Design and synthesis of doublecortin-like kinase 1 inhibitors and their bioactivity evaluation.

Doublecortin-like kinase 1 (DCLK) is a microtubule-associated serine/threonine kinase that is upregulated in a wide range of cancers and is believed to be related to tumour growth and development. Upregulated DCLK1 has been used to identify patients at high risk of cancer progression and tumours with chemotherapy-resistance. Moreover, DCLK1 has been identified as a cancer stem cell (CSC) biomarker in various cancers, which has received considerable attention recently. Herein, a series of DCLK1 inhibitors were prepared based on the previously reported XMD8-92 structure. Among all the synthesised compounds, D1, D2, D6, D7, D8, D12, D14, and D15 showed higher DCLK1 inhibitory activities (IC50 40-74 nM) than XMD8-92 (IC50 161 nM). Compounds D1 and D2 were selective DCLK1 inhibitors as they showed a rather weak inhibitory effect on LRRK2. The antiproliferative activities of these compounds were also preliminarily evaluated. The structure-activity relationship revealed by our compounds provides useful guidance for the further development of DCLK1 inhibitors.

Design, Synthesis, and Antitumor Activity Evaluation of Proteolysis-Targeting Chimeras as Degraders of Extracellular Signal-Regulated Kinases 1/2.

Inhibition of the extracellular signal-regulated kinases 1/2 (ERK1/2) alone or in combination with other targets has emerged as a promising treatment strategy for a variety of human tumors. In addition to the development of inhibitors, the development of ERK1/2 degraders is an alternative approach to decrease its activity. We synthesized proteolysis-targeting chimeras (PROTACs) as effective ERK1/2 degraders, among which B1-10J showed high degradative activity, with DC50 of 102 nM and cytotoxic IC50 of 2.2 µM against HCT116 cells. Moreover, B1-10J dose-dependently inhibited tumor cell migration. Xenograft experiments in nude mice demonstrated that B1-10J inhibited HCT116 tumor cell growth and achieved significant regression of tumors at a daily dose of 25 mg/kg.

Design, Synthesis, and Biological Evaluation of Proteolysis-Targeting Chimeras as Highly Selective and Efficient Degraders of Extracellular Signal-Regulated Kinase 5.

Extracellular signal-regulated kinase 5 (ERK5) is recognized as a key member of the mitogen-activated protein kinase family and is involved in tumor growth, migration, and angiogenesis. However, the results of ERK5 inhibition in multiple studies are controversial, and a highly specific ERK5-targeting agent is required to confirm physiological functions. Using proteolysis-targeting chimera technology, we designed the selective ERK5 degrader PPM-3 and examined its biological effect on cancer cells. Interestingly, the selective degradation of ERK5 with PPM-3 did not influence tumor cell growth directly. Based on proteomics analysis, the ERK5 deletion may be associated with tumor immunity. PPM-3 influences tumor development by affecting the differentiation of macrophages. Therefore, PPM-3 is an effective small-molecule tool for studying ERK5 and a promising immunotherapy drug candidate.

Scutellarin attenuates oxidative stress and neuroinflammation in cerebral ischemia/reperfusion injury through PI3K/Akt-mediated Nrf2 signaling pathways.

Cerebral ischemia/reperfusion injury (CIRI) seriously threatens human life and health. Scutellarin (Scu) exhibits neuroprotective effects, but little is known about its underlying mechanism. Therefore, we explored its protective effect on CIRI and the underlying mechanism. Our results demonstrated that Scu rescued HT22 cells from cytotoxicity induced by oxygen and glucose deprivation/reoxygenation (OGD/R). Scu also showed antioxidant activity by promoting nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation, upregulating heme oxygenase-1 (HO-1) expression, increasing superoxide dismutase (SOD) activity, and inhibiting reactive oxygen species (ROS) generation in vitro. Additionally, Scu reduced nuclear factor-kappa B (NF-κB) activity and the levels of pro-inflammatory factors. Interestingly, these effects were abolished by Nrf2 inhibition. Furthermore, Scu reduced infarct volume and blood-brain barrier (BBB) permeability, improved sensorimotor functions and depressive behaviors, and alleviated oxidative stress and neuroinflammation in rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). Mechanistically, Scu-induced Nrf2 nuclear accumulation and inactivation of NF-κB were accompanied by an enhanced level of phosphorylated protein kinase B (p-AKT) both in vitro and in vivo. Pharmacologically inhibiting the phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathway blocked Scu-induced Nrf2 nuclear translocation and inactivation of NF-κB, as well as its antioxidant and anti-inflammatory activities. In summary, these results suggest that Scu exhibits antioxidant, anti-inflammatory, and neuroprotective effects in CIRI through Nrf2 activation mediated by the PI3K/Akt pathway.

Intranasal 15d-PGJ2 inhibits the growth of rat lactotroph pituitary neuroendocrine tumors by inducing PPARγ-dependent apoptotic and autophagic cell death.

PPARγ agonists have been reported to induce cell death in pituitary neuroendocrine tumor (PitNET) cell cultures. However, the therapeutic effects of PPARγ agonists in vivo remain unclear. In the present study, we found that intranasal 15d-PGJ2, an endogenous PPARγ agonist, resulted in growth suppression of Fischer 344 rat lactotroph PitNETs induced by subcutaneous implantation with a mini-osmotic pump containing estradiol. Intranasal 15d-PGJ2 reduced the volume and weight of the pituitary gland and the level of serum prolactin (PRL) in rat lactotroph PitNETs. 15d-PGJ2 treatment attenuated pathological changes and significantly decreased the ratio of PRL/pituitary-specific transcription factor 1 (Pit-1) and estrogen receptor α (ERα)/Pit-1 double-positive cells. Moreover, 15d-PGJ2 treatment induced apoptosis in the pituitary gland characterized by an increased ratio of TUNEL-positive cells, cleavage of caspase-3, and elevated activity of caspase-3. 15d-PGJ2 treatment decreased the levels of cytokines, including TNF-α, IL-1ß, and IL-6. Furthermore, 15d-PGJ2 treatment markedly increased the protein expression of PPARγ and blocked autophagic flux, as evidenced by the accumulation of LC3-II and SQSTM1/p62 and the decrease in LAMP-1 expression. Importantly, all these effects mediated by 15d-PGJ2 were abolished by cotreatment with the PPARγ antagonist GW9662. In conclusion, intranasal 15d-PGJ2 suppressed the growth of rat lactotroph PitNETs by inducing PPARγ-dependent apoptotic and autophagic cell death. Therefore, 15d-PGJ2 may be a potential new drug for lactotroph PitNETs.

Discovery of 3,5-diaryl-1H-pyrazol-based ureas as potent RET inhibitors.

Rearranged during transfection (RET) is a promising target for antitumor drug development. Multikinase inhibitors (MKI) have been developed for RET-driven cancers but displayed limited efficacy in disease control. Two selective RET inhibitors were approved by FDA in 2020 and proved potent clinical efficacy. However, the discovery of novel RET inhibitors with high target selectivity and improved safety is still highly desirable. Herein, we reported a class of 3,5-diaryl-1H-pyrazol-based ureas as new RET inhibitors. The representative compounds 17a/b displayed high selectivity to other kinases, and potently inhibited isogenic BaF3-CCDC6-RET cells harboring wild-type, or gatekeeper mutation (V804M). They also displayed moderate potency against BaF3-CCDC6-RET-G810C with solvent-front mutation. Compound 17b showed better pharmacokinetics properties and demonstrated promising oral in vivo antitumor efficacy in a BaF3-CCDC6-RET-V804M xenograft model. It may be utilized as a new lead compound for further development.

Increasing brain glucose uptake by Gypenoside LXXV ameliorates cognitive deficits in a mouse model of diabetic Alzheimer's disease.

We have previously reported that Gypenoside LXXV (GP-75), a novel natural PPARγ agonist isolated from Gynostemma pentaphyllum, ameliorated cognitive deficits in db/db mice. In this study, we further investigated the beneficial effects on cognitive impairment in APP/PS1 mice and a mouse model of diabetic AD (APP/PS1xdb/db mice). Interestingly, intragastric administration of GP-75 (40 mg/kg/day) for 3 months significantly attenuated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice. GP-75 treatment markedly reduced the levels of glucose, HbA1c and insulin in serum and improved glucose tolerance and insulin sensitivity in APP/PS1xdb/db mice. Notably, GP-75 treatment decreased the ß-amyloid (Aß) burden, as measured by 11 C-PIB PET imaging. Importantly, GP-75 treatment increased brain glucose uptake as measured by 18 F-FDG PET imaging. Moreover, GP-75 treatment upregulated PPARγ and increased phosphorylation of Akt (Ser473) and GLUT4 expression levels but decreased phosphorylation of IRS-1 (Ser616) in the hippocampi of both APP/PS1 and APP/PS1xdb/db mice. Furthermore, GP-75-induced increases in GLUT4 membrane translocation in primary hippocampal neurons from APP/PS1xdb/db mice was abolished by cotreatment with the selective PPARγ antagonist GW9662 or the PI3K inhibitor LY294002. In summary, GP-75 ameliorated cognitive deficits in APP/PS1 and APP/PS1xdb/db mice by enhancing glucose uptake via activation of the PPARγ/Akt/GLUT4 signaling pathways.

Increasing brain glucose metabolism by ligustrazine piperazine ameliorates cognitive deficits through PPARγ-dependent enhancement of mitophagy in APP/PS1 mice.

PPARγ agonists have been proven to be neuroprotective in vitro and in vivo models of Alzheimer's disease (AD). In the present study, we identified ligustrazine piperazine derivative (LPD) as a novel PPARγ agonist, which was detected by a dual-luciferase reporter assay system. LPD treatment dose-dependently reduced Aß40 and Aß42 levels in PC12 cells stably transfected with APP695swe and PSEN1dE9. Intragastric administration of LPD for 3 months dose-dependently reversed cognitive deficits in APP/PS1 mice. LPD treatment substantially decreased hippocampal Aß plaques in APP/PS1 mice and decreased the levels of Aß40 and Aß42 in vivo and in vitro. Moreover, LPD treatment induced mitophagy in vivo and in vitro and increased brain 18F-FDG uptake in APP/PS1 mice. LPD treatment significantly increased OCR, ATP production, maximal respiration, spare respiratory capacity, and basal respiration in APP/PS1 cells. Mechanistically, LPD treatment upregulated PPARγ, PINK1, and the phosphorylation of Parkin (Ser65) and increased the LC3-II/LC3-I ratio but decreased SQSTM1/p62 in vivo and in vitro. Importantly, all these protective effects mediated by LPD were abolished by cotreatment with the selective PPARγ antagonist GW9662. In summary, LPD could increase brain glucose metabolism and ameliorate cognitive deficits through PPARγ-dependent enhancement of mitophagy in APP/PS1 mice.

Alterations of the Gut Microbiome in Patients With Pituitary Adenoma.

Pituitary adenoma (PA) includes invasive pituitary adenoma (IPA) and noninvasive pituitary adenoma (NIPA), which are associated with the endocrine system. The gut microbiome plays an important role in human metabolism, but the association between the gut microbiome and pituitary adenoma remains unclear. A total of 44 subjects were enrolled in this study. Of these, 29 PA patients were further divided into IPA patients (n = 13) and NIPA patients (n = 16), while 15 healthy age-matched subjects were defined as control subjects. We collected faecal samples and characterized the gut microbial profiles by metagenomic sequencing using the Illumina X-ten platform. PLS-DA showed different microbial clusters among the three groups, and slightly different microbial ecological networks were observed. LEfSe analysis revealed significant alterations in the microbial community among PA patients. In particular, the enrichment of Clostridium innocuum, along with the reduced abundance of Oscillibacter sp. 57_20 and Fusobacterium mortiferum, were observed both in the IPA and NIPA groups compared to the control group. Moreover, PA patients could be effectively classified based on these bacteria using a support vector machine algorithm. In summary, this study demonstrated significant differences in the gut microbiome between PA patients and healthy controls. Future mechanistic experiments are needed to determine whether such alterations are a cause or consequence of pituitary adenoma.

A novel natural PPARγ agonist, Gypenoside LXXV, ameliorates cognitive deficits by enhancing brain glucose uptake via the activation of Akt/GLUT4 signaling in db/db mice.

Targeting the PPARγ might be a potential therapeutic strategy for diabetes-associated cognitive decline (DACD). In this study, Gypenoside LXXV (GP-75), a dammarane-type triterpene compound isolated from Gynostemma pentaphyllum, was found to be a novel PPARγ agonist using a dual-luciferase reporter assay system. However, whether GP-75 has protective effects against DACD remains unknown. Interestingly, intragastric administration of GP-75 (40 mg/kg/day) for 12 weeks significantly attenuated the cognitive deficit in db/db mice. GP-75 treatment significantly improved the glucose tolerance and lipid metabolism, and suppressed neuroinflammation. Notably, GP-75 treatment dramatically increased the uptake of glucose by the brain, as detected by 18 F-FDG PET. Incubation of primary cortical neurons with GP-75 significantly increased 2-deoxyglucose uptake. In addition, GP-75 treatment markedly increased the p-Akt (Ser 473)/total Akt levels and the expression levels of PPARγ and GLUT4, while decreasing the levels of p-IRS-1 (Ser 616)/total IRS-1. Importantly, all of these protective effects mediated by GP-75 were abolished by cotreatment with the PPARγ antagonist, GW9662. However, GP-75-mediated PPARγ upregulation was not affected by coincubation with the phosphatidylinositol 3-kinase inhibitor, LY294002. Collectively, GP-75 might be a novel PPARγ agonist that ameliorates cognitive deficit by enhancing brain glucose uptake via the activation of Akt/GLUT4 signaling in db/db mice.

Folium Ginkgo extract and tetramethylpyrazine sodium chloride injection (Xingxiong injection) protects against focal cerebral ischaemia/reperfusion injury via activating the Akt/Nrf2 pathway and inhibiting NLRP3 inflammasome activation.

CONTEXT: Folium Ginkgo extract and tetramethylpyrazine sodium chloride injection (Xingxiong injection) is a compound preparation commonly used for treating cerebral ischaemia/reperfusion injury in ischaemic stroke in China. However, its potential mechanisms on ischaemic stroke remain unknown. OBJECTIVE: This study explores the potential mechanisms of Xingxiong injection in vivo or in vitro. MATERIALS AND METHODS: Sprague-Dawley (SD) rats were randomly assigned to five groups: the sham (normal saline), the model (normal saline) and the Xingxiong injection groups (12.5, 25 or 50 mL/kg). The rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 14 d. Xingxiong injection was administered via intraperitoneal (i.p.) injection immediately after ischaemia induction for 14 d. Afterwards, rats were sacrificed at 14 d induced by administration of Xingxiong injection. RESULTS: Xingxiong injection significantly reduces infarct volume (23%) and neurological deficit scores (93%) compared with the MCAO/R group. Additionally, Xingxiong injection inhibits the loss in mitochondrial membrane potential (43%) and reduces caspase-3 level (44%), decreases NOX (41%), protein carbonyl (29%), 4-HNE (40%) and 8-OhdG (41%) levels, inhibits the expression of inflammatory factors, such as TNF-α (26%), IL-1ß (34%), IL-6 (39%), MCP-1 (36%), CD11a (41%) and ICAM-1 (43%). Moreover, Xingxiong injection can increase p-Akt/Akt (35%) and Nrf2 (47%) protein expression and inhibit NLRP3 (42%) protein expression. CONCLUSIONS: Xingxiong injection prevents cerebral ischaemia/reperfusion injury via activating the Akt/Nrf2 pathway and inhibiting NLRP3 inflammasome. These findings provide experimental evidence for clinical use of drugs in the treatment of ischaemic stroke.

Notoginsenoside R1 activates the NAMPT-NAD+-SIRT1 cascade to promote postischemic angiogenesis by modulating Notch signaling.

Nicotinamide phosphoribosyltransferase (NAMPT) maintains mitochondrial function and protects against cerebral ischemic injury by improving energy metabolism. Notoginsenoside R1 (R1), a unique constituent of Panax notoginseng, has been shown to promote the proliferation and tube formation of human umbilical vein endothelial cells. Whether R1 has proangiogenesis on the activation of NAMPT in ischemic stroke remains unclear. The purpose of this study was to investigate the pharmacodynamic effect and mechanism of R1 on angiogenesis after ischemic stroke. We used male Sprague-Dawley (SD) rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). R1 was administered via intraperitoneal (i.p.) injection immediately after ischemia induction. The promotion of R1 on angiogenesis were detected by immunofluorescence staining, 3D stereoscopic imaging and transmission electron microscopy detection. HBMEC cells were pretreated with different concentrations of R1 for 12 h before oxygen-glucose deprivation/reoxygenation (OGD/R) exposure. Afterward, scratch assay, EdU staining and tube formation were determined. Western blot analyses of proteins, including those involved in angiogenesis, NAMPT-SIRT1 cascade, VEGFR-2, and Notch signaling, were conducted. We showed that R1 significantly restored cerebral blood flow, improved mitochondrial energy metabolism and promoted angiogenesis. More importantly, incubation with 12.5-50 µM R1 significantly increased the migration, proliferation and tube formation of HBMECs in vitro. The promotion of R1 on angiogenesis were associated with the NAMPT-NAD+-SIRT1 cascade and Notch/VEGFR-2 signaling pathway, which was partially eliminated by inhibitors of NAMPT and SIRT1. We demonstrated that R1 promotes post-stroke angiogenesis via activating NAMPT-NAD+-SIRT1 cascade. The modulation of Notch signaling and VEGFR-2 contribute to the post-stroke angiogenesis. These findings offer insight for exploring new therapeutic strategies for neurorestoration via R1 treatment after ischemic stroke.

Cassane diterpenoid derivative induces apoptosis in IDH1 mutant glioma cells through the inhibition of glutaminase in vitro and in vivo.

BACKGROUND: Glioblastoma multiforme (GBM) is the most frequent, lethal and aggressive tumour of the central nervous system in adults. The discovery of novel anti-GBM agents based on the isocitrate dehydrogenase (IDH) mutant phenotypes and classifications have attracted comprehensive attention. PURPOSE: Diterpenoids are a class of naturally occurring 20-carbon isoprenoid compounds, and have previously been shown to possess high cytotoxicity for a variety of human tumours in many scientific reports. In the present study, 31 cassane diterpenoids of four types, namely, butanolide lactone cassane diterpenoids (I) (1-10), tricyclic cassane diterpenoids (II) (11-15), polyoxybutanolide lactone cassane diterpenoids (III) (16-23), and fused furan ring cassane diterpenoids (IV) (24-31), were tested for their anti-glioblastoma activity and mechanism underlying based on IDH1 mutant phenotypes of primary GBM cell cultures and human oligodendroglioma (HOG) cell lines. RESULTS: We confirmed that tricyclic-type (II) and compound 13 (Caesalpin A, CSA) showed the best anti-neoplastic potencies in IDH1 mutant glioma cells compared with the other types and compounds. Furthermore, the structure-relationship analysis indicated that the carbonyl group at C-12 and an α, ß-unsaturated ketone unit fundamentally contributed to enhancing the anti-glioma activity. Studies investigating the mechanism demonstrated that CSA induced oxidative stress via causing glutathione reduction and NOS activation by negatively regulating glutaminase (GLS), which proved to be highly dependent on IDH mutant type glioblastoma. Finally, GLS overexpression reversed the CSA-induced anti-glioma effects in vitro and in vivo, which indicated that the reduction of GLS contributed to the CSA-induced proliferation inhibition and apoptosis in HOG-IDH1-mu cells. CONCLUSION: Therefore, the present results demonstrated that compared with other diterpenoids, tricyclic-type diterpenoids could be a targeted drug candidate for the treatment of secondary IDH1 mutant type glioblastoma through negatively regulating GLS.

Xuesaitong injection (lyophilized) combined with aspirin and clopidogrel protect against focal cerebral ischemic/reperfusion injury in rats by suppressing oxidative stress and inflammation and regulating the NOX2/IL-6/STAT3 pathway.

BACKGROUND: Combination of aspirin (ASA) and clopidogrel (CLP) [dual antiplatelet therapy (DAPT)] has been limited in reducing early recurrent stroke events. Xuesaitong injection (lyophilized) (XST) made of total saponins from P. notoginseng, which significantly improves cerebral circulation and has been widely used in clinical applications for decades to treat and prevent ischemic stroke. Here, we confirmed the protective effect and mechanism of XST combined with DAPT (XST+ASA+CLP) on cerebral ischemia/reperfusion injury, exploring their better pharmacological action for clinical patients. METHODS: Sprague-Dawley rats (SD rats) (n=9 in each group) were randomly assigned to three groups and pretreated with XST, ASA+CLP, or XST+ASA+CLP for 7 days. Then rats were subjected to 2 h of middle cerebral artery occlusion (MCAO) followed by reperfusion for 24 h. Therapeutic effect of XST+ASA+CLP was measured by infarct volume, neurological behavior and regional cerebral blood flow (rCBF). Inhibition of neuronal apoptosis and glial cells was determined by immunofluorescent staining. We studied the protein levels of neurotrophic factors, neuroplasticity-related factors, oxidative stress indicators and inflammatory factors by ELISA assay. RESULTS: XST+ASA+CLP group showed significant reduction in infarct volumes and neurological deficit scores. XST+ASA+CLP group also had higher levels in rCBF and synaptic growth, and showed remarkable inhibition of microglia and astrocytes activation and the neuronal apoptosis. In addition, XST+ASA+CLP group had lower levels of NADPH, protein carbonyl, 4-hydroxynonenal (4-HNE), 8-hydroxydeoxyguanosine (8-OHdG) and several inflammatory cytokines. Moreover, XST+ASA+CLP group also had lower levels of NOX2, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, and p-STAT3/STAT3. CONCLUSIONS: These results demonstrate that a combination of XST, ASA, and CLP effectively protected rats against middle cerebral artery occlusion/reperfusion (MCAO/R) injury by suppressing the NOX2/IL-6/ STAT3 pathway. These novel findings provide theoretical basis and experimental evidence for the rationality of clinical combined use of drugs in the treatment of ischemic stroke.

Notoginseng Leaf Triterpenes Ameliorates OGD/R-Induced Neuronal Injury via SIRT1/2/3-Foxo3a-MnSOD/PGC-1α Signaling Pathways Mediated by the NAMPT-NAD Pathway.

BACKGROUND: Cerebral ischemic stroke (CIS) is a common cerebrovascular disease whose main risks include necrosis, apoptosis, and cerebral infarction. But few therapeutic advances and prominent drugs seem to be of value for ischemic stroke in the clinic yet. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stem and leaf have been confirmed to have neuroprotective effects against mitochondrial damages caused by cerebral ischemia in vivo. However, the potential mechanisms of mitochondrial protection have not been fully elaborated yet. METHODS: The oxygen and glucose deprivation and reperfusion (OGD/R)-induced SH-SY5Y cells were adopted to explore the neuroprotective effects and the potential mechanisms of PNGL in vitro. Cellular cytotoxicity was measured by MTT, viable mitochondrial staining, and antioxidant marker detection in vitro.Mitochondrial functions were analyzed by ATP content measurement, MMP determination, ROS, NAD, and NADH kit in vitro. And the inhibitor FK866 was adopted to verify the regulation of PNGL on the target NAMPT and its key downstream. RESULTS: In OGD/R models, treatment with PNGL strikingly alleviated ischemia injury, obviously preserved redox balance and excessive oxidative stress, inhibited mitochondrial damage, markedly alleviated energy metabolism dysfunction, improved neuronal mitochondrial functions, obviously reduced neuronal loss and apoptosis in vitro, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL markedly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions and OGD/R-induced SH-SY5Y cells and regulated the downstream SIRT1/2-Foxo3a and SIRT1/3-MnSOD/PGC-1α pathways. And FK866 further verified that the protective effects of PNGL might be mediated by the NAMPT in vitro. CONCLUSIONS: The mitochondrial protective effects of PNGL are, at least partly, mediated via the NAMPT-NAD+ and its downstream SIRT1/2/3-Foxo3a-MnSOD/PGC-1α signaling pathways. PNGL, as a new drug candidate, has a pivotal role in mitochondrial homeostasis and energy metabolism therapy via NAMPT against OGD-induced SH-SY5Y cell injury.

Disrupting phosphatase SHP2 in macrophages protects mice from high-fat diet-induced hepatic steatosis and insulin resistance by elevating IL-18 levels.

Chronic low-grade inflammation plays an important role in the pathogenesis of type 2 diabetes. Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2) has been reported to play diverse roles in different tissues during the development of metabolic disorders. We previously reported that SHP2 inhibition in macrophages results in increased cytokine production. Here, we investigated the association between SHP2 inhibition in macrophages and the development of metabolic diseases. Unexpectedly, we found that mice with a conditional SHP2 knockout in macrophages (cSHP2-KO) have ameliorated metabolic disorders. cSHP2-KO mice fed a high-fat diet (HFD) gained less body weight and exhibited decreased hepatic steatosis, as well as improved glucose intolerance and insulin sensitivity, compared with HFD-fed WT littermates. Further experiments revealed that SHP2 deficiency leads to hyperactivation of caspase-1 and subsequent elevation of interleukin 18 (IL-18) levels, both in vivo and in vitro Of note, IL-18 neutralization and caspase-1 knockout reversed the amelioration of hepatic steatosis and insulin resistance observed in the cSHP2-KO mice. Administration of two specific SHP2 inhibitors, SHP099 and Phps1, improved HFD-induced hepatic steatosis and insulin resistance. Our findings provide detailed insights into the role of macrophagic SHP2 in metabolic disorders. We conclude that pharmacological inhibition of SHP2 may represent a therapeutic strategy for the management of type 2 diabetes.

Nano-encapsulated tryptanthrin derivative for combined anticancer therapy via inhibiting indoleamine 2,3-dioxygenase and inducing immunogenic cell death.

Aim: We developed a polycaprolactone-based nanoparticle (NP) to encapsulate tryptanthrin derivative CY-1-4 and evaluated its antitumor efficacy. Materials & methods: CY-1-4 NPs were prepared and evaluated for their cytotoxicity and associated mechanisms, indoleamine 2,3-dioxygenase (IDO)-inhibitory ability, immunogenic cell death (ICD)-inducing ability and antitumor efficacy. Results: CY-1-4 NPs were 123 nm in size. In vitro experiments indicated that they could both induce ICD and inhibit IDO. In vivo studies indicated that a medium dose reduced 58% of the tumor burden in a B16-F10-bearing mouse model, decreased IDO expression in tumor tissues and regulated lymphocytes subsets in spleen and tumors. Conclusion: CY-1-4 is a potential antitumor candidate that could act as a single agent with combined functions of IDO inhibition and ICD induction.

SHP2 inhibition triggers anti-tumor immunity and synergizes with PD-1 blockade.

Tyrosine phosphatase SHP2 is a promising drug target in cancer immunotherapy due to its bidirectional role in both tumor growth promotion and T-cell inactivation. Its allosteric inhibitor SHP099 is known to inhibit cancer cell growth both in vitro and in vivo. However, whether SHP099-mediated SHP2 inhibition retards tumor growth in vivo via anti-tumor immunity remains elusive. To address this, a CT-26 colon cancer xenograft model was established in mice since this cell line is insensitive to SHP099. Consequently, SHP099 minimally affected CT-26 tumor growth in immuno-deficient nude mice, but significantly decreased the tumor burden in CT-26 tumor-bearing mice with intact immune system. SHP099 augmented anti-tumor immunity, as shown by the elevated proportion of CD8+IFN-γ + T cells and the upregulation of cytotoxic T-cell related genes including Granzyme B andPerforin, which decreased the tumor load. In addition, tumor growth in mice with SHP2-deficient T-cells was markedly slowed down because of enhanced anti-tumor responses. Finally, the combination of SHP099 and anti-PD-1 antibody showed a higher therapeutic efficacy than either monotherapy in controlling tumor growth in two colon cancer xenograft models, indicating that these agents complement each other. Our study suggests that SHP2 inhibitor SHP099 is a promising candidate drug for cancer immunotherapy.

Notoginsenoside R1 Ameliorates Diabetic Retinopathy through PINK1-Dependent Activation of Mitophagy.

: Accumulating evidence has indicated that inflammation, oxidative stress, apoptosis, and autophagy in retinal Müller cells are involved in diabetic retinopathy (DR). Notoginsenoside R1 (NGR1), a novel saponin extracted from Panax notoginseng, posesses pharmacological properties, including treating diabetic encephalopathy and improving microcirculatory disorders. Nevertheless, its beneficial effects on DR and the potential mechanism remain to be elucidated. In this study, we found retinal vascular degeneration, reduced retinal thickness, and impaired retinal function in db/db mice were all dramatically attenuated by oral treatment with NGR1 (30 mg/kg) for 12 weeks. NGR1 pretreatment also significantly inhibited apoptosis, markedly suppressed the VEGF expression, markedly increased PEDF expression and markedly inhibited oxidative stress and inflammation in rat retinal Müller cells (rMC-1) subjected to high glucose (HG) and in the retinas of db/db mice. Furthermore, NGR1 pre-treatment upregulated the level of PINK1 and Parkin, increased the LC3-II/LC3-I ratio, and downregulated the level of p62/SQSTM1 in rMC-1 cells induced by HG and in the retinas of db/db mice. Moreover, NGR1 administration enhanced the co-localization of GFP-LC3 puncta and MitoTracker in rMC-1 cells. Importantly, knockdown of PINK1 abolished the protective effects of NGR1. In conclusion, these phenomena suggested that NGR1 prevented DR via PINK1-dependent enhancement of mitophagy.

Exploring on the bioactive markers of Codonopsis Radix by correlation analysis between chemical constituents and pharmacological effects.

ETHNOPHARMACOLOGICAL RELEVANCE: Codonopsis Radix is a commonly used traditional Chinese medicine, and has the effect of strengthening spleen and tonifying lung, nourishing blood and engendering liquid. In addition, it is also used as important food materials. AIM OF THE STUDY: The aim of the study was to explain the underlying correlations between chemical constituents and pharmacological effects and explore the bioactive markers of Codonopsis Radix. MATERIALS AND METHODS: Codonopsis Radix samples from Min county, Gansu province processed with different methods were taken as the materials, UPLC-ESI-Q-TOF-MS/MS analysis was conducted to identify the compounds and establish UPLC fingerprint. Meanwhile, hematopoietic and immunologic functions of Codonopsis Radix were investigated to obtain relevant pharmacological index. Then, the correlation analysis between chemical constituents in UPLC fingerprints and pharmacological effects was carried out. The plant name was confirmed to the database "The Plant List" (www.theplantlist.org). RESULTS: According to the results of canonical correlation analysis, tryptophan, syringin, tangshenoside I, codonopyrrolidium A, lobetyolin and two unknown compounds might be the potential bioactive markers related to the hematopoietic and immunologic functions of Codonopsis Radix, which could be recommended as the index compounds. CONCLUSION: This study illustrated the underlying correlations between chemical constituents and pharmacological effects, explored the pharmacological material basis, and could lay a foundation for the improvement of quality standard of Codonopsis Radix.