RESUMO
The aim of this study is to explore the mechanism of ligustilide, the main active constituent of essential oils of traditional Chinese medicine Angelicae Sinensis Radix, on alleviating oxygen-glucose deprivation/reperfusion(OGD/R) injury in PC12 cells from the perspective of ferroptosis. OGD/R was induced in vitro, and 12 h after ligustilide addition during reperfusion, cell viability was detected by cell counting kit-8(CCK-8) assay. DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS). Western blot was employed to detect the expression of ferroptosis-related proteins, glutathione peroxidase 4(GPX4), transferrin receptor 1(TFR1), and solute carrier family 7 member 11(SLC7A11), and ferritinophagy-related proteins, nuclear receptor coactivator 4(NCOA4), ferritin heavy chain 1(FTH1), and microtubule-associated protein 1 light chain 3(LC3). The fluorescence intensity of LC3 protein was analyzed by immunofluorescence staining. The content of glutathione(GSH), malondialdehyde(MDA), and Fe was detected by chemiluminescent immunoassay. The effect of ligustilide on ferroptosis was observed by overexpression of NCOA4 gene. The results showed that ligustilide increased the viability of PC12 cells damaged by OGD/R, inhibited the release of ROS, reduced the content of Fe and MDA and the expression of TFR1, NCOA4, and LC3, and improved the content of GSH and the expression of GPX4, SLC7A11, and FTH1 compared with OGD/R group. After overexpression of the key protein NCOA4 in ferritinophagy, the inhibitory effect of ligustilide on ferroptosis was partially reversed, indicating that ligustilide may alleviate OGD/R injury of PC12 cells by blocking ferritinophagy and then inhibiting ferroptosis. The mechanism by which ligustilide reduced OGD/R injury in PC12 cells is that it suppressed the ferroptosis involved in ferritinophagy.
Assuntos
Ferroptose , Animais , Ratos , Células PC12 , Ferroptose/genética , Espécies Reativas de Oxigênio , Fatores de Transcrição , GlutationaRESUMO
This study explores the effect of total flavonoids of Rhododendra simsii(TFR) on middle cerebral artery occlusion(MCAO)-induced cerebral injury in rats and oxygen-glucose deprivation/reoxygenation(OGD/R) injury in PC12 cells and the underlying mechanism. The MCAO method was used to induce focal ischemic cerebral injury in rats. Male SD rats were randomized into sham group, model group, and TFR group. After MCAO, TFR(60 mg·kg~(-1)) was administered for 3 days. The content of tumor necrosis factor-α(TNF-α), interleukin-1(IL-1), and interleukin-6(IL-6) in serum was detected by enzyme-linked immunosorbent assay(ELISA). The pathological changes of brain tissue and cerebral infarction were observed based on hematoxylin and eosin(HE) staining and 2,3,5-triphenyltetrazolium chloride(TTC) staining. RT-qPCR and Western blot were used to detect the mRNA and protein levels of calcium release-activated calcium channel modulator 1(ORAI1), stromal interaction molecule 1(STIM1), stromal intera-ction molecule 2(STIM2), protein kinase B(PKB), and cysteinyl aspartate specific proteinase 3(caspase-3) in brain tissues. The OGD/R method was employed to induce injury in PC12 cells. Cells were randomized into the normal group, model group, gene silencing group, TFR(30 µg·mL~(-1)) group, and TFR(30 µg·mL~(-1))+gene overexpression plasmid group. Intracellular Ca~(2+) concentration and apoptosis rate of PC12 cells were measured by laser scanning confocal microscopy and flow cytometry. The effect of STIM-ORAI-regulated store-operated calcium entry(SOCE) pathway on TFR was explored based on gene silencing and gene overexpression techniques. The results showed that TFR significantly alleviated the histopathological damage of brains in MCAO rats after 3 days of admini-stration, reduced the contents of TNF-α, IL-1, and IL-6 in the serum, down-regulated the expression of ORAI1, STIM1, STIM2, and caspase-3 genes, and up-regulated the expression of PKB gene in brain tissues of MCAO rats. TFR significantly decreased OGD/R induced Ca~(2+) overload and apoptosis in PC12 cells. However, it induced TFR-like effect by ORAI1, STIM1 and STIM2 genes silencing. However, overexpression of these genes significantly blocked the effect of TFR in reducing Ca~(2+) overload and apoptosis in PC12 cells. In summary, in the early stage of focal cerebral ischemia-reperfusion injury and OGD/R-induced injury in PC12 cells TFR attenuates ischemic brain injury by inhibiting the STIM-ORAI-regulated SOCE pathway and reducing Ca~(2+) overload and inflammatory factor expression, and apoptosis.
Assuntos
Isquemia Encefálica , Flavonoides , Traumatismo por Reperfusão , Animais , Masculino , Ratos , Apoptose , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Caspase 3 , Interleucina-1 , Interleucina-6 , Ratos Sprague-Dawley , Traumatismo por Reperfusão/metabolismo , Fator de Necrose Tumoral alfa/genética , Flavonoides/farmacologia , Rhododendron/químicaRESUMO
This paper explored the protective effect of total flavonoids of Rhododendron simsii(TFR) on focal cerebral ischemia-reperfusion injury(CIRI) in rats and its relationship with the store-operated calcium entry(SOCE) pathway regulated by stromal intera-ction molecule(STIM) and calcium release-activated calcium modulator(Orai).Rats were randomly assigned into the sham group, model(middle cerebral artery occlusion, MCAO) group, TFR(60 mg·kg~(-1)) group, TFR(60 mg·kg~(-1))+SOCE pathway inhibitor 2-aminoethoxydiphenyl borate(2-APB, 2.5 mg·kg~(-1)) group, and 2-APB(2.5 mg·kg~(-1)) group.The rats in the sham group and MCAO group were administrated with normal saline, and those in the TFR group and TFR+2-APB group were administrated with TFR(60 mg·kg~(-1)) by gavage for 14 days until sampling.The rats in the 2-APB group and TFR+2-APB group were intraperitoneally injected with 2-APB(2.5 mg·kg~(-1)) after operation.The levels of interleukin-1(IL-1), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) in serum were measured by ELISA.The cerebral infarction and the pathological status of ischemic brain tissue were detected via TTC staining and HE staining, respectively.The protein and mRNA levels of STIM1, STIM2, Orai1, cysteinyl aspartate specific proteinase 3(caspase-3), and protein kinase B(PKB) in brain tissue were respectively determined by Western blot and RT-qPCR.The growth of brain neurons in each group was observed via immunofluorescence method.The results showed that compared with the MCAO group, TFR lowered the levels of IL-1, IL-6 and TNF-α in serum and the score of neurological function, ameliorated the pathological injury of brain tissue, and decreased the infarct size.Moreover, TFR up-regulated the mRNA and protein levels of STIM1, STIM2, Orai1, and PKB, down-regulated those of caspase-3 in brain tissue, and increased the double-labeled positive cells under fluorescence microscope.However, the above effects were significantly weakened by the addition of 2-APB, a SOCE inhibitor.The results suggested that TFR may play a protective role against focal cerebral ischemia-reperfusion injury by up-regulating the expression of SOCE-related signal molecules, promoting neurogenesis around the ischemic area, improving the survival state of neurons, and redu-cing the activity of inflammatory mediators.
Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Rhododendron , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Cálcio/metabolismo , Caspase 3 , Flavonoides , Interleucina-1 , Interleucina-6 , RNA Mensageiro/genética , Ratos , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
AIM: To investigate the effect of high concentration of glucose (HCG) on double stranded RNA-activated protein kinase-like ER kinase (PERK)-eukaryotic initiation factor-2α (eIF2α)-transcription factor C/EBP homologous protein (CHOP)-cysteine aspartate specific proteinase (caspase-12) signaling pathway activation and apoptosis in rabbit corneal epithelial cells (RCECs). METHODS: RCECs were treated by different concentrations of glucose for 0-48h. The expressions of PERK, p-PERK, eIF2α, p-eIF2α, 78 kDa glucose-regulated protein 78 (GRP78), CHOP, B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-2-associated X protein (Bax) and caspase-12 were determined by Western blot. Apoptosis was detected by TUNEL assay. Meanwhile, the function of PERK-eIF2α-CHOP-caspase-12 signaling pathway activation in high glucose-induced apoptosis was evaluated using PERK inhibitor, GSK2606414. RESULTS: HCG significantly promoted the expression of p-PERK, p-eIF2α, GRP78, CHOP, Bax and cleaved caspase-12 in RCECs (P<0.05), while remarkably decreased the expression of Bcl-2 and caspase-12 (P<0.05), and the alterations caused by glucose were in concentration- and time-dependent manners. Meanwhile, PERK and eIF2α expressions were not affected in all groups (P>0.05). TUNEL assay showed that the apoptosis rate of RCECs in the HCG group increased significantly in contrast with that in the normal concentration of glucose or osmotic pressure control group (P<0.05), and the apoptosis rate increased with the increase of glucose concentration within limits (P<0.05). GSK2606414 down-regulated the expression of p-PERK and p-eIF2α in the HCG group (P<0.05), while still did not affect the expression of PERK and eIF2α among groups (P>0.05). Correspondingly, GSK2606414 also significantly reduced the apoptosis rate induced by high glucose (P<0.05). CONCLUSION: HCG activates PERK-eIF2α-CHOP-caspase-12 signaling pathway and promotes apoptosis of RCECs.
RESUMO
Epidermolytic palmoplantar keratoderma (EPPK) is generally associated with dominant-negative mutations of the Keratin 9 gene (KRT9), and rarely with the Keratin 1 gene (KRT1). To date, a myriad of mutations has been reported with a high frequency of codon 163 mutations within the first exon of KRT9 in different populations. Notably, a distinct phenotypic heterogeneity, digital mutilation, was found recently in a 58-year-old female Japanese EPPK patient with p.R163W. Here, we report the most common mutation, c.C487T (p.R163W) of KRT9, in two large EPPK pedigrees from southeast China. The arginine residue in peptide position 163 remains almost constant in at least 47 intermediate filament proteins ranging from snail to human. A substitution in arginine alters both the charge and shape of the 1A rod domain and disrupts the function of the helix initiation motif of keratins, finally compromising the integrity of filaments and weakening their stability in the epidermis of palms and soles. We summarize the clinical symptoms of EPPK in Chinese and show that knuckle pads are associated with KRT9 mutations. We suggest that the frequency of p.R163W in Chinese EPPK patients (31.03%) is consistent with that in the general population (29.33%), and that codon 163 is truly a hotspot mutational site of KRT9.
Assuntos
Povo Asiático/genética , Queratina-9/genética , Ceratodermia Palmar e Plantar Epidermolítica/genética , Mutação/genética , Adulto , Arginina/genética , Feminino , Pé/patologia , Genes Dominantes , Mãos/patologia , Humanos , Ceratodermia Palmar e Plantar Epidermolítica/diagnóstico , Ceratodermia Palmar e Plantar Epidermolítica/patologia , Masculino , Linhagem , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genéticaRESUMO
Knuckle pads and camptodactyly are overlapping symptoms associated with many genetic and environmental factors. To the best of our knowledge, all reported cases of epidermolytic palmoplantar keratoderma (EPPK) with knuckle pads have been without accompanying camptodactyly. We here report a novel KRT9 mutation-EPPK family with combined knuckle pads and camptodactyly. All the EPPK-affected individuals in this southern Chinese pedigree suffered severe diffuse palmar and plantar hyperkeratosis including hyperhidrosis and cuticle splitting: 3 females presented EPPK only, 8 adult males had notably severe knuckle pads and camptodactyly as well as EPPK, and one 6-year-old boy manifested EPPK with knuckle pads. Haplotype analysis excluded the known candidate loci for camptodactyly and/or knuckle pad-like phenotypes on chromosomes 13q12, 3q11.2-q13.12, 1q24-q25, 4p16.3 and 16q11.1-q22, while only the markers D17S1787 and D17S579 flanking KRT9 showed co-segregation with EPPK. Then a novel c.T1373C (p.L458P) mutation within the sixth exon of KRT9 was validated, and this mutation presented a more severe pathogenicity than the previously reported p.L458F. We speculated that KRT9 plays a complicated role in the genesis of EPPK with knuckle pads and camptodactyly, which needs to be further investigated.
Assuntos
Anormalidades Múltiplas/genética , Dedos/anormalidades , Deformidades Congênitas da Mão/genética , Queratina-9/genética , Ceratodermia Palmar e Plantar Epidermolítica/genética , Mutação de Sentido Incorreto , Adulto , Povo Asiático/genética , Criança , Análise Mutacional de DNA , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , LinhagemRESUMO
BACKGROUND: Whole exome sequencing provides a labor-saving and direct means of genetic diagnosis of hereditary disorders in which the pathogenic gene harbors a large cohort of exons. We set out to demonstrate a suitable example of genetic diagnosis of MEN 2A/FMTC (multiple endocrine neoplasia type 2/familial medullary thyroid carcinoma) using this approach. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the whole exome of six individuals from a large Chinese MEN2A/FMTC pedigree to identify the variants of the RET (REarranged during Transfection) protooncogene and followed this by validation. Then prophylactic or surgical thyroidectomy with modified or level VI lymph node dissection and adrenalectomy were performed for the carriers. The cases were closely followed up. Massively parallel sequencing revealed four missense mutations of RET. We unexpectedly discovered that the proband's daughter with MEN 2A-related MTC presented a novel p.C634Y/V292M/R67H/R982C compound mutation, due to the involvement of p.C634Y in the proband with MEN 2A and p.V292M/R67H/R982C in the proband's husband with FMTC. In the maternal origin, p.C634Y caused bilateral MTC in all 5 cases and bilateral pheochromocytoma in 2 of the 5; the earliest onset age was 28 years. In the paternal origin, one of the six p.V292M/R67H/R982C carriers presented bilateral MTC (70 years old), one only had bilateral C-cell hyperplasia (44 years), two had bilateral multi-nodules (46 and 48 years) and two showed no abnormality (22 and 19 years). CONCLUSIONS/SIGNIFICANCE: The results confirmed the successful clinical utility of whole exome sequencing, and our data suggested that the p.C634Y/V292M/R67H/R982C mutation of RET exhibited a more aggressive clinical phenotype than p.C634Y or p.V292M/R67H/R982C, while p.V292M/R67H/R982C presented a relatively milder pathogenicity of MTC and likely predisposed to FMTC.