[Cryoprotective effects of deicing micro/nanomaterials with different sizes and shapes on cells].

Extensive studies have been conducted on deicing nanomaterials to improve the cryoprotective effects on cells, tissues, and organs. The nanomaterials with different composition, sizes, and shapes can inhibit the formation and growth of ice crystals, thereby reducing the damage to the cryopreserved samples. In this study, the carbon composite particles (CCPs) with different sizes and shapes were prepared by the hydrothermal method. The results demonstrated that the cryoprotective effect of CCPs enhanced with the decrease in particle size. Compared with spherical CCPs, Janus nanoparticles and WSP nanoflower with special shapes demonstrated improved protective effects on cryopreserved cells. In addition, the combination of deicing micro/nanomaterials at appropriate concentrations with commercial cryoprotectants exerted improved cryoprotective effects on cells. The prepared deicing micro/nanomaterials can improve cell cryopreservation, demonstrating great application potential in biomedical research and cryopreservation.

A genome-wide CRISPR/Cas9 knockout screen identifies TMEM239 as an important host factor in facilitating African swine fever virus entry into early endosomes.

African swine fever (ASF) is a highly contagious, fatal disease of pigs caused by African swine fever virus (ASFV). The complexity of ASFV and our limited understanding of its interactions with the host have constrained the development of ASFV vaccines and antiviral strategies. To identify host factors required for ASFV replication, we developed a genome-wide CRISPR knockout (GeCKO) screen that contains 186,510 specific single guide RNAs (sgRNAs) targeting 20,580 pig genes and used genotype II ASFV to perform the GeCKO screen in wild boar lung (WSL) cells. We found that knockout of transmembrane protein 239 (TMEM239) significantly reduced ASFV replication. Further studies showed that TMEM239 interacted with the early endosomal marker Rab5A, and that TMEM239 deletion affected the co-localization of viral capsid p72 and Rab5A shortly after viral infection. An ex vivo study showed that ASFV replication was significantly reduced in TMEM239-/- peripheral blood mononuclear cells from TMEM239 knockout piglets. Our study identifies a novel host factor required for ASFV replication by facilitating ASFV entry into early endosomes and provides insights for the development of ASF-resistant breeding.

[Active secondary metabolites from an endophytic fungus Fusarium solani MBM-5 of Datura arborea].

This study investigated the chemical and biological activity of the secondary metabolites from an endophytic fungus Fusa-rium solani MBM-5 of Datura arborea. A total of six alkenoic acid compounds, including a new compound and five known ones, were isolated from the ethyl acetate extract of F. solani MBM-5 by using the chromatographic methods(open ODS column chromatography, silica gel column chromatography, Sephadex LH-20, and semi-preparative HPLC). The structures of the compounds were identified by using their physical and chemical data, spectroscopic methods(UV, IR, NMR, and HR-ESI-MS), and Mosher's reaction, which were fusaridioic acid E(1), fusaridioic acid C(2), fusaridioic acid A(3), L660282(4), hymeglusin(5), and hymeglnone(6). Compound 1 is new. MTT assay and Griss method were used to evaluate the growth inhibition of all the compounds against two tumor cells, as well as their influence and anti-inflammatory action on the release of NO from LPS-induced RAW264.7 cells. The results showed that compound 5 had strong growth inhibition activity against A549 and HepG2 cell lines, with IC_(50) values of 4.70 and 13.57 µmol·L~(-1), respectively. Compounds 1 and 6 significantly inhibited the release of NO from LPS-induced RAW264.7 cells, with IC_(50) values of 77.00 and 70.33 µmol·L~(-1), respectively.

[Effects of tetramethylpyrazine on pharmacokinetics in plasma and brain dialysate and neuropathic pain in rat model of spared sciatic nerve injury].

This study aims to explore the effects of tetramethylpyrazine(TMP) on pharmacokinetics in plasma and brain dialysate and neuropathic pain in the rat model of partial sciatic nerve injury(SNI), and to investigate the correlation between the analgesic effect of TMP and its concentrations in the plasma and brain dialysate. Male SD rats were randomized into Sham, SNI, and SNI+TMP groups. Mechanical stimulation with von frey filaments and cold spray method were employed to evaluate the mechanical sensitivity and cold sensitivity of rats. Another two groups, Sham+TMP and SNI+TMP, were used to intubate the common jugular vein and implant microdialysis probes into the anterior cingulate gyrus(ACC), respectively.After intraperitoneal injection of TMP at a dose of 80 mg·kg~(-1), automatic blood collection and intracerebral microdialysis(perfusion rate of 1 µL·min~(-1)) systems were used to collect the blood and brain dialysate for 24 h. HSS T3 C_(18) reversed-phase chromatographic column(2.1 mm×50 mm, 2.5 µm) was used for liquid chromatographic separation. Gradient elution was carried out with the mobile phase of methanol-water(containing 0.005% formic acid) at a flow rate of 0.25 mL·min~(-1). Electrospray ion source was used for mass spectrometry, and the scanning mode was multi-reaction monitoring under the positive ion mode. The ion pairs for quantitative analysis were TMP m/z 137/122 and aspirin m/z 179/137, respectively. DAS 2.11 was used to calculate the pharmacokinetic parameters. The optimal time of TMP to exert the analgesia effect and inhibit cold pain sensitivity was 60 min after treatment. The TMP in the plasma and brain dialysate of SNI rats showed the T_(max) of 15 min and 30 min, the C_(max) of(2 866.43±135.39) and(1 462.14±197.38) µg·L~(-1), the AUC_(0-t) of(241 463.30±28 070.31) and(213 115.62±32 570.07) µg·min·L~(-1), the MRT_(0-t) of(353.13±47.73) and(172.16±12.72) min, and the CL_Z of 0.73 and 0.36 L·min·kg~(-1), respectively. The analgesic effect of TMP had a significant correlation with the blood drug concentration in the ACC, which indicated that this method was suitable for the detection of TMP in rat plasma and brain dialysate. The method is accurate, reliable, and sensitive and can realize the important value of the application of correlation analysis theory of "automatic blood collection-microdialysis/PK-PD" in the research on neuropathic pain.

Jiaohong pills attenuate neuroinflammation and amyloid-ß protein-induced cognitive deficits by modulating the mitogen-activated protein kinase/nuclear factor kappa-B pathway.

BACKGROUND: Jiaohong pills (JHP) consist of Pericarpium Zanthoxyli (PZ) and Radix Rehmanniae, two herbs that have been extensively investigated over many years due to their potential protective effects against cognitive decline and memory impairment. However, the precise mechanisms underlying the beneficial effects remain elusive. Here, research studies were conducted to investigate and validate the therapeutic effects of JHP on Alzheimer's disease. METHODS: BV-2 cell inflammation was induced by lipopolysaccharide. AD mice were administered amyloid-ß (Aß). Behavioral experiments were used to evaluate learning and memory ability. The levels of nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), and interleukin-10 (IL-10) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expressions of inducible nitric oxide synthase (iNOS) and the phosphorylation level of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) were detected using Western blot. Nissl staining was used to detect neuronal degeneration. RESULTS: The results demonstrated that an alcoholic extract of PZ significantly decreased the levels of NO, IL-1ß, TNF-α, and iNOS; increased the expression level of IL-10; and significantly decreased the phosphorylation levels of MAPK and NF-κB. These inhibitory effects were further confirmed in the AD mouse model. Meanwhile, JHP improved learning and memory function in AD mice, reduced neuronal damage, and enriched the Nissl bodies in the hippocampus. Moreover, IL-1ß and TNF-α in the cortex were significantly downregulated after JHP administration, whereas IL-10 showed increased expression. CONCLUSIONS: It was found that JHP reduced neuroinflammatory response in AD mice by targeting the MAPK/NF-κB signaling pathway.

Shenlian extract protected ox-LDL-loaded macrophages against ER stress by promoting LAL-LXRα mediated cholesterol flux.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlian (SL) extract is consisted of extracts from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm.f.) Nees, two herbs commonly used in Chinese clinical formula to treat atherosclerosis by removing blood stasis and clearing away heat. Pharmacologically, the anti-atherosclerotic effects of these two herbs are related to unresolved inflammation and the macrophage anergy or apoptosis in lesions led by the lipid flux blockage and ER stress. However, the deeper understanding of SL extract in protecting macrophage in plaques remains unknown. AIM OF THE STUDY: This study aimed to investigate the underlying mechanism of SL extract in protecting ER-stressed macrophages from apoptosis in atherosclerosis. METHODS: The ApoE-/- atherosclerotic mice model and ox-LDL loaded macrophages model were established to assess the effect of SL extract on ER stress in vivo and in vitro. Key markers related to ER stress in plaque were determined by immunohistochemical staining. Proteins involved in apoptosis and ER stress in macrophages loaded by ox-LDL were assessed by Western blot. ER morphology was observed by electron microscope. Lipid flux was temporally and quantitatively depicted by Oil red staining. The LAL and LXRα were blocked by lalistat and Gsk 2033 respectively to investigate whether SL extract protected the function of macrophages by the activation of LAL-LXRα axis. RESULTS: Our study reported that, in ApoE-/- atherosclerotic mice, SL extract effectively relieved ER stress of carotid artery plaque. In lipid-overloaded macrophage models, SL extract significantly alleviated ER stress by promoting cholesterol degradation and efflux, which finally prevented apoptosis of foam cells induced by ox-LDL. Blockage of ER stress by 4-Phenylbutyric acid (4-PBA), an inhibitor of Endoplasmic Reticulum (ER) stress, largely attenuated the protective effects of SL extract on macrophage. By utilizing the selective antagonists against both LAL and LXRα, this study further revealed that the beneficial effects of SL extract in macrophages was dependent on the proper functionalization of LAL-LXRα axis. CONCLUSIONS: By highlighting the therapeutic significance of macrophage protection in resolving atherosclerosis inflammation, our study pharmacologically provided convincing mechanistic evidence of SL extract in the activation LAL-LXRα axis and revealed its promising potential in the promotion of cholesterol turnover and prevention of ER stress induced apoptosis in lipid-loaded macrophages.

Plasma membrane lipid composition and metabolomics analysis of Yorkshire boar sperms with high and low resistance to cryopreservation.

The resistance of sperm to freezing varies widely among boars. The semen ejaculate of different boars can be grouped into poor freezability ejaculate (PFE) and good freezability ejaculate (GFE). In this study, five Yorkshire boars each of the GFE and PFE were selected by comparing the changes in sperm motility before and after cryopreservation. Firstly, we found that the sperm plasma membrane of the PFE group showed weak integrity after PI and 6-CFDA staining. Then the electron microscopy results verified that the plasma membrane condition of all segments of GFE was better than that of PFE segments. Furthermore, the lipid composition of sperm plasma membranes in GPE and PFE sperm was analyzed by using mass spectrometry, and 15 lipids showed differences between the two groups. Among those lipids, only phosphatidylcholine (PC) (14:0/20:4) and phosphatidylethanolamine (PE) (14:0/20:4) were higher in PFE. The remaining lipid contents, including those of dihydroceramide (18:0/18:0), four hexosylceramides (18:1/20:1, 18:0/22:1, 18:1/16:0, 18:1/18:0), lactosylceramide (18:1/16:0), two hemolyzed phosphatidylethanolamines (18:2, 20:2), five phosphatidylcholines (16:1/18:2, 18:2/16:1, 14:0/20:4, 16:0/18:3, 18:1/20:2), and two phosphatidylethanolamines (14:0/20:4, 18:1/18:3), were all positively correlated with resistance to cryopreservation (p < 0.05, r > 0.6). Moreover, we analyzed the metabolic profile of sperm using untarget metabolomic. KEGG annotation analysis revealed that the altered metabolites were mainly involved in fatty acid biosynthesis. Finally, we determined that the contents of oleic acid, oleamideetc, N8-acetylspermidine etc., were different between GFE and PFE sperm. In summary, the different lipid metabolism levels and long-chain polyunsaturated fatty acids (PUFAs) in plasma membrane may be key factors contributing to differences in sperm resistance to cryopreservation among boars.

Role of the inflammasome in insulin resistance and type 2 diabetes mellitus.

The inflammasome is a protein complex composed of a variety of proteins in cells and which participates in the innate immune response of the body. It can be activated by upstream signal regulation and plays an important role in pyroptosis, apoptosis, inflammation, tumor regulation, etc. In recent years, the number of metabolic syndrome patients with insulin resistance (IR) has increased year by year, and the inflammasome is closely related to the occurrence and development of metabolic diseases. The inflammasome can directly or indirectly affect conduction of the insulin signaling pathway, involvement the occurrence of IR and type 2 diabetes mellitus (T2DM). Moreover, various therapeutic agents also work through the inflammasome to treat with diabetes. This review focuses on the role of inflammasome on IR and T2DM, pointing out the association and utility value. Briefly, we have discussed the main inflammasomes, including NLRP1, NLRP3, NLRC4, NLRP6 and AIM2, as well as their structure, activation and regulation in IR were described in detail. Finally, we discussed the current therapeutic options-associated with inflammasome for the treatment of T2DM. Specially, the NLRP3-related therapeutic agents and options are widely developed. In summary, this article reviews the role of and research progress on the inflammasome in IR and T2DM.

Insect recognition based on complementary features from multiple views.

Insect pest recognition has always been a significant branch of agriculture and ecology. The slight variance among different kinds of insects in appearance makes it hard for human experts to recognize. It is increasingly imperative to finely recognize specific insects by employing machine learning methods. In this study, we proposed a feature fusion network to synthesize feature presentations in different backbone models. Firstly, we employed one CNN-based backbone ResNet, and two attention-based backbones Vision Transformer and Swin Transformer to localize the important regions of insect images with Grad-CAM. During this process, we designed new architectures for these two Transformers to enable Grad-CAM to be applicable in such attention-based models. Then we further proposed an attention-selection mechanism to reconstruct the attention area by delicately integrating the important regions, enabling these partial but key expressions to complement each other. We only need part of the image scope that represents the most crucial decision-making information for insect recognition. We randomly selected 20 species of insects from the IP102 dataset and then adopted all 102 kinds of insects to test the classification performance. Experimental results show that the proposed approach outperforms other advanced CNN-based models. More importantly, our attention-selection mechanism demonstrates good robustness to augmented images.

Collectively encoding protein properties enriches protein language models.

Pre-trained natural language processing models on a large natural language corpus can naturally transfer learned knowledge to protein domains by fine-tuning specific in-domain tasks. However, few studies focused on enriching such protein language models by jointly learning protein properties from strongly-correlated protein tasks. Here we elaborately designed a multi-task learning (MTL) architecture, aiming to decipher implicit structural and evolutionary information from three sequence-level classification tasks for protein family, superfamily and fold. Considering the co-existing contextual relevance between human words and protein language, we employed BERT, pre-trained on a large natural language corpus, as our backbone to handle protein sequences. More importantly, the encoded knowledge obtained in the MTL stage can be well transferred to more fine-grained downstream tasks of TAPE. Experiments on structure- or evolution-related applications demonstrate that our approach outperforms many state-of-the-art Transformer-based protein models, especially in remote homology detection.

OCT4 expression transactivated by GATA protein is essential for non-rodent trophectoderm early development.

Oct4 is exclusively expressed in rodent inner cell mass (ICM) but silenced in its trophectoderm (TE). However, for many non-rodent animals, including pig, cattle, rabbit, goat, and human, OCT4 has a remarkable expression in early TE. This study, applying pig as the main research model, proves that OCT4 expression in TE is supported by a unique GATA motif in the OCT4 upstream conserved regulatory region, and GATA4 is responsible for its activation. Moreover, OCT4 acts as a specific regulator of a narrow range of genes (including BCL2A1 and HNRNP2AB1) that are essential for the first wave of rapid proliferation in early TE. This study describes the regulatory mechanism to direct the OCT4 expression and its significance in TE of porcine preimplantation embryo.

Shenlian extract attenuates myocardial ischaemia-reperfusion injury via inhibiting M1 macrophage polarization by silencing miR-155.

CONTEXT: Shenlian extract (SL) is a combination of Salvia miltiorrhiza Bge. (Labiatae) and Andrographis paniculata (Burm. F.) Wall. Ex Nees (Acanthaceae) extracts, which promote blood circulation and clear endogenous heat toxins. Myocardial ischaemia-reperfusion injury (MI/RI) is aggravated myocardial tissue damage induced by reperfusion therapy after myocardial infarction. OBJECTIVES: This study explores the effect of SL on MI/RI and the underlying mechanism. MATERIALS AND METHODS: Primary peritoneal macrophages (pMACs) were treated with LPS and SL (5, 10 or 20 µg/mL) for 24 h. The myocardial ischaemia-reperfusion (MI/R) model was established after administration of different doses of SL (90, 180 or 360 mg/kg). Myocardial tissue injury was assessed by methylthiazolyl tetrazolium (TTC) staining and levels of creatine kinase (CK), lactate dehydrogenase (LDH) and superoxide dismutase (SOD) in mice. The double immunofluorescence staining of iNOS/F4/80 and CD86/F4/80 was used to detect macrophage M1 polarization. The levels of miR-155, inflammatory factors and chemokines were detected by qRT-PCR or ELISA. CD86, iNOS, SOCS3, JAK2, p-JAK2, STAT3 and p-STAT3 proteins expressions in macrophages were analyzed by western blotting. Conditioned medium transfer systems were designed to unite M1 macrophages with H/R cardiomyocytes, and cell apoptosis was detected by TUNEL staining, western blotting or immunohistochemistry. RESULTS: SL reduced apoptosis, diminished CK and LDH levels, raised SOD concentration and decreased infarct size in the MI/R model. Meanwhile, SL decreased miR-155 level, inhibited M1 macrophage polarization and inflammation. Furthermore, SL promoted SOCS3 expression and blocked JAK2/STAT3 pathway in vitro. CONCLUSIONS: SL may be a promising TCM candidate for MI/RI. The underlying mechanisms could be associated with inhibition of M1 macrophage polarization via down-regulating miR-155.

Pharmacokinetic Differences of Wuji Pill Components in Normal and Chronic Visceral Hypersensitivity Irritable Bowel Syndrome Rats Attributable to Changes in Tight Junction and Transporters.

The Wuji pill, also called Wuji Wan (WJW), is an effective traditional medicine for the clinical treatment of irritable bowel syndrome (IBS). It is principally composed of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, and Radix Paeoniae Alba. There have been no reports on the pharmacokinetics of WJW on IBS. Because it is more meaningful to study pharmacokinetics in relation to specific pathological conditions, our study investigated the pharmacokinetic differences of five representative components (berberine, palmatine, evodiamine, rutaecarpine, and paeoniflorin) in normal rats and chronic visceral hypersensitivity IBS (CVH-IBS) model rats after single dose and multiple doses of WJW using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Transmission electron microscopy, immunohistochemistry, and immunofluorescence were used to explore mechanisms behind the pharmacokinetic differences in terms of tight junction proteins (Occludin and ZO-1), myosin light chain kinase (MLCK), and transporters including P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1), and multidrug resistance associated protein 2 (MRP2) in rat colons. After a single dose, for all components except rutaecarpine, significant differences were observed between normal and model groups. Compared with normal group, T1/2 and AUC0-t of berberine and palmatine in model group increased significantly (562.5 ± 237.2 vs. 1,384.9 ± 712.4 min, 733.8 ± 67.4 vs. 1,532.4 ± 612.7 min; 5,443.0 ± 1,405.8 vs. 9,930.8 ± 2,304.5 min·ng/ml, 2,365.5 ± 410.6 vs. 3,527.0 ± 717.8 min·ng/ml), while Cl/F decreased (840.7 ± 250.8 vs. 397.3 ± 142.7 L/h/kg, 427.7 ± 89.4 vs. 288.9 ± 114.4 L/h/kg). Cmax and AUC0-t of evodiamine in model group increased significantly (1.4 ± 0.6 vs. 2.4 ± 0.7 ng/ml; 573 ± 45.3 vs. 733.9 ± 160.2 min·ng/ml), while T1/2, Tmax, Cl/F, and Vd/F had no significant difference. Tmax and AUC0-t of paeoniflorin in model group increased significantly (21.0 ± 8.2 vs. 80.0 ± 45.8 min; 15,428.9 ± 5,063.6 vs. 33,140.6 ± 5,613.9 min·ng/ml), while Cl/F decreased (110.5 ± 48.1 vs. 43.3 ± 9.5 L/h/kg). However, after multiple doses, all five components showed significant differences between normal and model groups. Moreover, these differences were related to tight junction damage and the differential expression of transporters in the colon, suggesting that dose adjustment might be required during administration of WJW in the clinical treatment of IBS.

Protective effects of curcumin against thyroid hormone imbalance after gas explosion-induced traumatic brain injury via activation of the hypothalamic-pituitary-thyroid axis in male rats.

Gas explosion (GE)-induced traumatic brain injury (TBI) can affect thyroid hormone (TH) homeostasis in miners. This study evaluated the effects of hepatic transthyretin and hypothalamic-pituitary-thyroid (HPT) axis on thyroids and explored the protective effect and mechanism of curcumin on GE-induced TBI. Thirty rats were randomly divided into three groups (10 per group): first group (control group)-rats received GE treatment once; second group (GE group)-rats received GE treatment (200 m from the source of the explosion once); third group (GE + Cur group)-rats received curcumin (Cur) by lavage at a dose of 100 mg/kg/day once every other day for 7 days after receiving GE. After GE, the pathological changes were analyzed by hemotoxylin and eosin staining, and the levels of serum reactive oxygen species (ROS), urine iodine (UI), THs, nuclear factor-kappa B (NF-κB), superoxide dismutase (SOD), glutathione peroxidase (Gpx), and malondialdehyde (MDA) were analyzed using ELISA. Expression of proteins in the HPT axis of rats was examined by immunohistochemistry and Western blotting. We found that GE could induce pathologic changes in rat thyroid and liver. Serum levels of THs, NF-κB and serum redox state became unbalanced in rats after GE. GE could inhibit the biosynthesis and biotransformation of THs by affecting key HPT axis proteins. Additionally, GE reduced the level of hepatic transthyretin. Serum THs levels and thyroid sections were almost recovered to normal after curcumin treatment. The aforementioned key HPT axis proteins in the curcumin group showed opposite expression trends. In summary, GE affected THs balance while curcumin can protect against these injury effects by affecting TH biosynthesis, biotransformation, and transport, and inducing oxidative stress and inflammatory responses.

Evaluation of porcine urine-derived cells as nuclei donor for somatic cell nuclear transfer.

BACKGROUND: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. OBJECTIVES: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. METHODS: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. RESULTS: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. CONCLUSIONS: Sow urine-derived cells could be used to produce SCNT embryos.

The NLRP3 Inflammasome in Non-Alcoholic Fatty Liver Disease and Steatohepatitis: Therapeutic Targets and Treatment.

Non-alcoholic fatty liver disease (NAFLD) is among the most prevalent primary liver diseases worldwide and can develop into various conditions, ranging from simple steatosis, through non-alcoholic steatohepatitis (NASH), to fibrosis, and eventually cirrhosis and hepatocellular carcinoma. Nevertheless, there is no effective treatment for NAFLD due to the complicated etiology. Recently, activation of the NLPR3 inflammasome has been demonstrated to be a contributing factor in the development of NAFLD, particularly as a modulator of progression from initial hepatic steatosis to NASH. NLRP3 inflammasome, as a caspase-1 activation platform, is critical for processing key pro-inflammatory cytokines and pyroptosis. Various stimuli involved in NAFLD can activate the NLRP3 inflammasome, depending on the diverse cellular stresses that they cause. NLRP3 inflammasome-related inhibitors and agents for NAFLD treatment have been tested and demonstrated positive effects in experimental models. Meanwhile, some drugs have been applied in clinical studies, supporting this therapeutic approach. In this review, we discuss the activation, biological functions, and treatment targeting the NLRP3 inflammasome in the context of NAFLD progression. Specifically, we focus on the different types of therapeutic agents that can inhibit the NLRP3 inflammasome and summarize their pharmacological effectiveness for NAFLD treatment.

Network pharmacology analysis and experimental validation to explore the mechanism of Shenlian extract on myocardial ischemia.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlian extract (SL), extracted from Salvia miltiorrhiza Bunge and Andrographis paniculata (Burm. f.) Nees, has been proved to be effective in the prevention and treatment of atherosclerosis. Recently, we have partially elucidated the mechanisms involved in the therapeutic effects of SL on myocardial ischemia (MI). However, the underlying mechanisms remain largely unclear. AIM OF THE STUDY: This study aims to explore the potential molecular mechanism of SL on MI on the basis of network pharmacology. MATERIALS AND METHODS: First, the main active ingredients of SL were screened in the Traditional Chinese Medicine Integrated Database, and the MI-associated targets were collected from the DisGeNET database. Then, we used compound-target and target-pathway networks to uncover the therapeutic mechanisms of SL. On the basis of network pharmacology analysis results, we assessed the effects of SL in MI rat model and oxygen glucose deprivation model of H9c2 cells and validated the possible molecular mechanisms of SL on myocardial injury in vivo and in vitro. RESULTS: The network pharmacology results showed that 37 potential targets were recognized, including TNF-α, Bcl-2, STAT3, PI3K and MMP2. These results revealed that the possible targets of SL were involved in the regulation of inflammation and apoptosis signaling pathway. Then, in vivo experiments indicated that SL significantly reduced the myocardial infarction size of MI rats. Serum CK-MB, cTnT, CK, LDH, and AST levels were significantly decreased by SL (P < 0.05 or P < 0.01). In vitro, SL significantly increased H9c2 cell viability. The levels of inflammation factors including TNF-α and MMP2 were significantly decreased by SL (P < 0.05 or P < 0.01). TUNEL and Annexin V/propidium iodide assays indicated that SL could significantly decrease the cell apoptotic rate in vivo and in vitro (P < 0.05 or P < 0.01). The remarkable upregulation of anti-apoptotic Bcl-2 and downregulation of pro-apoptotic Bax protein level further confirmed this result. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the PI3K-AKT and JAK2-STAT3 pathways were significantly enriched in SL. Compared with the model group, SL treatment significantly activated the PI3K-AKT and JAK2-STAT3 pathways in vivo and in vitro according to Western blot analyses. CONCLUSION: SL could protect the myocardium from MI injury. The underlying mechanism may be related to the reduction of inflammation and apoptosis by activating the PI3K/AKT and JAK2/STAT3 pathways.

Muyin extract inhibits non-small-cell lung cancer growth by inducing autophagy and apoptosis in vitro and in vivo.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer with a higher mortality rate. Both apoptosis and autophagy are crucial processes in the pathophysiology of NSCLC. Muyin extract (MSE) is a combination of Momordica cochinchinensis (Lour.) Spreng seeds and Epimedium brevicornu Maxim extract, with an optimal ratio of 1:1. Our previous research has firstly shown that MSE exerts a good anti-tumor activity, especially for NSCLC. PURPOSE: This study aims to evaluate the inhibitory effect of MSE on NSCLC and explore the underlying mechanism. METHODS: In vitro, cell proliferation was examined by MTT and colony formation. Apoptosis was detected by annexin V-FITC/PI assay while autophagy was assessed by Acridine orange (AO) and Monodansylcadaverine (MDC) staining. In vivo, Lewis lung cancer cell transplanted mice model was established to measure the effect of MSE on tumor growth. Hematoxylin eosin (H & E) staining was used to observe the pathological changes of the tumor after MSE treatment. The apoptosis in tumor tissue was detected by TUNEL assay. Meanwhile, the cellular proliferation marker Ki67 and autophagy marker LC3Ⅱ were observed by immunohistochemistry staining. The IL-4 and IFN-γ concentrations in blood were tested by Elisa. The apoptosis related factors (Bcl-2, Bax Caspase-3, cleaved Caspase-3, Caspase-9 and p53), autophagy marker proteins (Atg-5, Becline-1, LC3Ⅱ/Ⅰand p62) as well as Akt/mTOR pathway were detected by western blotting. RESULTS: Present study showed that MSE greatly inhibited the proliferation of NSCLC in vitro and in vivo, together with apoptotic rate increasing. P53 and cleaved Caspase-3 levels were up-regulated while Bcl-2/Bax ratio, Caspase-3 and Caspase-9 levels were significantly down-regulated treated with MSE. Meanwhile, MSE activated autophagy, Atg-5, Becline-1 as well as the ratio of LC3Ⅱ/Ⅰ were notably up-regulated while p62 was down-regulated after MSE treatment. Importantly, MSE significantly blocked Akt/mTOR pathway, which is a common upstream signal triggered by autophagy and apoptosis. Furthermore, when co-treated with specific autophagy inhibitor, the inhibitory rate and anti-apoptotic Bcl-2 level were significantly reversed. Impressively, MSE remarkably increased IFN-γ/ IL-4 ratio while VP16 did not in animal model, and the inhibition rate in tumor weight after MSE treatment was higher than xiaojin pill. CONCLUSION: Taken together, it is proved that MSE may be a promising oral TCM candidate for NSCLC therapy with immunity improvement. The underlying mechanisms could be associated with the induction of apoptosis and autophagy through blocking Akt/mTOR pathway, meanwhile, it may promote crosstalk between autophagy and apoptosis.

Generation and characterization of stable pig pregastrulation epiblast stem cell lines.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.