in T2DM rats. YD reduced blood glucose, improved glucose tolerance, and improved insulin secretion in the first phase of T2DM rats and partially restored the decreased expression of pancreatic GPR40 induced by high glucose and high fat. The effect of a high concentration of YD was close to that of the GPR40 agonist TAK-875. These results were supported by Nagasumi et al. , who fouIn vitro, the BIS of RINm5f rat pancreatic β cells and the early-phase insulin secretion function after glucose stimulation were impaired to varying degrees after high-glucose and high-fat injury, and intracellular GPR40 and IP3R-1 expressions were both decreased, as previously observed (in vivo and in vitro experiments were consistent.observed . In the observed . It indiTherefore, it could be hypothesized that high-glucose and high-fat injury inhibits the expression of GPR40 in the pancreatic islet β cell membrane, inhibiting the signal transduction of the GPR40-IP3R-1 pathway, resulting in decreased insulin secretion by β cells. YD could increase the expression of GPR40 and IP3R-1, leading to increased insulin secretion by islet β cells injured by high glucose and high fat.This study had limitations. YD is a complex mixture of four herbs and several active compounds. Whether a single compound or the additive or synergistic effect of several compounds is responsible for the effects is unknown. Although YD was shown to increase GPR40 and IP3R-1, how YD increases GPR40 and IP3R-1 could not be determined in the present study. Future studies using knockdown and overexpression of the upstream and downstream proteins involved in GPR40 and IP3R-1 signaling will be necessary. YD could increase the insulin secretion of pancreatic β-cells and reduce blood glucose through the GPR40-IP3R-1 signaling pathway. It can be hypothesized that the GPR40-IP3R-1 signaling pathway plays an important role in the impaired insulin secretion and pathogenesis of T2DM.


P < 0.05). In the groups negative for serum HBV-DNA (groups 3 and 5), the exosomal HBV-DNA levels were higher than the serum HBV-DNA levels . The exosomal and serum HBV-DNA levels were correlated in groups 2 (R2 = 0.84) and 4 (R2 = 0.98). The exosomal HBV-DNA levels were correlated with total bilirubin (R2 = 0.94), direct bilirubin (R2 = 0.82), and indirect bilirubin (R2 = 0.81) in group 5 . In patients with CHB and negative for serum HBV-DNA, exosomal HBV-DNA was detectable and could be used to monitor the treatment effects. Exosomal HBV-DNA could be used in patients with a high suspicion of HBV infection but negative for serum HBV-DNA.This study examined exosomal hepatitis B virus (HBV)-DNA levels in chronic HBV infection (CHB). Patients were grouped according to the European Association for the Study of the Liver classification . Exosomes were isolated, comparative analysis of exosomes and serum HBV-DNA. The HBV-DNA content was lower in exosomes than in serum for groups 1, 2, and 4 (all  China iPresently, diagnosing hepatitis B is mainly based on the HBV-DNA nucleic acid test, HBV serum marker test, liver function test, and other auxiliary tests . The latExosomes are small 40–200 nm) vesicles containing complex RNA, DNA, and proteins and are formed by the fusion and exocytosis of intracellular vesicles and plasma membrane through multiple vesicle bodies 0–200 nm ,6, firstBased on this assumption, this study aimed to examine the sensitivity of exosomal HBV-DNA levels and their correlation with serum biochemical indicators of HBV infection.22.1Serum HBV-DNA , immune indicators , and liver biochemical indicators  were determined in the serum samples of 39 patients at the Affiliated Hospital of North Sichuan Medical College. The study involved human participants and was reviewed and approved by North Sichuan Medical College-2020 NSMC, number 10. Written informed consent was obtained from all patients or their legal guardians when <18 years of age.The patients were grouped according to the European Association for the Study of the Liver 2017 Clinical Practice Guidelines on managing HBV infection   was used for NTA to determine the size and number of serum exosomes.2.4HepG2 cells  were cultured in DMEM with 10% fetal bovine serum without exosomes. The exosomes were fixed using 1 ml of 4% paraformaldehyde and stained with DIO (Dioc18(3)  at 2 mM in DMSO and diluted before use to 2 µM) for 20 min at 37°C. The exosomes were centrifuged at 1,000 rpm for 5 min, and the supernatant was removed. The exosomes were resuspended in DMEM and transferred to the HepG2 cells. After 24 h, the cell morphology was observed under fluorescence microscopy.2.5g. Next, 4 μl of MgCl2 and RNase and 5 μl of DNase I was added and incubated for 3–4 h at 37°C. Then, 200 μl of PEG8000 was added, mixed well, and incubated on ice for 40 min, followed by centrifugation at 12,000×g, 4°C for 7 min. The supernatant was discarded. Then, 400 μl of digestion solution was added, mixed, and added with 15 μl of proteinase K. After overnight digestion at 45°C, 400 μl of phenol chloroform isoamyl alcohol mixture (25:24:1) was added. The mixture was centrifuged at 14,000×g for 3 min. An equal volume of isopropanol was mixed with the supernatant, mixed, and centrifuged for 11 min at 16,500×g and 4°C. The supernatant was discarded, the pellet was washed with 70% ethanol, and resuspended in ddH2O after drying.First, 400 μl of HBV core particle DNA extract was added to the exosomes and incubated for 15 min at 37°C. The mixture was centrifuged for 3 min at 15,000×2.62O, and 2 μl of 1/100 diluted template DNA. The thermal cycling conditions were (1) 95°C for 10 min as an initial denaturation step and (2) 39 cycles of 95°C for 15 s and 60°C for 1 min. The absolute quantification of HBV-DNA was generated based on the Ct value with the set dissociation curve.An SYBR kit  was used for the fluorescence quantification of the exosomes. The primers for HBV-DNA were forward 5′-ACC GAC CTT GAG GCA TAC TT-3′ and reverse 5′-GCC TAC AGC CTC CTA GTA CA-3′. The reaction system included 12.5 μl of 2× UltraSYBR Mixture, 1 μl of each HBV-DNA primer 10 μM, 8.5 μl of ddH2.7P-values <0.05 were considered statistically significant.SPSS 23.0 (IBM) was used for statistical analysis. Continuous variables were presented as means ± standard error  or means ± standard deviation (other continuous variables) and compared with one-way analysis of variance; as the variances of the groups were not uniform, the Games-Howell method was used for pair-wise comparison among the six groups. In each group, exosomal HBV-DNA levels were compared with the serum HBV-DNA values using a non-parametric test. The correlations between exosomal HBV-DNA and the other clinical indexes were analyzed using Spearman’s correlation. Two-sided 33.1Patient grouping and characteristics are shown in Table S2. The groups are presented in Table S1.3.2Under electron microscopy, the serum exosomes showed an obvious saucer-like structure . The dia3.36 IU/ml in group 1, (3.13 ± 0.998) × 106 IU/ml in group 2, (1.02 ± 0.149) × 105 IU/ml in group 3, (5.08 ± 2.65) × 105 IU/ml in group 4, (3.10 ± 0.478) × 103 IU/ml in group 5, and 0 IU/ml in group 6. There were significant differences between each pair of groups , except for group 1 vs. group 2 and group 3 vs. group 4. The HBV-DNA content was lower in the exosomes than in the serum for groups 1, 2, and 4 but higher in the exosomes in groups 3 and 5  (The HBV-DNA copy numbers were (4.11 ± 1.17) × 10 < 0.05) . Therefo3.4P < 0.05) ) and 3.10 × 103 IU/ml ), respectively, showing that the former was exosomes and the latter was serum  and 5 . In groups 3 and 5, the exosomal HBV-DNA levels were higher than the serum HBV-DNA levels  . Figure as serum . These r3.5P < 0.05). Figure S1 presents the comparisons in each patient. All patients had higher serum HBV-DNA levels than the exosomal HBV-DNA. Still, some patients presented high exosomal HBV-DNA levels . ALT and AST levels indicate the degree of liver damage. Exosomes are derived from cells. Damaged and ruptured cells can release more exosomes. If the liver cells are not damaged, the release of exosomes should be limited, resulting in that HBV in the liver would not be related to the serum levels. The exosomal and serum HBV-DNA levels were correlated in groups 2 (R2 = 0.84) and 4 (R2 = 0.98). The results indicate that as serum HBV-DNA, exosomal HBV-DNA can represent the disease activity in patients with positive serum HBV-DNA. When the serum HBV-DNA is high , the serum has a larger volume to accommodate HBV-DNA than exosomes, and thus, more HBV-DNA is detected in the serum. Serum HBV-DNA in group 1 was 4.35–27.45 times that of exosomal HBV-DNA, while serum HBV-DNA in group 2 was 1.84–41.17 times that of exosomal HBV-DNA. When serum HBV-DNA levels were moderate, such as in group 4 (HBV-DNA > 2 × 103 IU/ml), the serum HBV-DNA levels were still higher than that of exosomes, and the difference was 1.73–40.80 times (P < 0.05). When serum HBV-DNA was negative, such as in groups 3 and 5 (HBV-DNA < 5 × 102 IU/ml), there was still virus replication even if the HBV-DNA in serum was undetectable, but exosomal HBV-DNA was detectable because of their key function in replication.In the HBV-DNA (+) group, only the exosomal HBV-DNA in groups 2 and 4 were correlated with the serum HBV-DNA, while there were no correlations in group 1 . 3.6R2 = 0.94) (R2 = 0.82) (R2 = 0.81) (P < 0.05). In the other groups, there were no correlations between exosomal HBV-DNA and liver function markers . Therefore, the results support the clinical usefulness of exosomal HBV-DNA levels for monitoring CHB patients with negative serum HBV-DNA.Since exosomal HBV-DNA could be clinically used in serum HBV-DNA-negative patients, the exosomal HBV-DNA levels were examined for correlation with liver function markers. The exosomal HBV-DNA levels were correlated with TBIL ( = 0.94) , DBIL (R = 0.82) , and IBI = 0.81)  in group4Serum HBV-DNA and ALT are routinely used as key clinical indicators to diagnose and evaluate patients with CHB ,18. The 5 and 3.10 × 103 IU/ml) were far higher than the detection limit of serum HBV-DNA (5 × 102 IU/ml) in groups 3 and 5, suggesting that exosomal HBV-DNA might be more sensitive than serum HBV-DNA. Exosomal HBV-DNA is more sensitive than serum HBV-DNA when serum HBV-DNA is negative. Interestingly, for patients whose serum HBV-DNA levels indicated a moderate-high replication level (>2 × 103 IU/ml) (group 4), the copy numbers of exosomal HBV-DNA (1.02 × 105 IU/ml) (group 3) were comparable to the exosomal HBV-DNA levels (5.08 × 105 IU/ml) (group 4) from the HBV-DNA-positive patients (P > 0.05). It suggests that the serum HBV-DNA levels might not reflect the true status of HBV replication in disease progression.Exosomes play crucial roles in communication among cells and might be diagnostic and prognostic indicators for various diseases ,24,25,26When serum HBV-DNA levels were high, there was no correlation between exosomal HBV-DNA and liver function-related indicators in group 1, while there were correlations in groups 2 and 4, which can be due to liver damage. On the other hand, when serum HBV-DNA is low, exosomal HBV-DNA has a certain correlation with liver function-related indicators, such as in the HBV-DNA-negative group (group 5). This study showed that exosomal HBV-DNA was positively correlated with TBIL, DBIL, and IBIL, indicating that the exosomal HBV-DNA levels could reflect liver injury to a certain extent. It should also be noted that ALT, an important biochemical indicator of liver injury, showed no correlation to the HBV-DNA levels in serum and exosomes.5 IU/ml and ALT is normal, there is a controversy about the use of antiviral treatment [6 IU/ml, i.e., 1.3 times higher than the exosomal HBV-DNA copy number in group 2. On the other hand, eight patients (HBeAg (+), serum HBV-DNA >2 × 103 IU/ml, and ALT > ULN) in group 2 absolutely met the guideline criteria for antiviral treatment, but their exosomal HBV-DNA levels were only 1.10 × 106 IU/ml, far lower than that of group 1. Based on this inference, antiviral therapy may be given to patients in group 1 when exosomal HBV-DNA is >3.13 × 105 IU/ml. Meanwhile, the patients in groups 3 and 5 did not meet the indications for clinical antiviral therapy. The average copy numbers of HBV-DNA in the exosomes and serum were 1.02 × 105 and 3.10 × 103 IU/ml, respectively, about 6–200 times higher in exosomes than in serum. Therefore, the lower limit of exosomal HBV-DNA could be >1.56 × 105 IU/ml for antiviral therapy. Exosomes could be used as a simple and non-invasive detection indicator for monitoring CHB [Combined with clinical diagnosis and treatment standards, when HBV-DNA is >2 × 10reatment . Comprehring CHB ,28,29.Recent studies showed that exosomes could regulate the immune response during HBV infection. Immune escape by down-regulating IL-12 to inhibit the activity of NK cells is one of the important reasons for persistent virus infection ,31. The 5in vivo. In serum HBV-DNA-negative patients, exosomal HBV-DNA were strongly and positively correlated with TBIL, DBIL, and IBIL.This study suggests that when a patient’s serum HBV-DNA is negative, exosomal HBV-DNA can accurately reflect the level of HBV replication


Furthermore, PcSA@Lip was able to accumulate selectively in tumors after intravenous injection with the fluorescence intensity ratio of tumors to livers was 4.1:1. The significant tumor inhibition effects resulted in a 98% tumor inhibition rate after PcSA@Lip was injected intravenously at an ultra-low PcSA@Lip dose (0.8 nmol g−1 PcSA) and light dose (30 J cm−2). Therefore, the liposomal PcSA@Lip is a prospective nanophotosensitizer possessing hybrid type I and type II photoreactions with efficient photodynamic anticancer effects.Phthalocyanines are potentially promising photosensitizers (PSs) for photodynamic therapy (PDT), but the inherent defects such as aggregation-caused quenching effects and non-specific toxicity severely hinder their further application in PDT. Herein, we synthesized two zinc(II) phthalocyanines (PcSA and PcOA) monosubstituted with a sulphonate group in the alpha position with “O bridge” and “S bridge” as bonds and prepared a liposomal nanophotosensitizer (PcSA@Lip) by thin-film hydration method to regulate the aggregation of PcSA in the aqueous solution and enhance its tumor targeting ability. PcSA@Lip exhibited highly efficient production of superoxide radical (O Cancer continues to serve as one of the deadliest diseases, causing millions of deaths every year ,2. Curre1O2) produced by the type II mechanism −, 281.0049; found 281.0060.Notably, 4-nitrophthalonitrile , sodium 3-mercapto-1-propanesulfonate , and K2CO3  and Zn(OAc)2  was heated to 100 °C in 30 mL n-pentanol, and then added 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) (0.5 mL). The reaction solution was stirred at 145 °C overnight. After a short cooling, the volatiles were removed with rotary evaporation in vacuum. The residue was purified by silica gel column chromatography using ethyl acetate (EA) and N,N-dimethylformamide (DMF) as eluent. The crude product was further purified by size exclusion chromatography using DMF as eluent to obtain compound PcSA . 1H NMR  δ 9.27–9.01 , 8.86–8.85 , 8.23–8.15 , 7.98–7.91 , 3.59 , 2.94 , 2.41–2.35 . HRMS (ESI): m/z calcd for C35H21N8O3S2Zn [M-Na]−, 729.0464; found 792.0496.A mixture of PTSA , phthalonitrile , K2CO3  was stirred in 10 mL DMSO at 100 °C for 48 h. The subsequent treatment procedure was consistent with that of PTSA to obtain the product PTOA . 1H NMR  δ 6.93–6.89 , 6.49–6.45 ,2.90–2.89 , 2.51–2.47 , 1.56–1.53 . HRMS (ESI): m/z calcd for C11H9N2O4S [M-Na]−, 265.0278; found 265.0288.A mixture of 4-nitrophthalonitrile , sodium 3-hydroxy-1-propanesulfonate , and K2CO3 , and Zn(OAc)2  was heated to 100 °C in 30 mL n-pentanol, and then added DBU (0.5 mL). The reaction was stirred at 145 °C overnight. The purification procedure was in accordance with PcSA to obtain compound PcOA . 1H NMR  δ 9.55–8.99 , 8.26–8.01 , 3.23–3.22 , 2.18–2.17 , 1.69–1.73 . HRMS (ESI): m/z calcd for C35H21N8O4SZn [M-Na]−, 713.0692; found 713.0762.A mixture of PTOA , phthalonitrile , K3 solution containing lecithin and cholesterol. The solution was dried by rotary evaporation under vacuum to form a thin film, and further evacuated for 1 h. Then, the thin film was sonicated with distilled water hydration for 30 min to form a liposome suspension. Then, it was sonicated at 4 °C for 15 min using an ultrasonic cell crusher and squeezed with 450 nm and 220 nm filter membranes at room temperature.PcSA@Lip was prepared using thin-film hydration method and then squeezed as previously reported . The molThe prepared nanoliposome was characterized using dynamic light scattering (Anton Paar Litesizer 500) and transmission electron microscopy (TECNAI G2 F20) .2∙− and 1O2 were monitored with the dihydroethidium (DHE) probe and the Singlet Oxygen Sensor Green (SOSG) probe, respectively. PSs  and DHE (50 μM) were dissolved in water containing 250 μg/mL ctDNA. Using water containing only DHE and ctDNA as control. Fluorescence spectra (excited at 510 nm) were recorded at different irradiated times after the mixtures were upon light irradiation . Consistent with the method used to detect O2∙−, SOSG and PSs were prepared in water at 4 μM and the aqueous solution containing only SOSG was used as control. The fluorescence spectra (excited at 488 nm) of the mixtures after being light irradiation  at different times were determined with an Edinburgh FL900/FS900 fluorescence spectrophotometer .The O2 for 10 min, and the temperature change was recorded with a near-infrared thermal imaging camera . Water was used as control.To measure the photothermal conversion ability of PcSA@Lip and PcSA, an aqueous solution of PSs  was continuously irradiated with a 680 nm laser at 0.5 W/cm2 atmosphere.Human hepatocarcinoma (HepG2) cells  were incubated in DMEM (GENVIEW) culture medium containing 10% fetal bovine serum, penicillin (50 units/mL), and streptomycin (50 mg/mL) at 37 °C under a humidified 5% CO4 cells per well) were hatched overnight at 37 °C under a 5% CO2 atmosphere in 96-well plate. Then, the cells were incubated with 100 μL of different concentrations of PSs (PcSA@Lip and PcSA) for 2 h in the dark, respectively. After removing the PSs, the cells were rinsed with PBS and re-incubated with 100 μL of culture medium, followed by illumination of 27 J/cm2 . Cell viability was determined by the colorimetric 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay as described previously after 24 h continued incubation without light.The cell phototoxicity assay was performed as previously reported . Briefly4 cells per dish) were inoculated at 37 °C under 5% CO2 and cultured overnight in confocal dishes. After 24 h the old medium was aspirated and the cells were incubated with PSs  for 2 h in a dark incubator. Cells were then rinsed with PBS and observed under a Leica TCS SPE confocal microscope. The fluorescence signal of PSs was collected at 640–750 nm upon 635 nm excitation. Images were processed with SPE ROI fluorescence analysis software.HepG2 cells (7.5 × 104 cells per well) were maintained overnight at 37 °C under 5% CO2 atmosphere in 96-well plates for cell apposition and growth. Then, cells were under different treatments: (i) incubation in medium containing PcSA@Lip ([PcSA] = 0.25 μM) for 2 h in the dark (ii) incubation in medium containing PcSA@Lip ([PcSA] = 0.25 μM) for 2 h in the dark followed by light irradiation  (iii) incubation in medium containing 0.25 μM PcSA for 2 h in the dark (iv) incubation in medium containing 0.25 μM PcSA for 2 h in the dark followed by light irradiation . HepG2 cells were stained with Calcein-AM/PI Double Stain Kit after the different treatments described above[HepG2 cells  were seeded at 37 °C under 5% CO2 atmosphere and cultured overnight on confocal dishes. After removing the medium, the cells were incubated in medium containing photosensitizer  under the same condition for 1.5 h, and then DCFH-DA (10 μM) as a ROS probe was added to co-incubated for 0.5 h. Finally, cells were rinsed with PBS. Under 488 nm excitation, fluorescence images were detected from 500 nm to 600 nm with and without light irradiation  using a confocal microscope (Leica). The assays for 1O2 and O2∙− were consistent with the ROS procedure, with SOSG probe and DHE probe concentrations of 5 μM and 20 μM, respectively.Notably, 2,7-dichlorofluorescein diacetate (DCFH-DA), SOSG, and DHE were used as fluorescent probes to detect intracellular ROS, 7 cells 200 μL) were inoculated subcutaneously on the right axilla of ICR mice (20–25 g).Hepatoma H22 cells were purchased from the China Center for Type Culture Collection ; Female ICR mice were purchased from Wushi Animal Co., Ltd. . All animal studies were carried out in compliance with guidelines of the Animal Ethics Committee of Fuzhou University (2023-SG-001), and also approved by the committee. To establish a subcutaneous tumor model, H22 cells . After in vivo imaging studies, mice were euthanized at 24 h post-injection. Tumors and main organs were collected from mice at 24 h after intravenous injection and their fluorescence imaging was quantified (n = 3).PcSA@Lip and PcSA  aqueous solutions were injected intravenously into mice with tumor sizes of 200 mm3, the mice were divided into six groups randomly (n = 5): (i) phosphate buffer saline (PBS), (ii) PBS followed by laser irradiation , (iii) PcSA, (iv) PcSA followed by laser irradiation , (v) PcSA@Lip, (vi) PcSA@Lip followed by laser irradiation  . The mice in the (ii) (iv) (vi) group were treated with laser irradiation  at 8 h post-intravenous injection. The tumor size was measured with calipers every other day for 14 d and calculated using the following formula: volume = (tumor length) × (tumor width)2 × 0.5. The relative tumor volume was calculated as Vt/V0 .After the tumor volume reached 100 mm50 value as low as 0.16 ± 0.04 μM. In addition, PcSA@Lip demonstrated significantly enhanced tumor accumulation and improved photodynamic antitumor efficacy in H22 tumor-bearing mice with a tumor inhibition rate of up to 98.0% after intravenous injection. Therefore, the liposomal PcSA@Lip is a prospective nanophotosensitizer possessing hybrid type I and type II photoreactions with efficient photodynamic anticancer effects.In summary, we successfully synthesized two monosubstituted sulfonate-modified zinc(II) phthalocyanines (PcSA and PcOA), among which PcSA with an “S” bridge linkage displayed superior ROS generation capability. To improve the hydrophilicity and tumor targeting, we prepared PcSA as a liposomal nanophotosensitizer (PcSA@Lip) by a facile thin film hydration method. PcSA@Lip significantly inhibited the aggregation behavior of PcSA in water, and enhanced the ROS production generated by type I and type II mechanisms. PcSA@Lip exhibited increased cellular phototoxicity with the IC


Haemonchus contortus excretory-secretory proteins (HcESPs) that interact with host Th9 cells. In the present study, the effects of the Haemonchus contortus ribosomal protein L6 domain DE-containing protein (HcL6) on IL-9 secretion, Th9 differentiation, and IL-9 transcription were assessed by employing ELISA, flow cytometry, and qPCR methodologies. The observations revealed the transcriptional upregulation of several key genes within the Th9 immune response pathway. Moreover, silencing STAT6, PU.1, and NF-κB was found to attenuate the Th9 immune response. In this study, we unveiled the Th9 immune response-inducing capabilities of HcL6 and elucidated some of its underlying mechanisms. These findings suggest that HcL6 is an immunostimulatory antigen capable of inducing the Th9 immune response. These insights could prove instrumental in identifying potential candidate antigens for the development of immunoprophylactic strategies against H. contortus infections.Th9 cells play a crucial role in parasite immunity. The development of Th9 cells is facilitated by several cytokines. Key transcription factors, such as STAT6, STAT5, and PU.1, are known to enhance IL-9 expression during the Th9 immune response. NF-κB-mediated transduction pathways participate in the induction of IL-9. In a previous study, we unveiled a unique ribosomal protein derived from The online version contains supplementary material available at 10.1186/s13567-023-01214-5. H. contortus excretory-secretory proteins (ESPs) have not been elucidated.IL-9 is a typical cytokine produced by Th9 cells. Several studies report that IL-9 and Th9 immune responses play protective and central roles in parasitic nematode infections –4. HowevIRF-4, PU.1, TGF-β1 and Smad3 genes implicated in the TGF-β/Smad signaling pathway were significantly increased when the concentration of ESPs was greater than 10 μg/mL compared with levels in the control group. Th9 cell development requires transcription factors downstream of IL-4, including STAT6, IRF4, and GATA3 [A cytokine pool in the culture conditions is necessary to differentiate Th9 cells. The main cytokines that promote Th9 cell differentiation are IL-4 and TGF-β, as indicated in our previous study . The trand GATA3 , 10. Furnd GATA3 , 12. T cnd GATA3 . CGRP prPlasmodium berghei ANKA, protects the host against malaria by acting as an optimal antigen for generating hepatic TRM cells. In a single-dose vaccination targeting RPL6, sterilizing immunity was effective and prolonged against high-dose sporozoite challenges [Ribosomal proteins perform other functions independent of the translation machinery in addition to their role in ribosome biogenesis. Previous findings have indicated that PbRPL6120-127, a putative 60S ribosomal protein L6 (RPL6) in allenges . HoweverH. contortus excretory-secretory proteins (HcESPs) interacting with goat Th9 cells were identified by liquid chromatography-tandem mass spectrometry (LC‒MS/MS) analysis. H. contortus ribosomal protein L6 domain DE-containing protein (HcL6) was detected among the proteins interacting with Th9 cells [In a previous study, 218 h9 cells . ResearcThe treatments of animals in our research conformed with the guidelines of the Animal Welfare Board of Nanjing Agricultural University, China. The protocols were approved by the Animal Ethics Committee of Nanjing Agricultural University, China. The Science and Technology Agency of Jiangsu Province authorized the trials  2010–0005).Wistar rats  were obtained from the Experimental Animal Center of Jiangsu, China (SCXK 2018–004), and were fed in the Animal Experimental Laboratory of Nanjing Agricultural University under aseptic conditions.Five- to seven-month-old goats were reared in ventilated cages under clean and hygienic conditions to prevent accidental nematode infections. They were also provided with hay, water and alfalfa pellets at all times. During the study, no nematode eggs were found in the goat feces. As described previously, peripheral blood mononuclear cells (PBMCs) from goats were isolated and cultured using peripheral venous blood samples (10 mL per goat) obtained by venipuncture .H. contortus adult worms using TRIzol reagent  [Total RNA was extracted from rk, USA) . The RNAE. coli BL21 (DE3) and incubated in Luria-Bertini medium with kanamycin at 37 ℃ until the OD600 ranged from 0.6 to 0.8. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to induce protein expression. The His-tag-fused recombinant proteins were purified with a His Trap™ FF kit  following the manufacturer’s protocol. A ToxinEraser™ Endotoxin Removal kit  was used to remove endotoxin from the sample. Afterwards, 0.22-µm filters were used to filter the rHcL6. A BCA method was used to determine the protein concentration (776.09 μg/mL) [The expression of recombinant HcL6 protein was performed as described . In brie9 μg/mL) , and the23) was determined by indirect enzyme-linked immunosorbent assay (ELISA) as previously described [Wistar rats were given 300 g of rHcL6 protein blended with Freund’s complete adjuvant  to generate polyclonal antibodies. After the primary immunization, the rats were boosted 4 times with the same dose of rHcL6 protein with Freund’s incomplete adjuvant  at one-week intervals. In rat sera, the specific IgG titer  at 37 ℃ for 1 h to block the nonspecific binding sites. The blots of rHcL6 samples were treated with rat anti-rHcL6 serum (1:300 in TBST) or normal rat serum (control) overnight at 4 ℃. After the membranes were washed five times in TBST, horseradish peroxidase (HRP)-conjugated rabbit anti-rat IgG secondary antibody (Sigma‒Aldrich) in TBST (1:5000) was incubated with the membranes for 1 h at 37 ℃. Finally, immunoreactions were detected by a DAB kit (Sigma‒Aldrich) with 3–5 min of colour development.2 incubator. Following that, phorbol myristate acetate  and ionomycin  were added to cells containing brefeldin A solution  for another 4–6 h before intracellular dyeing was performed [Total Th9 cells in goat PBMCs were sorted by the flow-sorting cytometry method as described elsewhere . Brieflyerformed . Then, terformed . The Th9The collected Th9 cells were subjected to three washes using PBS (pH 7.4). Subsequently, Tide Quencher™ 3WS acid, Tide Quencher™ 2WS acid, and Tide Quencher™ 5WS acid  were introduced to quench fluorescence. This process was conducted over 5 h until no residual fluorescence was detectable. After several additional washes, the cell suspensions were loaded onto adhesion microscope slides and allowed to settle for 15–20 min before the supernatant was discarded. The Th9 cells were fixed using 4% paraformaldehyde (PA) for 15 min at room temperature. This step was followed by incubation with 5% bovine serum albumin (BSA) in PBS, with added 0.1% Tween-20 (PBST), at 37 ℃ for 30 min. The slides were then incubated overnight at 4 ℃ with rat anti-rHcL6 IgG (1:300) or normal rat IgG (serving as a control). Following three washes with PBST, Cy3-labelled goat anti-rat IgG (1:500) was applied to the slides and incubated for 1 h at 37 °C. Three additional PBST washes preceded the nuclear staining with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride  for 5 min at room temperature. An anti-fade medium (Sigma‒Aldrich) was utilized to prevent fluorescence quenching of the samples prior to microscopic examination. The final stage involved imaging the slides using an LSM780 laser scanning confocal microscope  at 100 × magnification. Digital images were processed and analysed using ZEN 2012 software (Zeiss).6 cells/mL) were incubated with various concentrations of rHcL6  in vitro in 24-well culture plates supplied with RPMI-1640 medium for 24 h at 37 ℃. Subsequently, RNA was extracted from these cells using a Total RNA Kit  according to the manufacturer’s instructions. The mRNA transcription of IL-9 was assessed as described elsewhere [−∆∆Ct method [The transcription of IL-9 was detected by real-time PCR. PBMCs (1 × 10lsewhere . Specifi6 cells/mL) were stimulated with lipopolysaccharide . Two hours following stimulation, 2 mL of cells (1 × 106 cells/mL) was added to each well of a 6-well plate. Then, the cells were incubated with different concentrations of rHcL6 to determine whether the protein could stimulate IL-9 secretion. All the samples were incubated at 37 ℃ and 5% CO2. After 24 h of incubation, the supernatants were collected by centrifugation. The IL-9 concentration in the supernatants was measured by ELISA. Different concentrations of IL-9 protein  and 100 μL of each cell supernatant were placed into a 96-well plate containing IL-9 monoclonal antibody at 37 ℃ for 90 min. After removing the supernatant, rat anti-IL-9 IgG polyclonal antibodies coupled with biotin (1:200) were added to each well at 37 ℃ for 1 h. After being washed three times with TBST, the avidin–biotin-peroxidase complex was added at 37 ℃ for 30 min. After three washes with TBST, TMB was added for 15–20 min in the dark, and then ELISA stop solution was added to terminate the reaction. The optical density at 450 nm was measured using a microplate reader . Three independent experiments were performed in the test with three technical replicates for each group.ELISA was used to examine the effects of rHcL6 on IL-9 secretion. PBMCs  in vitro in 12-well culture plates supplied with RPMI-1640 medium for 12 h at 37 ℃ with 5% CO6 cells/mL) were incubated with 20 μg/mL rHcL6 in vitro in 24-well culture plates supplied with RPMI-1640 medium for 24 h at 37 ℃. Subsequently, the method was the same as that described in the subsection “Detection of IL-9 transcription”. The primers for these nine key genes are listed in Table This study investigated the transcription of key genes in Th9 immune response signaling pathways, such as STAT1, STAT5, GATA3, SMAD, NFAT, IRF4, PU.1, STAT6 and NF-ĸB, by real-time PCR. PBMCs  of STAT6/NF-ĸB/PU.1 siRNA were procured Table . Transfe*p < 0.05, **p < 0.01, and ***p < 0.001. Flow cytometry data were analysed employing FlowJo software .Statistical analyses were carried out using GraphPad Prism 7.0 . All data are presented as the mean ± SD. Differences between groups were evaluated by one-way ANOVA followed by Tukey’s post hoc test. The thresholds for statistical significance were set at The complete coding sequence of the HcL6 gene (567 bp), encoding a protein composed of 189 amino acids, was cloned from adult worms through polymerase chain reaction (PCR) , 3.59 (p < 0.0001) and 6.94 (p < 0.0001), respectively, when compared with the blank control , while those treated with 5 μg/mL  or 10 μg/mL  did not show significant differences when compared with levels in the control group  , IRF4 , PU.1 , SMAD , STAT1 , STAT6 , STAT5  and NF-ĸB  compared to those in the blank control group . Nevertheless, NFAT exhibited no significant changes  compared with that in the blank control group  . The introduction of increasing concentrations of STAT6 siRNA-2  into peripheral blood mononuclear cells (PBMCs) led to a decrease in STAT6 transcription. The relative mRNA levels in the experimental groups were reduced to 0.72 (p > 0.05), 0.40 (p < 0.001), 0.53 (p < 0.01), and 0.68 (p < 0.05) at 10, 20, 40, and 80 nM, respectively, when compared to levels in the control  , IRF4 (p < 0.01), and SMAD (p < 0.001) upon adding 20 nM siRNA-2/STAT6. However, NFAT, PU.1, STAT1, STAT5, and NF-κB did not show significant alterations compared to the control  compared to those in the control  . Increasing concentrations  of NF-κB siRNA-1 transfected into PBMCs resulted in a decrease in the transcription of NF-κB. The relative mRNA fold changes in the groups were reduced to 0.52 (p < 0.0001), 0.21 (p < 0.0001), 0.39 (p < 0.0001) and 0.43 (p < 0.0001) for NF-κB transcription at 10, 20, 40 and 80 nM, respectively, when compared with that in the control  , IRF4 (p < 0.01), STAT6 (p < 0.05) and STAT5 (p < 0.001) were observed after the addition of 20 nM siRNA-1/NF-ĸB compared to the control (ns siRNA). Nevertheless, NFAT, PU.1, SMAD and STAT1 had no significant effects compared with the control (ns siRNA)  when compared with that of the control   Figure B. It wasp < 0.0001). Increasing concentrations  of PU.1 siRNA-1 transfected into PBMCs resulted in a decrease in the transcription of PU.1. The relative mRNA fold changes in the groups were reduced to 0.53 (p < 0.01), 0.52 (p < 0.01), 0.28 (p < 0.0001) and 0.38 (p < 0.001) for PU.1 transcription at 10, 20, 40 and 80 nM, respectively, when compared with the level of the control  , IRF4 (p < 0.01), SMAD (p < 0.0001) and STAT6 (p < 0.05) compared to that of the control (ns siRNA). Nevertheless, NFAT, STAT1 STAT5 and NF-κB had no significant effects compared with the control (ns siRNA)  when compared with that of the control   Figure B. It was) Figure C. We tes) Figure D.Figure The excretory-secretory products (ESPs) released by parasitic helminths promote infection and modulate host immune responses –24. It iIL-9-producing Th9 cells play a role in enhancing immunity against parasites and cancer but are highly affected by allergic disease and colitis. The CD4 + T-cell subset can produce IL-9 alone or IL-9 in combination with IL-10 but not IL-4, IL-5 or other cytokines , 26. TheSeveral cytokines promote Th9 cell development. In addition to TGF-β and IL-4, transcription factors such as NF-κB, NFAT, STAT6, IRF4, STAT5, PU.1, SMAD, GATA3 and STAT1 are required for Th9 cell development , 28, 29.The Janus kinases JAK1 and JAK3 are activated when IL-4 induces IL-4Ra. Subsequently, STAT6 is recruited and phosphorylated, and genes that regulate Th2 cell development are expressed . NotablyTGF-β-induced expression of PU.1 is required for IL-9 production . PU.1 suRelA, c-Rel, RelB, NF-κB1 (p50 and its precursor p105) and NF-κB2 (p52 and its precursor p100) are members of the mammalian Rel/NF-κB transcription factor family. These proteins play important roles in the immune system by regulating the development, survival, immune responses, and malignant transformation of lymphocytes and lymphoid organs. The p50-RelA and p52-RelB proteins are implicated in the NF-κB signaling pathway . The NF-In this study, we employed small interfering RNA (siRNA) to investigate the transcription levels of pivotal genes in Th9 immune response signaling pathways associated with STAT6/NF-κB/PU.1 synthesis. The outcomes showed that following treatment with siRNA, the expression levels of GATA3, IRF4, SMAD, STAT6, and PU.1 were downregulated. These findings suggest that HcL6 may stimulate the Th9 immune response via activation of the IL-4/STAT6/IRF4-GATA3 and TGF-β/PU.1 signaling-mediated transduction pathways Figure .Figure 8Additional file 1: Sequence of the HcL6 gene. The complete coding sequence of the HcL6 gene (567 bp).


Breakfast cereals (n:27), cornmeal (n:41), extruded maize (n:32), and oatmeal (n:40) purchased from these alternative shopping areas created to meet the food needs of low-income people in the suburbs formed the sample set of the study. These foods, which are sold in areas that are out of legal control and greatly affected by external environmental conditions, are more open to health risks. Mycotoxins, chemicals of a biological origin, are some of the most important of these risks. In terms of public health, it is important to investigate the presence of mycotoxins in foods, which can cause acute and chronic diseases such as immunosuppression, genotoxic, estrogenic, teratogenic effect, cancer, and liver and kidney dysfunctions. Grain-based foods are often contaminated with a large number of mycotoxins, but legal regulations have not been prepared that consider the health risks associated with the co-existence of mycotoxins. Many of the studies have focused on the presence of a single mycotoxin and the risks it poses. As a result, aflatoxin B1 levels in 28.57% of the samples and total aflatoxin (B1 + B2 + G1 + G2) levels in 26.43% of the samples were determined to exceed the limits defined in the “Turkish Food Codex Contaminants Regulation”. Citrinin could not be detected in any of the samples. The rate of mycotoxin occurrences above the limit of detection (LOD) in grain-based food samples ranged from 22.86% to 99.29%. Total aflatoxin (TAF) + Total Fumonisin (FUM) were found in 83.57% of the samples; TAF + FUM + OTA in 82.14%; TAF + FUM + OTA + T-2 in 44.29%; TAF + FUM + OTA + DON + HT-2, TAF + FUM + OTA + DON + T-2, and TAF + FUM + OTA + DON + ZEN in 22.86% of the samples.In this study, a total of 140 cereal-based foods sold in temporary open-air markets were analyzed by LC-MS/MS for aflatoxin B Today, the demographic structure of cities is changing with intense internal and external migration. Demands vary depending on culture, habits, and income level of people in cities with increasing populations. Alternative shopping areas are created in cities, especially in suburbs, to meet the food needs of people of different socio-economic levels . Open-aiProduction of mycotoxins in cereals is largely dependent on factors such as pre- and/or post-harvest temperature, moisture content, and insect damage. Climate is the main determinant of fungal formation and mycotoxin production ,5,6. SomMany countries have made important regulations on “acceptable health risks” to control mycotoxin contamination in foods and prohibit the trade of contaminated products. These regulations generally depend on a country’s level of economic development, the rate of consumption of high-risk products, and the susceptibility of crops to contamination ,10. TherMost of the studies in the literature ,13,14,151, B2, G1, G2, ochratoxin, zearalenone (ZEN), deoxynivalenol (DON), fumonisin B1 (FB1), fumonisin B2 (FB2), citrinin (CIT), and HT-2 and T-2 toxins) in cereal-based foods with an economical, fast, and reliable LC-MS/MS technique.This study aimed to determine the presence, amount, and co-occurrence of multi-mycotoxins , extruded maize snacks (n:32), maize-based breakfast cereals (n:27), and oatmeal (n:40)) samples were purchased from temporary open-air markets in suburbs of İstanbul. Samples were collected in nylon bags (100 g) from May 2022 to August 2022. The samples were delivered to the laboratory in their original packages and were stored at 7 °C until analysis.As part of our research, we took a closer look at the techniques utilized by Kim et al. , De SantThe extraction solution was prepared by mixing 800 mL of methanol  with 200 mL of LC-MS-grade water . The mobile phases were constituted of 0.252 g of ammonium formate  in 1 L of LC-MS-grade water containing 1 mL formic acid (mobile phase A) and 0.252 g of ammonium formate in 1 L of LC-MS-grade methanol containing 1 mL of formic acid  (mobile phase B).g for 5 min, passed through a 0.45 µm filter  and collected in a 15 mL centrifuge tube. The filtrate was transferred into a 1.5 mL vial, with a volume of 800 µL. After adding 200 µL of mobile phase A, the solution was analysed. Two replicates were performed in one day.The samples were homogenised in the grinder . Five grams of the samples were weighed. The weighed samples were mixed with 12 mL of the extraction solution for two hours while shaken. At the end of the period, the samples were centrifuged at 3000× −1 and the injection volume was 20 µL. The LC flow was directed into the MS detector for between 1.0 and 7.0 min. Delta EMV for MS analysis: 400(+), 400(−); Gas Temperature: 350 °C; Gas Flow: 11 L·min−1, Nebulizer: 30 psi; and Capillary: 3500 V (+), 2500 V (−). Optimised parameters are summarised in LC-MS/MS analysis was performed using an Agilent 6420 Triple Quad/G6420A LC-MS system. Chromatographic separation was performed on an Athena C18-WP column   at a column temperature of 35 °C. A gradient program was set up as follows: 0–3 min with 75% A, 25% B, 3.0–5.0 min linear gradient down to 30% A, 70% B; hold at 0% A for 4 min; return to 100% A in 0.1 min . The flow rate was 0.35 mL·min1, AFB2, AFG1, AFG2, OTA, FB1, and FB2 were purchased from the Pa group ; DON, ZEN, CIT, T-2, and HT-2 were from Trilogy  in liquid form 1000 mg·L−1 and they were stored at 4 °C. The standard solutions for AFB1, AFB2, AFG1, AFG2, and OTA were diluted 0.1–1–2.5–5–10 mg·L−1; for ZEN, HT-2 toxin, T-2 toxin; for CIT 1–5–10–25–50–100 mg·L−1; for FB2 25–50–125–250–500 mg·L−1; and for DON and FB1 50–100–250–500–1000 mg·L−1. The LC-MS/MS method was validated to investigate performance characteristics such as linearity, the limit of detection (LOD), the limit of quantification (LOQ), and accuracy, according to the European Commission [1, AFB2, AFG1, AFG2, FB1, FB2, T-2, HT-2, OTA, DON, and ZEN under optimised LC-MS/MS conditions. External calibration was performed. To ensure the peak measurements’ accuracy, we ensured the signal area was at least three times larger than the noise area. During the LOD and LOQ studies, oatmeal, wheat flour, maize flour, and breakfast cereals containing oats were used. We conducted a blank sample analysis to verify that no mycotoxins were present in the samples. Upon observation, 100 μg·kg−1 for DON; 0.3 μg·kg−1 for AFB1 and AFB2; 0.4 μg·kg−1 for AFG1 and AFG2; 0.8 μg·kg−1 for OTA; 25 μg·kg−1 for FB1; 60 μg·kg−1 for FB2; 100 μg·kg−1 for CIT; and 10 μg·kg−1 for ZEN, T-2, and HT-2 spiking levels were performed. Spiking was performed to determine LOD and LOQ. This study was conducted by two different technicians . Ten measurements with two replicates were taken for each mycotoxin in matrices. The standard deviation (SD) of the measurement data, LOD, and LOQ were calculated using specific Formulas (1)–(3).LOD: limit of detection;LOQ: limit of quantification;SD: standard deviation;xmean: the arithmetic mean of the data;xi: each value of the dataset;n: the total number of data.Mycotoxin standards of AFBmmission . Linear For the determination of measurement uncertainties, ISO  and Elli1 and 26.43% in terms of total aflatoxin.The results for our cereal-based products are presented in 1, 45% with AFB2, 36.43% with AFG1, and 40.71% with AFG2 at levels above the detectable limits. While this rate was 22.86% for FB1, contamination with FB2 was detected in 90% of samples. On the other hand, 87.86% of the samples were contaminated with OTA, 44.29% with T-2, 40.71% with HT-2, and 23.57% with ZEN and DON. CIT was not detected in any of the samples.It was determined that 74.29% of the samples were contaminated with AFB1 was calculated as 2.01 ± 1.6 µg·kg−1, and the median was determined as 1.65 µg·kg−1. The mean AFB2, AFG1, and AFG2 values of the positive samples were calculated as 2.39 ± 0.18 µg·kg−1, 2.41 ± 0.07 µg·kg−1, and 2.29 ± 1.84 µg·kg−1, respectively. The positive samples’ mean TAF concentration was 3.79 ± 4.14 μg·kg−1.The mean concentration of the samples with AFB1 was calculated as 146.17 ± 7.75 µg·kg−1, and the median was determined as 142.46 µg·kg−1. The mean FB2 concentration was 97.97 ± 58.02 µg·kg−1 and the median was 59.99 µg·kg−1.The mean concentration of the samples with FB−1, 171.26 ± 2.63 µg·kg−1, 16.41 ± 0.34 µg·kg−1, and 35.17 ± 8.65 µg·kg−1, respectively. None of the samples’ T-2 values were above the LOQ. The positive samples’ median concentration of that were 1.30 μg·kg−1, 170.64 μg·kg−1, 16.32 μg·kg−1, and 38.91 μg·kg−1.OTA, DON, ZEN, and HT-2 values of the positive samples were calculated as 1.3 ± 0.002 µg·kgIn our study, multiple mycotoxin analyses in cereal-based foods (n:140) were performed using the liquid chromatography–tandem mass chromatography method, which provides a fast, reliable, and economic analysis (n:140). LC-MS/MS is mostly preferred for mycotoxin analysis due to its high selectivity and sensitivity. According to a report by Amirahmadi et al. , the mat−1 for AFB1 and more than 4 µg·kg−1 for total aflatoxin [The EU stated that no direct human consumption product should be present at levels higher than 2 µg·kgflatoxin ,26. The flatoxin .1 and TAF. On the other hand, 95% and 90% of oatmeal samples were found to be above the limit values for AFB1 and TAF, respectively. The sample rate exceeding the AFB1 and TAF limit values in extruded maize snack products was determined as 3.13%. However, 3.7% of maize-based breakfast cereal products exceeded the AFB1 limit while none of the samples exceeded the TAF limit value.In our study, it was observed that none of the cornmeal samples exceeded the limit value reported in TFC for AFB−1 of aflatoxin in their study on 60 cornmeal and corn grain samples. Similarly, Zhou et al. [2 in the range of 0.67–1.3 μg·kg−1 in 16 corn samples they examined. In another study, the mean AFB2 level was measured in 79 different types of corn samples, with very little difference, but still close (1.5 μg·kg−1) [There are many studies in the literature about the presence of aflatoxins in corn and oat-based samples. Alborch et al.  found thu et al.  also detμg·kg−1) . When thμg·kg−1) ,30,31,32μg·kg−1) ,34,35.1, while other aflatoxin types could not be detected. Juan et al. [1 was found in only 0.1% of the samples they analysed, while other types were not present. This has been associated with the correct functioning of the food safety system in the processing of products. In addition, in some studies, it has been reported that the total amount of aflatoxins in the oat-based samples examined was below the detection limits, in other words, no type of aflatoxins was found in these products [2 at the level of 1.1–2.6 μg·kg−1 and AFG2 at the level of 0.4–1.7 μg·kg−1 in 40% of the 20 breakfast samples they examined. Additionally, Kuzdralinski et al. [−1.In terms of oat-based products, there is a slight difference in the presence of aflatoxin compared to corn-based products. For example, in some studies, it is reported that the types of aflatoxins detected in oat-based products are more limited, but also their levels are lower. According to Ortiz et al. ’s study,n et al.  also staproducts ,39,40,41products  reportedi et al.  stated t1, FB2, and FB3, and FB1 is the most common type in foods, especially in corn and corn-based products. The fact that fumonisin B1 (average 146.17 μg·kg−1) was detected only in corn-based samples among the samples examined within the framework of our study is a finding that confirms this information. In the literature review, it is seen that the studies mostly focus on FB1. However, although it is not as strong as FB1 regarding toxicity potential, it is also known that if FB2 is present in the flora, it increases toxicity by showing a synergistic effect with FB1. Among the samples in this study, an average of 58–60 μg·kg−1 was detected in corn-based samples and an average of 166.55 μg·kg−1 in oat-based samples. Many studies in the literature also support our findings from corn products [Fumonisin, one of the most toxic types of mycotoxins known, unlike aflatoxins, multiplies by contaminating the developing crop prior to harvest, so that, generally, it is difficult to control or prevent its development. There are three types of fumonisin, FBproducts ,44. On tproducts ,42,45.A. ochraceus, A. steyni, A. westerdijkiae) and OTA, classified in Group 2, are the most known toxins for humans in this regard. The presence of these toxins, as with the toxins we mentioned before, is mostly affected by errors in agricultural practices, storage defects, insect attacks, genetic factors, and negative climatic conditions.In addition to the toxins we mentioned in corn and oat-based agricultural products, other toxins can be found. For example, heterogeneous trichothecenes and mycooestrogens  produced by some Fusarium species fungi and different Aspergillus species (−1) in 33.3% of 60 corn-based products they collected from open markets, while Kim et al. [−1 DON in 13% of 507 corn samples in their study. In another study, researchers reported that corn samples (n:37) were contaminated with DON  and OTA  equivalent to our data [According to the data of our study, it was observed that those toxins instead of OTA were found at higher rates and values in oat-based products compared to corn-based ones, while OTA was found to be higher only in corn-based products . Moreovem et al.  reportedour data .−1, 0.6–11.8 μg·kg−1, and 1.2–13.6 μg·kg−1, respectively [In a study on oat-based products, it was reported that approximately 50% of the 20 oat breakfast samples were contaminated with DON, OTA, and ZEN at levels of 2.5–146.5 μg·kgectively . There aectively ,37,39,44ectively ,47,48,49Another important issue that needs to be emphasized is that these toxins can be found in different and multiple combinations in foods . This can cause toxins to interact with each other in undesirable ways. TAF and FUM, DON and ZEN, TAF and OTA, and FUM and ZEN are mostly observed in the literature in cereals and grain product samples. However, due to the synergistic negative effects of co-occurring mycotoxins on human and animal health, it is important to evaluate their co-occurrence . UnfortuThis research contributes to eliminating the deficiency observed in the literature regarding the presence and concentration of mycotoxins in cereal-based products marketed in open-air markets in Türkiye. Mycotoxin contaminations, mycotoxin combinations, contamination status, and frequency of occurrence in these foodstuffs were determined. It has been revealed that effective control of mycotoxins, which may pose a risk to human health even at low doses, should be carried out with monitoring systems in this and similar types of foodstuffs. On the other hand, considering the additive and synergistic interactions of different mycotoxins, new legal regulations will be needed regarding the presence and limit values of mycotoxin combinations in foods. It is highly recommended to continuously monitor the mycotoxins that were studied in vulnerable food items and to conduct research aimed at identifying masked mycotoxins and other fungal metabolites. Additionally, efforts should be focused on reducing and managing mycotoxins within the food supply chain.