Silibinin ameliorates Aβ<Subscript>25-35</Subscript>-induced memory deficits in rats by modulating autophagy and attenuating neuroinflammation as well as oxidative stress

Alzheimer’s disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that inflammatory response, oxidative stress and autophagy are involved in amyloid β (Aβ)-induced memory deficits. Silibinin (silybin), a flavonoid derived from the herb milk thistle, is well known for its hepatoprotective activities. In this study, we investigated the neuroprotective effect of silibinin on Aβ_25-35-injected rats. Results demonstrated that silibinin significantly attenuated Aβ_25-35-induced memory deficits in Morris water maze and novel object-recognition tests. Silibinin exerted anxiolytic effect in Aβ_25-35-injected rats as determined in elevated plus maze test. Silibinin attenuated the inflammatory responses, increased glutathione (GSH) levels and decreased malondialdehyde (MDA) levels, and upregulated autophagy levels in the Aβ_25-35-injected rats. In conclusion, silibinin is a potential candidate for AD treatment because of its anti-inflammatory, antioxidant and autophagy regulating activities.     

Assessment of Zinc Status in School-Age Children from Rural Areas in China Nutrition and Health Survey 2002 and 2012

Zinc is an essential trace element for growth and development in children, but zinc deficiency is a serious nutritional problem worldwide. Our study aimed to assess the zinc status of school-age children living in rural areas of China and to examine the change of zinc status based on the China Nutrition and Health Survey 2002 and 2012. We used the probability proportional to size sampling method for subject selection, and a total of 3407 school-age children were included in this study. Zinc status was assessed by three items of indicators recommended by the World Health Organization (WHO), the United Nations Children’s Fund (UNICEF), the International Atomic Energy Agency (IAEA), and the International Zinc Nutrition Consultative Group (IZiNCG). The concentration of serum zinc was 718.2 μg/L, and 44.4% of children being zinc deficiency in 2002, while 846.8 μg/L and 10.4% in 2012. Zinc intake was 7.8 mg/day with a 7.6% inadequate zinc intake in 2002, together with 6.9 mg/day and 38.2% in 2012. Height-for-age Z score was ?1.06 and 19.1% of children being stunting in 2002, as well as ?0.15 and 6.8% in 2012. In conclusion, the zinc status of school-age children living in rural areas of China has been significantly improved in addition to zinc intake over the past 10 years. However, the zinc deficiency still observed in poor rural areas of China in 2012. In addition, we suggested that the zinc bioavailability should be taken into account when assessing zinc status in population.     

Magnesium Ions Promote the Biological Behaviour of Rat Calvarial Osteoblasts by Activating the PI3K/Akt Signalling Pathway

Magnesium has been investigated as a biodegradable metallic material. Increased concentrations of Mg²⁺ around magnesium implants due to biodegradation contribute to its satisfactory osteogenic capacity. However, the mechanisms underlying this process remain elusive. We propose that activation of the PI3K/Akt signalling pathway plays a role in the Mg²⁺-enhanced biological behaviours of osteoblasts. To test this hypothesis, 6, 10 and 18 mM Mg²⁺ was used to evaluate the stimulatory effect of Mg²⁺ on osteogenesis, which was assessed by evaluating cell adhesion, cell viability, ALP activity, extracellular matrix mineralisation and RT-PCR. The expression of p-Akt was also determined by western blotting. The results showed that 6 and 10 mM Mg²⁺ elicited the highest stimulatory effect on cell adhesion, cell viability and osteogenic differentiation as evidenced by cytoskeletal staining, MTT assay results, ALP activity, extracellular matrix mineralisation and expression of osteogenic differentiation-related genes. In contrast, 18 mM Mg²⁺ had an inhibitory effect on the behaviour of osteoblasts. Furthermore, 10 mM Mg²⁺ significantly increased the phosphorylation of Akt in osteoblasts. Notably, the aforementioned beneficial effects produced by 10 mM Mg²⁺ were abolished by blocking the PI3K/Akt signalling pathway through the addition of wortmannin. In conclusion, these results demonstrate that 6 mM and 10 mM Mg²⁺ can enhance the behaviour of osteoblasts, which is at least partially attributed to activation of the PI3K/Akt signalling pathway. Furthermore, a high concentration (18 mM Mg²⁺) showed an inhibitory effect on the biological behaviour of osteoblasts. These findings advance the understanding of cellular responses to biodegradable metallic materials and may attract greater clinical interest in magnesium.     

RS-1提高CRISPR-Cas9系统介导的人乳铁蛋白基因敲入效率

尝试利用CRISPR-Cas9系统敲除山羊基因组中β-乳球蛋白(BLG)基因,以实现在BLG基因座敲入人乳铁蛋白(h LF)基因,并进一步探讨了不同浓度RAD51蛋白激活剂(RS-1)对同源重组效率的影响。首先针对山羊BLG的第一外显子设计并构建了sg RNA和Cas9共表达载体p Cas9-sg BLG,将该载体转染至山羊耳成纤维细胞,利用PCR和T7EN1法验证了其基因组编辑活性;然后进一步构建了BLG基因打靶载体p BHA-h LF-NIE(包含NEO/EGFP);将该打靶载体与p Cas9-sg BLG载体共转染至山羊耳成纤维细胞,分别用0、5、10和20μmol/L RS-1处理细胞,分析了绿色荧光蛋白的表达效率;同时用800μg/m L G418对不同浓度RS-1处理后的细胞进行筛选,挑取EGFP阳性细胞克隆,进一步通过PCR和测序鉴定h LF定点敲入的阳性细胞克隆。结果显示:设计的sg RNA编辑山羊BLG位点的效率为25%-31%;报告基因的表达效率提示RS-1可以促进基因敲入效率的提高,其效率与RS-1浓度呈正相关,20μmol/L RS-1处理组的效率是对照组的3.5倍;利用G418筛选h LF敲入阳性细胞克隆后,当RS-1浓度为0-10μmol/L时,h LF敲入效率随着RS-1浓度增加而升高,在10μmol/L时阳性克隆率最高为32.61%,然而在20μmol/L时敲入阳性克隆率下降至22.22%,且衰老细胞克隆增多。以上结果表明,利用CRISPR-Cas9系统可以实现在山羊耳成纤维细胞中敲除BLG基因和敲入h LF基因,且适宜浓度的RS-1可以显著提升基因敲入效率,本试验为高效利用CRISPR-Cas9系统获得基因敲入的细胞提供了参考依据。     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Anthraquinone G503 Induces Apoptosis in Gastric Cancer Cells through the Mitochondrial Pathway

G503 is an anthraquinone compound isolated from the secondary metabolites of a mangrove endophytic fungus from the South China Sea. The present study elucidates the anti-tumor activity and the underlying mechanism of G503. Cell viability assay performed in nine cancer cell lines and two normal cell lines demonstrated that the gastric cancer cell line SGC7901 is the most G503-sensitive cancer cells. G503 induced SGC7901 cell death via apoptosis. G503 exposure activated caspases-3, -8 and -9. Pretreatment with the pan-caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK, but not caspase-8 inbibitor Z-IETD-FMK, attenuated the effect of G503. These results suggested that the intrinsic mitochondrial apoptosis pathway, rather than the extrinsic pathway, was involved in G503-induced apoptosis. Furthermore, G503 increased the ratio of Bax to Bcl-2 in the mitochondria and decreased the ratio in the cytosol. G503 treatment resulted in mitochondrial depolarization, cytochrome c release and the subsequent cleavage of caspase -9 and -3. Moreover, it is reported that the endoplasmic reticulum apoptosis pathway may also be activated by G503 by inducing capase-4 cleavage. In consideration of the lower 50% inhibitory concentration for gastric cancer cells, G503 may serve as a promising candidate for gastric cancer chemotherapy.     

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1–RIP1 interaction is dispensable for necroptosis; RIP1–RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1–RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3–RIP3 interaction is required for necroptosis and RIP3–RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1–RIP3 heterodimer itself cannot induce necroptosis, the RIP1–RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1–RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture,^{1, 2, 3} and dependence of receptor-interacting protein (RIP)1⁴ and RIP3.^{5, 6, 7} Physiological function of necroptosis has been illustrated in host defense,^{8, 9, 10, 11} inflammation,^{12, 13, 14, 15, 16} tissue injury,^{10, 17, 18} and development.^{19, 20, 21}Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD,^{22, 23, 24} caspase-8, RIP1, and RIP3, and the cells undergo necroptosis.^{25, 26} Caspase-8 and FADD negatively regulates necroptosis,^{27, 28, 29, 30} because RIP1, RIP3, and CYLD are potential substrates of caspase-8.^{31, 32, 33, 34} Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (MLKL) is downstream of RIP3,^{35, 36} and phosphorylation of MLKL is required for necroptosis.^{37, 38, 39, 40, 41, 42}Apoptosis inducing complex (complex II) and necrosome are both supramolecular complexes.^{43, 44, 45} A recent study showed that RIP1 and RIP3 form amyloidal fibrils through their RIP homotypic interaction motif⁴⁶ (RHIM)-mediated polymerization, and suggested that amyloidal structure is essential for necroptosis signaling.⁴⁷ The RIP1–RIP3 heterodimeric amyloid complex is believed to function as a scaffold that brings signaling proteins into proximity to permit their activation. However, RIP1 and RIP3 also can each form fibrils on their own RHIM domains in vitro. It is unclear how the homo- and hetero-interactions are coordinated and organized on the amyloid scaffold to execute their functions in necroptosis. Here, we used inducible dimerization systems to study the roles of RIP1–RIP1, RIP1–RIP3, and RIP3–RIP3 interactions in necroptosis signaling. Our data suggested that it is the RIP1–RIP3 interaction in the RIP1–RIP3 heterodimeric amyloid complex that empowers to recruit other free RIP3; homodimerization of RIP3 triggers its autophosphorylation and only the phosphorylated RIP3 can recruit MLKL to execute necroptosis.     

Morphological and Physiological Responses of Cotton (Gossypium hirsutum L.) Plants to Salinity

Salinization usually plays a primary role in soil degradation, which consequently reduces agricultural productivity. In this study, the effects of salinity on growth parameters, ion, chlorophyll, and proline content, photosynthesis, antioxidant enzyme activities, and lipid peroxidation of two cotton cultivars, [CCRI-79 (salt tolerant) and Simian 3 (salt sensitive)], were evaluated. Salinity was investigated at 0 mM, 80 mM, 160 mM, and 240 mM NaCl for 7 days. Salinity induced morphological and physiological changes, including a reduction in the dry weight of leaves and roots, root length, root volume, average root diameter, chlorophyll and proline contents, net photosynthesis and stomatal conductance. In addition, salinity caused ion imbalance in plants as shown by higher Na⁺ and Cl⁻ contents and lower K⁺, Ca²⁺, and Mg²⁺ concentrations. Ion imbalance was more pronounced in CCRI-79 than in Simian3. In the leaves and roots of the salt-tolerant cultivar CCRI-79, increasing levels of salinity increased the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), but reduced catalase (CAT) activity. The activities of SOD, CAT, APX, and GR in the leaves and roots of CCRI-79 were higher than those in Simian 3. CAT and APX showed the greatest H₂O₂ scavenging activity in both leaves and roots. Moreover, CAT and APX activities in conjunction with SOD seem to play an essential protective role in the scavenging process. These results indicate that CCRI-79 has a more effective protection mechanism and mitigated oxidative stress and lipid peroxidation by maintaining higher antioxidant activities than those in Simian 3. Overall, the chlorophyll a, chlorophyll b, and Chl (a+b) contents, net photosynthetic rate and stomatal conductance, SOD, CAT, APX, and GR activities showed the most significant variation between the two cotton cultivars.