Kellerin alleviates cerebral ischemic injury by inhibiting ferroptosis via targeting Akt-mediated transcriptional activation of Nrf2.

BACKGROUND: Ischemic stroke (IS) is characterized as a detrimental cerebrovascular disease with high mortality and disability. Ferroptosis is a novel mechanism involved in neuronal death. There is a close connection between IS and ferroptosis, and inhibiting ferroptosis may provide an effective strategy for treating IS. Our previous investigations have discovered that kellerin, the active compound of Ferula sinkiangensis K. M. Shen, possesses the capability to shield against cerebral ischemia injury. PURPOSE: Our objective is to clarify the relationship between the neuroprotective properties of kellerin against IS and its ability to modulate ferroptosis, and investigate the underlying regulatory pathway. STUDY DESIGN: We investigated the impact and mechanism of kellerin in C57BL/6 mice underwent middle cerebral artery occlusion/reperfusion (MCAO/R) as well as SH-SY5Y cells exposed to oxygen-glucose deprivation/ re-oxygenation (OGD/R). METHODS: The roles of kellerin on neurological severity, cerebral infarction and edema were investigated in vivo. The regulatory impacts of kellerin on ferroptosis, mitochondrial damage and Akt/Nrf2 pathway were explored. Molecular docking combined with drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) were performed to analyze the potential target proteins for kellerin. RESULTS: Kellerin protected against IS and inhibited ferroptosis in vivo. Meanwhile, kellerin improved the neuronal damage caused by OGD/R and suppressed ferroptosis by inhibiting the production of mitochondrial ROS in vitro. Further we found that kellerin directly interacted with Akt and enhanced its phosphorylation, leading to the increase of Nrf2 nuclear translocation and its downstream antioxidant genes expression. Moreover, kellerin's inhibitory effect on ferroptosis and mitochondrial ROS release was eliminated by inhibiting Akt/Nrf2 pathway. CONCLUSIONS: Our study firstly demonstrates that the neuroprotective properties of kellerin against IS are related to suppressing ferroptosis through inhibiting the production of mitochondrial ROS, in which its modulation on Akt-mediated transcriptional activation of Nrf2 plays an important role. This finding shed light on the potential mechanism that kellerin exerts therapeutic effects in IS.

Loureirin C inhibits ferroptosis after cerebral ischemia reperfusion through regulation of the Nrf2 pathway in mice.

BACKGROUND: Ischemic stroke (IS) is considered as a serious cerebral vascular disease. Ferroptosis is a novel type of regulated cell death (RCD), that closely related to the occurrence and progress of IS. Loureirin C, a type of dihydrochalcone compound derived from the Chinese Dragon's blood (CDB). The effective components extracted from CDB have shown neuroprotective effects in ischemia reperfusion models. However, the role of Loureirin C in mice after IS is not well understood. Thus, it is worth to identify the effect and mechanism of Loureirin C on IS. PURPOSE: The present research aims to prove the existence of ferroptosis in IS and explore whether Loureirin C can inhibit ferroptosis by regulating nuclear factor E2 related factor 2 (Nrf2) pathway in mice and exert neuroprotective effects on IS models. METHODS: Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established to evaluate the occurrence of ferroptosis and the potential Loureirin C brain-protective effect in vivo. The analysis of free iron, glutamate content, reactive oxygen species (ROS) and lipid peroxidation levels, along with transmission electron microscope (TEM) was applied to prove the existence of ferroptosis. The function of Loureirin C on Nrf2 nuclear translocation was verified by immunofluorescence staining. In vitro, primary neurons and SH-SY5Y cells were processed with Loureirin C after oxygen and glucose deprivation-reperfusion (OGD/R). ELISA kits, western blotting, co-immunoprecipitation (Co-IP) analysis, immunofluorescence, and quantitative real-time PCR were devoted to proving the neuroprotective effects of Loureirin C on IS via regulating ferroptosis and Nrf2 pathways. RESULTS: The results showed that Loureirin C not only dramatically alleviated brain injury and inhibited neurons ferroptosis in mice after MCAO/R, but also dose-dependently reduce ROS accumulation in ferroptosis after OGD/R. Further, Loureirin C inhibits ferroptosis by activating Nrf2 pathway, and promoting nuclear translocation of Nrf2. Besides, Loureirin C increases heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1) and glutathione peroxidase 4 (GPX4) content after IS. Intriguingly, the anti-ferroptosis effect of Loureirin C is weakened by Nrf2 knockdown. CONCLUSION: Our discoveries first revealed that the inhibitory action of Loureirin C on ferroptosis may greatly depend on its adjusting effect on the Nrf2 pathway, suggesting that Loureirin C could act as a novel anti-ferroptosis candidate and play a therapeutic role in IS. These novel discoveries on the role of Loureirin C on IS models reveal an innovative method that may contribute to neuroprotection for the prevention of IS.

Profiling aromatic constituents of Chimonanthus nitens Oliv. leaf granule using mass spectrometry.

RATIONALE: The chemical constituents of Chinese patent medicine are usually different from those of crude medicine because of specific preparation processes. Chimonanthus nitens Oliv. leaf granule is widely used for prevention against COVID-19 in China. However, no research has been reported on the chemical constituents of the granule and their variation during the preparation process. METHODS: Fragmentation patterns of reference compounds were investigated using electrospray ionization mass spectrometry, and the new gas-phase reaction was demonstrated by electronic and steric effects and calculated chemistry. Then, a strategy based on new fragmentation patterns was used to profile aromatic constituents. In addition, based on untargeted metabolomics analytical workflow, a comparison was made on the chemical constituents of the leaf and granule. RESULTS: New fragmentation patterns related to two competing reactions, ring-opening and ring-closing reactions for coumarin, have been proposed and investigated in depth. The newly established diagnostic ion at m/z 81.0331 worked strongly in the assignment of OH-7 and substituent at C-8 of coumarin. McLafferty rearrangement occurring in coumarin glycoside while sugar group locating at C-4 was first observed, and new diagnostic ions at m/z 147.0440, 119.0488, and 91.0543 were constructed. CONCLUSIONS: Aromatic constituents of the granule were first profiled. A total of 114 aromatic compounds were identified; of these 85 compounds were identified first. Kaempferol-7-O-neohesperidoside and its homologues were mostly enriched in the granule. Considering their reported bioactivities, these analogues possibly contribute greatly to clinical efficacy. Our results provided a new fragmentation theory for coumarins and a new material basis for the quality control of the granule.

Effects of forest age on soil erosion and nutrient loss in Dianchi watershed, China.

Soil erosion and nutrient loss are important environmental and ecological problems in the Dianchi watershed in southwestern China. Woodlands-the primary land type in the Dianchi watershed-play an important ecological role in controlling soil and water loss. In this study, we compared soil erosion and loss of total organic carbon (TOC), total nitrogen (TN), and total phosphorus (TP) in woodlands of different ages, i.e., young forest, medium forest, and near-mature forest, at the Dongda River catchment in south-western Dianchi watershed. Furthermore, changes in stoichiometries in soil were analyzed. The average degree of erosion of each forest age stage was below moderate. Based on the non-arable soil erosion modulus models of 137Cs and 210Pbex, the soil erosion rates decreased gradually with the increasing forest age. The forest age affected soil nutrient distribution and loss. The losses of TOC and TP gradually decreased, while the losses of TN first increased and then decreased with the growth of forest age. TOC, TN, and TP were enriched in the topsoil. Forest age affected soil stoichiometry and soil nutrient supply level. In general, the forest can effectively reduce soil erosion and nutrient loss in the red soil area with the forest age increasing.

Response of sedimentation rate to environmental evolution in Da River Reservoir in Southwest China.

Lake sediment records the evolution process of the interaction between human and nature. It is important to master the lacustrine sedimentation rate for the ecological environment assessment of catchment. A 60-cm sediment core was collected in the Da River Reservoir during 2019 to analyze radionuclides (210Pb and 137Cs) massic activities, grain size, total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), and metals (Mn, Cu, Al, and Pb) mass fractions to reconstruct the response of sedimentation rate to environmental evolution. The environmental changes in the small catchment were classified into the following three stages through cluster analysis (CA) for geochemical parameters in the sediment core: phase I (1881-1985), phase II (1987-1999), and phase III (2000-2018). The average depth sedimentation rates (ADSRs) of the three stages were 0.33, 0.90, and 1.50 cm/year, respectively. The sedimentation rates increased from the bottom to the surface layer, indicating that the exogenous inputs into the reservoir have been occurring. The sediment deposition in phase III was strongly disturbed by the environmental changes (such as warmer climate and intensified land use). Therefore, sedimentation rates showed a rapid increase. Both Pearson correlation analysis and redundancy analysis (RDA) showed that sedimentation rates were positively correlated with climatic factors, particle size, nutrients and metals mass fractions, elemental ratios, and socioeconomic parameters. Sedimentation rates show high sensitivity to anthropogenic activities and climatic change, which can be used to reconstruct the environmental evolution process at a small catchment scale.

Sedimentary record of nutrients and sources of organic matter in the Shuanglong reservoir, Dianchi watershed, China.

In the last few decades, the eutrophication of lakes has been a serious issue in the middle and lower reaches of the Yangtze River watershed. To explore the relationship between lake systems and anthropogenic activities, sediments were collected from the Shuanglong reservoir in the Dianchi watershed in Southwest China. Total nitrogen (TN), total phosphorus (TP), total organic carbon (TOC), and the carbon isotopic ratio (δ13C) were analyzed in sediment cores to reconstruct the effects of natural succession and human activities on the past lacustrine environmental conditions. A reliable chronology of the sediment core was established by using the 210Pb dating technique, which indicated that the age span of the 70-cm sediment core is from the years 1871 to 2011. Above - 31 cm depth in the core, TN, TP, TOC, C/N, and δ13C increased significantly, indicating that eutrophication has occurred since the 1980s. By combining the indicators of δ13C and C/N, it was shown that terrestrial and lacustrine components were the main sources of organic matter (OM) in the reservoir, which was mostly controlled by terrestrial C3 plants and algae. Since the 1980s, increased sewage discharge, fish aquaculture, fertilizer application, population, and economic strength have sped up the eutrophication process, and the eutrophication was further intensified in 2001.

Lipoprotein lipase in hypothalamus is a key regulator of body weight gain and glucose homeostasis in mice.

AIMS/HYPOTHESIS: Regulation of energy balance involves the participation of many factors, including nutrients, among which are circulating lipids, acting as peripheral signals informing the central nervous system of the energy status of the organism. It has been shown that neuronal lipoprotein lipase (LPL) participates in the control of energy balance by hydrolysing lipid particles enriched in triacylglycerols. Here, we tested the hypothesis that LPL in the mediobasal hypothalamus (MBH), a well-known nucleus implicated in the regulation of metabolic homeostasis, could also contribute to the regulation of body weight and glucose homeostasis. METHODS: We injected an adeno-associated virus (AAV) expressing Cre-green fluorescent protein into the MBH of Lpl-floxed mice (and wild-type mice) to specifically decrease LPL activity in the MBH. In parallel, we injected an AAV overexpressing Lpl into the MBH of wild-type mice. We then studied energy homeostasis and hypothalamic ceramide content. RESULTS: The partial deletion of Lpl in the MBH in mice led to an increase in body weight compared with controls (37.72 ± 0.7 g vs 28.46 ± 0.12, p < 0.001) associated with a decrease in locomotor activity. These mice developed hyperinsulinaemia and glucose intolerance. This phenotype also displayed reduced expression of Cers1 in the hypothalamus as well as decreased concentration of several C18 species of ceramides and a 3-fold decrease in total ceramide intensity. Conversely, overexpression of Lpl specifically in the MBH induced a decrease in body weight. CONCLUSIONS/INTERPRETATION: Our study shows that LPL in the MBH is an important regulator of body weight and glucose homeostasis.

Role of activating transcription factor 4 in the hepatic response to amino acid depletion by asparaginase.

The anti-leukemic agent asparaginase activates the integrated stress response (ISR) kinase GCN2 and inhibits signaling via mechanistic target of rapamycin complex 1 (mTORC1). The study objective was to investigate the protective role of activating transcription factor 4 (ATF4) in controlling the hepatic transcriptome and mediating GCN2-mTORC1 signaling during asparaginase. We compared global gene expression patterns in livers from wildtype, Gcn2 -/-, and Atf4 -/- mice treated with asparaginase or excipient and further explored selected responses in livers from Atf4 +/- mice. Here, we show that ATF4 controls a hepatic gene expression profile that overlaps with GCN2 but is not required for downregulation of mTORC1 during asparaginase. Ingenuity pathway analysis indicates GCN2 independently influences inflammation-mediated hepatic processes whereas ATF4 uniquely associates with cholesterol metabolism and endoplasmic reticulum (ER) stress. Livers from Atf4 -/- or Atf4 +/- mice displayed an amplification of the amino acid response and ER stress response transcriptional signatures. In contrast, reduction in hepatic mTORC1 signaling was retained in Atf4 -/- mice treated with asparaginase. CONCLUSIONS: GCN2 and ATF4 serve complementary roles in the hepatic response to asparaginase. GCN2 functions to limit inflammation and mTORC1 signaling whereas ATF4 serves to limit the amino acid response and prevent ER stress during amino acid depletion by asparaginase.

[Influence of scum of algal bloom on the release of N and P from sediments of Lake Taihu].

Sediment cores were sampled in macrophyte dominated zone, phytoplankton dominated zone and river mouth in Lake Taihu and incubated with one half of them added algae in laboratory in August to reveal the influence of algal accumulation on the release of nitrogen and phosphorus in sediments. Concentrations of dissolved oxygen (DO), total nitrogen (TN), total phosphorus (TP), ammonium, phosphate and other parameters were determined during cultivation of the sediment cores in dark. There were differences in release of TN and TP in sediments and significant variations in changes of DO, ammonium, phosphate in overlying water after adding algae. When DO declined to nearly zero, the changes of release of TN, TP in sediments differed from different ecological types of Lake Taihu with minus numbers of -0.1, -3.01 mg x (m2 x d)(-1) in macrophyte dominated zone, larger numbers of 31.1, 0.75 mg x (m2 x d)(-1) in phytoplankton dominated zone and 9.5, 2.46 mg x (m2 x d)(-1) in river mouth. The concentrations of ammonium in overlying water increased 3.62, 5.10, 6.57 mg/L and phosphate increased 53, 219, 418 microg/L in macrophyte dominated zone, phytoplankton dominated zone, river mouth, respectively. The changes of nutrients have good correlations with the decrease of DO and duration. The release of nutrients from sediments caused by accumulation and decay of algae may be the way that algal bloom satisfied itself.

Icariin exterts negative effects on human gastric cancer cell invasion and migration by vasodilator-stimulated phosphoprotein via Rac1 pathway.

Cellular movement is mainly orchestrated by actin-dependent cytoskeleton in which Rho GTPase Rac1 or vasodilator-stimulated phosphoprotein (VASP) closely collaborates. In the present in vitro study, we investigated the inhibitory effect and underlying molecular mechanism of icariin, a pure extract of the traditional Chinese medicine Herba epimedii, on the invasive and migration properties of human gastric cancer cell line BGC-823. At 50% growth-inhibiting concentration, icariin significantly suppressed tumor cells migration and invasion, which were traceable to down-regulation of Rac1 and VASP. Together with icariin, the selected siRNA targeting Rac1 or VASP reinforced these inhibitory effects. Rac1-siRNA-dependent down-regulation of Rac1 led to a large drop in VASP expression, whereas VASP-siRNA led to a slight fall in Rac1 expression, implying that the amount of Rac1 may influence VASP expression level. Moreover, transfection with Rac1 plasmids pcDNA3-EGFP-Rac1-Q61L led to the enhancement in expression level of both Rac1 and VASP. These results indicate that icariin exerts negative effects on tumor cell invasion and migration via the Rac1-dependent VASP pathway and may be a potential anti-cancer drug.

Phytoplankton community from Lake Taihu, China, has dissimilar responses to inorganic and organic nutrients.

To evaluate the response of phytoplankton from Lake Taihu to different types of nutrients, the phytoplankton responses were measured after adding inorganic nitrogen (N) and phosphorus (P) or decomposed algal scum (Microcystis spp.) into the lake water. Both types of nutrients promoted an increase in phytoplankton biomass as determined by chlorophyll a and algal wet weight. The addition of decomposed algal scum resulted in a significantly greater phytoplankton response than the addition of inorganic N and P alone. The dissolved inorganic N and P in the inorganic nutrient treatment were found not limit phytoplankton growth. The higher algal biomass obtained in the treatment with decomposed algal scum indicated the importance of other organic nutrients besides N and P such as trace elements, as well as the importance of the form of N since the levels of ammonia nitrogen (NH4(+)-N) from the decomposed algal treatment were actually higher than that of the inorganic N and P addition. Microcystis spp. (Cyanobacteria), Scenedesmus spp. (Chlorophyta) and Synechocystis spp. (Cyanobacteria) were the dominant taxa in the control, inorganic N and P treatment, and the decomposed algal scum treatment, respectively. Microcystis never bloomed in response to both types of nutrient additions indicating that the bloom propagation is not solely related to nutrient additions, but may be related to the absence of selective grazing from zooplankton.

PTP1B regulates leptin signal transduction in vivo.

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.