Development and evaluation of a triplex droplet digital PCR method for differentiation of M. tuberculosis, M. bovis and BCG.

Introduction: Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis, M. bovis, and BCG, poses a potential challenge. Methods: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively. Results: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 103 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis, M. bovis, and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively. Discussion: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

Cr(VI) induces ferroptosis in DF-1 cells by simultaneously perturbing iron homeostasis of ferritinophagy and mitophagy.

Hexavalent chromium [Cr(VI)] is an environmental pollutant known for its strong oxidizing and carcinogenic effects. However, its potential to induce ferroptosis in poultry remains poorly understood. This study aims to investigate the induction of ferroptosis by Cr(VI) in DF-1 cells and elucidate the underlying mechanisms. DF-1 cells exposed to Cr(VI) showed increased lipid reactive oxygen species and changes in ferroptosis marker genes (decreased expression of GPX4 and increased expression of COX2). Notably, the addition of the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) can reverse this effect. During the cell death process, Cr(VI) induced ferritinophagy, disrupting iron homeostasis and releasing labile iron ions. We predicted by docking that these iron ions would bind to mitochondrial membrane proteins through virtual docking. This binding was validated through colocalization analysis. In addition, Cr(VI) caused mitophagy, which releases additional ferrous ions. Therefore, Cr(VI) can induce the simultaneous release of ferrous ions through these pathways, thereby exacerbating lipid peroxidation and ultimately triggering ferroptosis in DF-1 cells. This study demonstrates that Cr(VI) can induce ferroptosis in DF-1 cells by disrupting intracellular iron homeostasis and providing valuable insights into the toxic effects of Cr(VI) in poultry and potentially other organisms.

Platycodon grandiflorus polysaccharide inhibits the inflammatory response of 3D4/21 cells infected with PCV2.

Porcine circovirus type 2 (PCV2) infection cause multi-systemic inflammation in pigs. Platycodon grandiflorus polysaccharide (PGPSt) has been reported to have the effects of immune regulation and disease resistance. Nevertheless, the role and mechanism of PGPSt in the inflammatory response of 3D4/21 cells induced by PCV2 infection remain unclear. The present study aims to investigate effects of PGPSt on inflammatory response and its possible underlying mechanisms in vitro models. Cells were treated with PCV2 for 36 h to construct a cell inflammation model. The 3D4/21 cell lines were pretreated with or without PGPSt, and the changes of inflammation-related markers and the signaling pathway were detected by CCK-8, ELISA, qPCR and Western blot. The results showed that PGPSt was non-toxic to cells and protected PCV2-infected cells from inflammatory damage. PGPSt could significantly inhibit the high acetylation of histone H3 (AcH3) and histone H4 (AcH4), down-regulate HAT and up-regulate HDAC activity, and reduce the expression of pro-inflammatory enzymes iNOS and COX-2 proteins levels. Then the levels of IL-1ß, IL-6 and TNF-α were significantly inhibited, and the level of IL-10 was promoted. We also observed that PGPSt inhibited the phosphorylation of p65, p38 and Erk1/2, which subsequently inhibited nuclear translocation of NF-κB p65 to express pro-inflammatory factors. In conclusion, PGPSt can reduce the inflammatory response by regulating histone acetylation, reducing the release of inflammatory factors, reducing the expression of pro-inflammatory enzymes, and inhibiting the activation of NF-κB and MAPKs signaling pathways. This suggests that PGPSt had an anti-inflammatory effect on the inflammatory response caused by PCV2 infection, which provided theoretical data support for the research.

Role of PERK-Mediated Endoplasmic Reticulum Stress in Ferroptosis Caused by Hexavalent Chromium in Chicken Hepatocytes.

This study aimed to investigate whether Cr(VI) can induce ferroptosis in chicken hepatocytes and determine the role of PERK-mediated endoplasmic reticulum stress (ERS). First, a model of Cr(VI) poisoning was established by exposing chicken hepatocytes to Cr(VI). The levels of ferroptosis-related proteins, meanwhile, GSH, SOD, MDA, and lipid ROS, were measured. Furthermore, the expression of GRP78 and PERK proteins was examined. Changes in ERS and ferroptosis were evaluated by silencing the PERK gene. Results showed that Cr(VI) led to the accumulation of lipid ROS, decreased expression of GPX4 and HSP27, increased expression of COX2, and induced ferroptosis in chicken hepatocytes. Exposure to Cr(VI) increased the protein expression of GRP78 and PERK, and silencing of PERK worsened Cr(VI)-induced ferroptosis. In conclusion, Cr(VI) can induce ferroptosis in chicken hepatocytes, and PERK plays an important role as a negative regulator.

Research on Bacterial Diversity and Antibiotic Resistance in the Dairy Farm Environment in a Part of Shandong Province.

Antimicrobials are extensively utilized in dairy farms to prevent and control diseases in cattle. However, their use contributes to the emergence of antimicrobial-resistant bacteria (ARB) and antimicrobial-resistant genes (ARG), and these can be transmitted to the environment. Regular monitoring of antimicrobial resistance (AMR) is crucial for implementing effective mitigation strategies. This research aimed to assess the environmental microbial species present on dairy farms in Shandong Province and characterize the antimicrobial resistance profiles of the isolates. Five dairy farms located in Shandong Province were selected, representing the prevalent large-scale farming patterns in the area. Sampling took place from April to June 2022, with a total of 223 isolates collected from various environmental locations within each farm (bedding, sports field, and milking parlor). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was employed to identify the species of the clinical isolates. The main pathogens isolated were Aerococcus viridans (5.38%, n = 12), Corynebacterium xerosis (4.93%, n = 11), and Acinetobacter lwoffii (4.03%, n = 9). Among the bacterial isolates, resistance to lincomycin was highest at 91%, and 88% were resistant to sulfadiazine. Antimicrobial resistance genes were detected in only a small proportion of the isolates, the most common of which was sul1. These findings highlight the necessity for careful evaluation of antimicrobial usage in maintaining their effectiveness in human medicine. Understanding the microbial species present and their antimicrobial resistance profiles aids in focusing efforts toward sustainable antimicrobial use and safeguarding human health.

The surface morphology of Atractylodes macrocephala polysaccharide and its inhibitory effect on PCV2 replication.

BACKGROUND: Porcine infection with Porcine circovirus type 2 (PCV2) causes immunosuppression, which is easy to cause concurrent or secondary infection, making the disease complicated and difficult to treat, and causing huge economic losses to the pig industry. Total polysaccharide from the rhizoma of Atractylodes macrocephala Koidz. (PAMK) is outstanding in enhancing non-specific immunity and cellular immunity, and effectively improving the body's disease resistance, indicating its potential role in antiviral immunotherapy. RESULTS: PAMK had the characteristics of compact, polyporous and agglomerated morphology, but does not have triple helix conformation. PCV2 infection led to the increase in LC3-II, degradation of p62 and the increase of viral Cap protein expression and viral copy number. PAMK treatment significantly alleviated PCV2-induced autophagy and inhibited PCV2 replication. Moreover, PAMK treatment significantly attenuated the increase of PINK1 protein expression and the decrease of TOMM20 protein expression caused by PCV2 infection, alleviated Parkin recruitment from cytoplasm to mitochondria and intracellular reactive oxygen species accumulation, restored mitochondrial membrane charge, alleviated viral Cap protein expression. CONCLUSION: PAMK alleviates PCV2-induced mitophagy to suppress PCV2 replication by inhibiting the Pink 1/Parkin pathway. These findings may provide new insights into the prevention and treatment of PCV2. © 2023 Society of Chemical Industry.

Diagnosis of a Rabbit Hemorrhagic Disease Virus 2 (RHDV2) and the Humoral Immune Protection Effect of VP60 Vaccine.

Rabbit hemorrhagic disease (RHD) is known as rabbit plague and hemorrhagic pneumonia. It is an acute, septic, and highly fatal infectious disease caused by the Lagovirus rabbit hemorrhagic disease virus (RHDV) in the family Caliciviridae that infects wild and domestic rabbits and hares (lagomorphs). At present, RHDV2 has caused huge economic losses to the commercial rabbit trade and led to a decline in the number of wild lagomorphs worldwide. We performed a necropsy and pathological observations on five dead rabbits on a rabbit farm in Tai'an, China. The results were highly similar to the clinical and pathological changes of typical RHD. RHDV2 strain was isolated and identified by RT-PCR, and partial gene sequencing and genetic evolution analysis were carried out. There were significant differences in genetic characteristics and antigenicity between RHDV2 and classical RHDV strain, and the vaccine prepared with the RHDV strain cannot effectively prevent rabbit infection with RHDV2. Therefore, we evaluated the protective efficacy of a novel rabbit hemorrhagic virus baculovirus vector inactivated vaccine (VP60) in clinical application by animal regression experiment. The result showed that VP60 could effectively induce humoral immunity in rabbits. The vaccine itself had no significant effect on the health status of rabbits. This study suggested that the clinical application of VP60 may provide new ideas for preventing the spread of RHD2.

The seroprevalence of canine dirofilariosis in dogs in the eastern coastal areas of China.

Background and objectives: The present study was carried out to assess the presence of canine Dirofilaria immitis infection in pet dogs in China. Materials and methods: From October 2018 to November 2019, a total of 216 sera were collected from pet hospitals in Shandong, Jiangsu, Shanghai, Zhejiang, and Fujian regions of Eastern China. The sera were tested by using a commercial canine heartworm antibody ELISA test kit. Results: 70.8% of the pets had significant clinical symptoms resembled to heartworm infection; the overall dirofilariosis positivity found was 12.5% (27/216); Significant positive rates differences were observed between symptomatic and asymptomatic dogs (P < 0.05) (i.e. 15.7% and 4.7% respectively).The prevalence of infection in Shandong Province (15.5%) was the highest among the surveyed areas, but the difference among the geographic regions was not statistically significant (P > 0.05). Furthermore, the prevalence detected in summer (28.2%) was significantly higher than in other seasons (P < 0.05). In addition, no significant difference was observed between male and female sex (P > 0.05). Conclusions: Altogether, these results suggest that an epidemic of dirofilariosis exists in eastern coastal China, as such preventive measures should be taken to control the spread of dirofilariosis to reduce the risk of human and pet infection with heartworm.

Staphylococcus aureus extracellular vesicles induce apoptosis and restrain mitophagy-mediated degradation of damaged mitochondria.

Extracellular vesicles (EVs) are nano-sized bilayer EVs with various components. EV secretion in pathogenic Gram-positive bacteria is a universal feature that can cause disease and damage the targeted host. In this study, we isolated and purified Staphylococcus aureus (S. aureus) EVs, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyzed Ev's protein composition. After that, the pathway of EVs internalized into MAC-T cells was evaluated. Moreover, the activation of mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NF-κB) pathway was measured by Western blot. Meanwhile, Western blot and confocal microscopy detected mitochondrial damage, apoptosis, and Parkin-mediated mitophagy. Results showed that purified S. aureus EVs exhibited a typical cup-shaped structure and internalized into MAC-T cells by lipid raft-mediated endocytic pathway. S. aureus EVs caused mitochondrial damage and apoptosis in MAC-T cells. However, degradation of the damaged mitochondria was impeded due to the Parkin-mediated mitophagy pathway being restrained by the disruption of the acidic environment of lysosomes by S. aureus EVs. Hence, our study reveals the role of S. aureus EVs in immune stimulation, disruption of mitochondria, and lysosomal acidic environment in bovine mammary epithelial cells. These findings help us understand the role of EVs in the pathogenic mechanism of S. aureus.

Protective effect of Platycodon grandiflorus polysaccharide on apoptosis and mitochondrial damage induced by pseudorabies virus in PK-15 cells.

Previous studies have confirmed that Platycodon grandiflorus polysaccharide (PGPSt) has the effects of regulating immunity and anti-apoptosis, but its effect on mitochondrial damage and apoptosis caused by PRV infection is still unclear. In this research, the effects of PGPSt on the cell viability, mitochondria morphology, mitochondrial membrane potential and apoptosis caused by PRV based on PK-15 cells were respectively examined by CCK-F assay, Mito-Tracker Red CMXRos, JC-1 staining method and Western blot etc. CCK-F test results showed that PGPSt had a protective effect on the decrease of cell viability caused by PRV. The results of morphological observation found that PGPSt can improve mitochondrial morphology damage, mitochondrial swelling and thickening, and cristae fracture. Fluorescence staining test results showed that PGPSt alleviated the decrease of mitochondrial membrane potential and apoptosis in infected cells. The expression of apoptosis-related proteins showed that PGPSt down-regulated the expression of the pro-apoptotic protein Bax and up-regulated the expression of the anti-apoptotic protein Bcl-2 in infected cells. These results indicated that PGPSt protected against PRV-induced PK-15 cell apoptosis by inhibiting mitochondrial damage.

Discovery of a novel nucleoside immune signaling molecule 2'-deoxyguanosine in microbes and plants.

INTRODUCTION: Beneficial microorganisms play essential roles in plant growth and induced systemic resistance (ISR) by releasing signaling molecules. Our previous study obtained the crude extract from beneficial endophyte Paecilomyces variotii, termed ZNC (ZhiNengCong), which significantly enhanced plant resistance to pathogen even at 100 ng/ml. However, the immunoreactive components of ZNC remain unclear. Here, we further identified one of the immunoreactive components of ZNC is a nucleoside 2'-deoxyguanosine (2-dG). OBJECTIVES: This paper intends to reveal the molecular mechanism of microbial-derived 2'-deoxyguanosine (2-dG) in activating plant immunity, and the role of plant-derived 2-dG in plant immunity. METHODS: The components of ZNC were separated using a high-performance liquid chromatography (HPLC), and 2-dG is identified using a HPLC-mass spectrometry system (LC-MS). Transcriptome analysis and genetic experiments were used to reveal the immune signaling pathway dependent on 2-dG activation of plant immunity. RESULTS: This study identified 2'-deoxyguanosine (2-dG) as one of the immunoreactive components from ZNC. And 2-dG significantly enhanced plant pathogen resistance even at 10 ng/ml (37.42 nM). Furthermore, 2-dG-induced resistance depends on NPR1, pattern-recognition receptors/coreceptors, ATP receptor P2K1 (DORN1), ethylene signaling but not salicylic acid accumulation. In addition, we identified Arabidopsis VENOSA4 (VEN4) was involved in 2-dG biosynthesis and could convert dGTP to 2-dG, and vne4 mutant plants were more susceptible to pathogens. CONCLUSION: In summary, microbial-derived 2-dG may act as a novel immune signaling molecule involved in plant-microorganism interactions, and VEN4 is 2-dG biosynthesis gene and plays a key role in plant immunity.

Platycodon grandiflorus Polysaccharides Alleviate Cr(VI)-Induced Apoptosis in DF-1 Cells via ROS-Drp1 Signal Pathway.

Hexavalent chromium (Cr(VI)) is a widespread heavy metal that has been identified as a human carcinogen, and acute or chronic exposure to Cr(VI) can cause organ damage. Platycodon grandiflorus polysaccharide (PGPS) is a constituent extracted from the Chinese herb Platycodon grandiflorus, which has various pharmacological effects. Therefore, the author investigated the role of PGPSt in Cr(VI)-induced apoptosis in chicken embryo fibroblast cell lines (DF-1 cells). Firstly, this study infected DF-1 cells using Cr(VI) to set up a model for cytotoxicity and then added PGPSt. Then, the intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and apoptosis rate were evaluated. The results showed that PGPSt could inhibit Cr(VI)-induced mitochondrial damage and increase the apoptosis rate. For further exploration of the mechanism of regulation of PGPSt, the ROS-Drp1 pathway was investigated. The antioxidant N-acetyl-L-cysteine (NAC) and mitochondrial division inhibitor 1(Mdivi-1) were added, respectively. The results showed that the NAC and Mdivi-1 restored abnormal mitochondrial fission and cell apoptosis. Thus, PGPSt can alleviate Cr(VI)-induced apoptosis of DF-1 cells through the ROS-Drp1 signaling pathway, which may suggest new research ideas for developing new drugs to alleviate Cr(VI) toxicity.

Regulation of Parkin in Cr (VI)-induced mitophagy in chicken hepatocytes.

The large amount of heavy metal chromium emissions from industrial production, ore smelting and sewage treatment plants have made chromium one of the most widespread heavy metal pollutants, with Cr (VI) being the most toxic. In recent years, people have gradually recognized the great harm of heavy metal chromium pollution, but the research on its pathogenic mechanism is still not deep enough. In this study, we treated the Primary cells of chicken liver with Cr (VI) to establish a model of toxicity. The optimal treatment time and Cr (VI) concentration were screened using the CCK-8 test. The intracellular mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were measured qualitatively and quantitatively by laser confocal and flow cytometry, respectively. This result was confirmed by the fact that Cr (VI) could cause mitophagy by causing damage to mitochondria. Subsequently, this study used LMH cells to construct a Parkin silencing model to further investigate that Parkin exerts the function on the Cr (VI)-induced mitophagy in chicken hepatocytes. The results showed that the knockdown of Parkin effectively blocked p62 degradation and LC3 lipidation and that PINK1 expression was significantly inhibited in LMH cells, further suggesting that the knockdown of Parkin effectively inhibited mitophagy. Mitochondrial morphology, MMP, and ROS were observed using laser confocal. The results showed that Parkin knockdown resulted in mitochondrial fission and increased levels of reactive oxygen species, together with increased depolarization of the mitochondrial membrane potential. These changes led to increased mitochondrial damage. In conclusion, this study showed that Cr (VI) could cause the occurrence of mitophagy by damaging mitochondria, and Parkin played a crucial role in Cr (VI)-induced mitophagy in chicken hepatocytes.

The Protective Effect of Lycium Ruthenicum Murr Anthocyanins in Cr (VI)-Induced Mitophagy in DF-1 Cells.

Cr (VI) is an extremely toxic environment and professional pollutant that seriously damages mitochondrial dysfunction when it enters a cell. Anthocyanins possess anti-oxidant, antiaging, and antifatigue properties. The regulatory effect of Lycium ruthenicum Murr anthocyanin (LRMA) on Cr (VI)-induced mitophagy in DF-1 cells was determined. The experimental design was divided into blank group, groups subjected to Cr (VI) and Cr (VI), and LRMA co-treatment groups. Cell viability was determined by the CCK-8 assay. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were assessed by flow cytometry and immunofluorescence. Mitophagy was monitored by ELISA and Western blot. Data showed that Cr (VI) caused the overexpression of autophagy-related proteins (LC3, Beclin-1) and reduced the expressions of autophagy protein p62 and TOMM20. Compared with the Cr (VI) group, the LRMA group showed considerably decreased mitochondrial damage and mitophagy. LRMA decreased the mitochondrial protein expression of PINK1 and Parkin's transfer from the cytoplasm to mitochondria. LRMA may confer protective effects by reducing PINK1/Parkin-mediated mitophagy in Cr (VI)-induced DF-1 cell models.

Effects of Artemisinin on Escherichia coli-Induced Mastitis in Bovine Mammary Epithelial Cells and Mice.

Bovine mastitis is an important disease affecting dairy farming, and it causes large economic losses to the dairy industry. Escherichia coli (E. coli) is considered to be a causative environmental pathogen and frequently enters into mammary glands, causing inflammation. Artemisinin is a highly effective malaria remedy and is not easy to develop drug resistance to. In recent years, other effects of artemisinin (including antitumor, anti-inflammatory, antifungal, etc.) have been increasingly discovered and applied. The current study aimed to investigate whether artemisinin could attenuate E. coli-induced inflammation. Through the E. coli mastitis model in MAC-T cells and mice, the protective effects of artemisinin were analyzed by CCK-8 (Cell Counting Kit-8), Western blot, and RT-qPCR. The results showed that artemisinin reversed the decrease of cell viability and upregulated TLR4 (toll-like receptor 4)/NF-κB (nuclear factor κB) and MAPK (mitogen activated protein kinase)/p38 signaling pathways, as well as restrained the expression of TNF-α, IL-6, and IL-1ß mRNA caused by E. coli. Meanwhile, artemisinin also alleviated mammary tissue damage, reduced inflammatory cells' infiltration, and decreased the levels of inflammatory factors in a mice mastitis model. This study demonstrated that artemisinin alleviated the inflammatory response of mouse mastitis and MAC-T cells induced by E. coli, thus providing a practical approach for the clinical control of mastitis.

Effect of Platycodon grandiflorus Polysaccharide on M1 Polarization Induced by Autophagy Degradation of SOCS1/2 Proteins in 3D4/21 Cells.

M1-polarized macrophages can improve the body's immune function. This study aimed to explore the mechanism of Platycodon grandiflorus polysaccharide (PGPSt) degrading SOCS1/2 protein through autophagy and promoting M1 polarization in 3D4/21 cells. Immunoprecipitation, confocal laser scanning microscopy, flow cytometry, and intracellular co-localization were used to detect the expression of related phenotypic proteins and cytokines in M1-polarized cells. The results showed that PGPSt significantly promoted the mRNA expression of IL-6, IL-12, and TNF-α and enhanced the protein expression of IL-6, IL-12, TNF-α, IL-1ß, iNOS, CD80, and CD86, indicating that PGPSt promoted M1 polarization in 3D4/21 cells. Next, the effect of the PGPSt autophagy degradation of SOCS1/2 on the M1 polarization of 3D4/21 cells was detected. The results showed that PGPSt significantly downregulated the expression level of SOCS1/2 protein, but had no obvious effect on the mRNA expression level of SOCS1/2, indicating that PGPSt degraded SOCS1/2 protein by activating the lysosome system. Further research found that under the action of 3-MA and BafA1, PGPSt upregulated LC3B II and downregulated SOCS1/2 protein expression, which increased the possibility of LC3B, the key component of autophagy, bridging this connection and degrading SOCS1/2. The interaction between SOCS1/2 and LC3 was identified by indirect immunofluorescence and Co-IP. The results showed that the co-localization percentage of the two proteins increased significantly after PGPSt treatment, and LC3 interacted with SOCS1 and SOCS2. This provides a theoretical basis for the application of PGPSt in the treatment or improvement of diseases related to macrophage polarization by regulating the autophagy level.

The surface morphology of Platycodon grandiflorus polysaccharide and its anti-apoptotic effect by targeting autophagy.

BACKGROUND: Fumonisin B1 is categorised as possible carcinogenic to humans which commonly contaminate maize and maize-based products worldwide, FB1, like other environmental pollutants, may activate apoptosis, autophagy, the inflammatory response and oxidative stress. Platycodon grandiflorus polysaccharide (PGPSt) is prepared from a traditional herbal medicine in Asia with tremendous pharmacological activities. However, whether PGPSt could relieve FB1-induced apoptosis has not been elucidated. The study aimed to evaluate the surface morphology of PGPSt and its protective effect on fumonisin B1-induced apoptosis. METHODS: The surface morphology of PGPSt was evaluated by SEM and AFM. Expressions of proteins involved in autophagy and apoptosis were detected by western blot analysis. Western blot, transient transfection, JC-1 and Annexin V-FITC/PI staining, CCK8, Live-cell imaging and autophagy inhibitor were used to observe the effect and explore the mechanism of PGPSt on FB1-induced apoptosis of 3D4/21 cells. RESULTS: PGPSt had triple helix conformation, and had the characteristics of compact, polyporous and agglomerated morphology. PGPSt promoted the expression of LC3-II and Beclin1, reduced the expression of p62, and significantly activated autophagy. PGPSt inhibited the Akt/mTOR signaling pathway at 24 h. Besides, PGPSt increased the expression of Bcl-2 and decreased the expression of Cleaved Caspase-3. PGPSt-mediated autophagy was inhibited by 3-MA, accompanied by the upregulation of Caspase-3 and Cleaved Caspase-3, suggesting that enhanced autophagy inhibited apoptosis. CONCLUSION: PGPSt can activate autophagy, which in turn protects FB1-induced apoptosis. Targeting autophagy may provide a new way to improve the health of humans or animals in FB1 contaminated areas.