Oridonin ameliorates caspase-9-mediated brain neuronal apoptosis in mouse with ischemic stroke by inhibiting RIPK3-mediated mitophagy.

Neuronal loss is a primary factor in determining the outcome of ischemic stroke. Oridonin (Ori), a natural diterpenoid compound extracted from the Chinese herb Rabdosia rubescens, has been shown to exert anti-inflammatory and neuroregulatory effects in various models of neurological diseases. In this study we investigated whether Ori exerted a protective effect against reperfusion injury-induced neuronal loss and the underlying mechanisms. Mice were subjected to transient middle cerebral artery occlusion (tMCAO), and were injected with Ori (5, 10, 20 mg/kg, i.p.) at the beginning of reperfusion. We showed that Ori treatment rescued neuronal loss in a dose-dependent manner by specifically inhibiting caspase-9-mediated neuronal apoptosis and exerted neuroprotective effects against reperfusion injury. Furthermore, we found that Ori treatment reversed neuronal mitochondrial damage and loss after reperfusion injury. In N2a cells and primary neurons, Ori (1, 3, 6 µM) exerted similar protective effects against oxygen-glucose deprivation and reoxygenation (OGD/R)-induced injury. We then conducted an RNA-sequencing assay of the ipsilateral brain tissue of tMCAO mice, and identified receptor-interacting protein kinase-3 (RIPK3) as the most significantly changed apoptosis-associated gene. In N2a cells after OGD/R and in the ipsilateral brain region, we found that RIPK3 mediated excessive neuronal mitophagy by activating AMPK mitophagy signaling, which was inhibited by Ori or 3-MA. Using in vitro and in vivo RIPK3 knockdown models, we demonstrated that the anti-apoptotic and neuroprotective effects of Ori were RIPK3-dependent. Collectively, our results show that Ori effectively inhibits RIPK3-induced excessive mitophagy and thereby rescues the neuronal loss in the early stage of ischemic stroke.

Pyroptosis-related prognosis model, immunocyte infiltration characterization, and competing endogenous RNA network of glioblastoma.

BACKGROUND: Glioblastoma (GBM) has a high incidence rate, invasive growth, and easy recurrence, and the current therapeutic effect is less than satisfying. Pyroptosis plays an important role in morbidity and progress of GBM. Meanwhile, the tumor microenvironment (TME) is involved in the progress and treatment tolerance of GBM. In the present study, we analyzed prognosis model, immunocyte infiltration characterization, and competing endogenous RNA (ceRNA) network of GBM on the basis of pyroptosis-related genes (PRGs). METHODS: The transcriptome and clinical data of 155 patients with GBM and 120 normal subjects were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). Lasso (Least absolute shrinkage and selection operator) Cox expression analysis was used in predicting prognostic markers, and its predictive ability was tested using a nomogram. A prognostic risk score formula was constructed, and CIBERSORT, ssGSEA algorithm, Tumor IMmune Estimation Resource (TIMER), and TISIDB database were used in evaluating the immunocyte infiltration characterization and tumor immune response of differential risk samples. A ceRNA network was constructed with Starbase, mirtarbase, and lncbase, and the mechanism of this regulatory axis was explored using Gene Set Enrichment Analysis (GSEA). RESULTS: Five PRGs (CASP3, NLRP2, TP63, GZMB, and CASP9) were identified as the independent prognostic biomarkers of GBM. Prognostic risk score formula analysis showed that the low-risk group had obvious survival advantage compared with the high-risk group, and significant differences in immunocyte infiltration and immune related function score were found. In addition, a ceRNA network of messenger RNA (CASP3, TP63)-microRNA (hsa-miR-519c-5p)-long noncoding RNA (GABPB1-AS1) was established. GSEA analysis showed that the regulatory axis played a considerable role in the extracellular matrix (ECM) and immune inflammatory response. CONCLUSIONS: Pyroptosis and TME-related independent prognostic markers were screened in this study, and a prognosis risk score formula was established for the first time according to the prognosis PRGs. TME immunocyte infiltration characterization and immune response were assessed using ssGSEA, CIBERSORT algorithm, TIMER, and TISIDB database. Besides a ceRNA network was built up. This study not only laid foundations for further exploring pyroptosis and TME in improving prognosis of GBM, but also provided a new idea for more effective guidance on clinical immunotherapy to patients and developing new immunotherapeutic drugs.

Characterization of circRNA-Associated-ceRNA Networks Involved in the Pathogenesis of Postoperative Cognitive Dysfunction in Aging Mice.

Postoperative cognitive dysfunction (POCD) is a clinical entity associated with declined cognitive function following surgery. It occurs more frequently in elderly patients. Recent studies have shown that circRNA-associated-ceRNA networks, constructed based on interactions between circRNA-miRNA and miRNA-mRNA, provide key insight into the molecular mechanisms underlying the pathogenesis of several neurological diseases. However, the mechanism of POCD remains undetermined. In this study, laparotomies were performed under isoflurane anesthesia on young (2-month-old) and aging (17-month-old) male C57BL/6 mice. The results showed that the aging mice were more likely than the young mice to develop POCD. Subsequently, differentially expressed circRNAs, miRNAs, and mRNAs were characterized by RNA sequencing the hippocampi of young and aging mice under control and surgery conditions. Six circRNAs, 6 miRNAs, and 203 mRNAs were identified to construct the circRNA-associated-ceRNA network for the control condition, while 13 circRNAs, 8 miRNAs, and 189 mRNAs were used for the circRNA-associated-ceRNA network for the surgery condition. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of these two networks revealed that the circRNA-associated-ceRNA networks are involved in POCD pathogenesis though modulating the Wnt and VEGF signaling pathways, as well as neural processes associated with long-term synaptic depression and synaptic transmission. In particular, the mmu-miR-298-5P regulatory pathway identified in this study's mouse model suggests that mm9_circ_009789- and mm9_circ_004229-associated-ceRNA networks as closely related to the occurrence of POCD through regulating PKC signaling pathway, neural cell apoptosis and glycolipid metabolism pathway. These findings provide possible insight into the role of the circRNA-associated-ceRNA networks, helping to unravel the complexity of the molecular pathogenesis of POCD.

Hematopoietic Stem-Cell Transplantation versus Immunosuppressive Therapy in Patients with Adult Acquired Severe Aplastic Anemia: A Cost-Effectiveness Analysis.

OBJECTIVE: Controversy remains regarding which therapy to initially select for severe aplastic anemia (SAA) patients aged 35-50. This cost-effectiveness analysis aimed to use the Markov model to compare immunosuppressive therapy (IST) with hematopoietic stem-cell transplantation (HSCT) in age-stratified patients with SAA. METHODS: A cost-effectiveness analysis using a Markov model compared IST with HSCT in age-stratified patients with SAA. Baseline data were derived from a systematic literature review and collected from Huashan Hospital, Fudan University. The primary outcome was an incremental cost-effectiveness ratio (ICER). RESULTS: The HSCT strategy dominated in patients aged 18-35 even though it was $146,970 more expensive than IST, and the ICER of HSCT to IST was $14,054.19/quality-adjusted life-year (QALY), which was less than the willingness-to-pay value of $25,397.57/QALY. The IST strategy dominated in patients aged 35-50, because it was $72,009 less expensive than HSCT and yielded 3.24 QALYs more than HSCT. The model was vigorous in the sensitivity analyses of the key variables tested through the plausible ranges that were acquired from costing sources and previously published literature. CONCLUSION: The preferred induction strategy for patients aged 18-35 with SAA appears to be HSCT, and the preferred strategy for patients aged 35-50 is IST, which minimizes costs while maximizing QALYs.

[Effect of auricular point sticking therapy on perioperative pain in patients with partial lung resection].

OBJECTIVE: To observe the analgesic effect of auricular point sticking therapy during the perioperative stage in the patients with partial lung resection. METHODS: A total of 92 patients with partial lung resection were randomized into an auricular point group (31 cases, 1 case dropped off), the sham-auricular point group (30 cases) and a medication group (31 cases, 1 case dropped off). The routine medication for analgesia was provided in all of the three groups. In the auricular point group, 1 day before operation, the auricular point sticking therapy was applied at shenmen (TF4), sympathetic (AH6a), subcortex (AT4), brain stem (AT3,4i), anterior ear lobe (LO4), chest (AH10) and lung (CO14), retained till the third day after operation. In the sham-auricular point group, the auricular adhesive tape without semen vaccariae was used at the same points as the auricular point group. Separately, the scores of visual analogue scale (VAS) in 8, 16, 24, 48 h and 72 h after operation, the time for obtaining the mean of the postoperative VAS score<3 points, the scores of hospital anxiety and depression scale (HAD) in 8, 72 h after operation and the plasma concentration of ß-endorphin in 24, 48 h after operation, as well as the adverse reactions after operation were recorded in the patients of each group. RESULTS: In 8, 16, 24, 48 h and 72 h after operation, VAS scores in the auricular point group were lower than those in the sham-auricular point group and the medication group separately (P<0.05). In the auricular point group, 16 h after operation the mean of VAS score was less than 3 points, but in the sham-auricular point group and the medication group, 48 h after operation, such a mean score of VAS was obtained. In 8, 72 h after operation, HAD score in the auricular point group was lower than that in the sham-auricular point group and the medication group respectively. In 24, 48 h after operation, the concentration of plasmaß-endorphin was all higher than that in the sham-auricular point group and the medication group respectively (P<0.05). In 24 h after operation, the plasma concentration ofß-endorphin in the sham-auricular point group was higher than the medication group (P<0.05). The incidence of adverse reaction in the auricular point group was lower than that in the sham-auricular point group and the medication group respectively (P<0.05). CONCLUSION: Auricular point sticking therapy relieves perioperative pain, shortens analgesic time, releases anxious and depressive emotions and reduces postoperative adverse reaction in the patients with partial lung resection. The analgesic mechanism is probably related to the increase of plasma concentration of ß-endorphin.

The MADS-Box Transcription Factor EjAGL65 Controls Loquat Flesh Lignification via Direct Transcriptional Inhibition of EjMYB8.

Loquat fruit accumulates lignin in its flesh when undergoing chilling injury during postharvest storage, making it a suitable model for the study of flesh lignification. Transcriptional regulation of lignin biosynthesis is principally controlled by the NAC-MYB transcriptional cascade in model plants. Previous research has demonstrated that EjMYB8 activates lignin biosynthesis through direct interaction with the promoter of Ej4CL1. However, the classic NAC-MYB gene regulation network has not been established. Here, the MADS-box gene EjAGL65 was discovered by screening a cDNA library using the EjMYB8 promoter as bait in yeast. A phylogenetic analysis and structural comparisons revealed that EjAGL65 belongs to the Mδ subgroup of the MADS-box family, whose members have not been reported as being involved in the regulation of lignin deposition. EjAGL65 transcription was downregulated at 0°C compared to 5°C, indicating a negative correlation with the change of lignin content. A dual-luciferase assay indicated that EjAGL65 is capable of inhibiting the promoter activity of EjMYB8 in vivo. These results showed that the Mδ MADS-box gene EjAGL65 transcriptionally regulates EjMYB8 during postharvest chilling induced flesh lignification, which differs from the classical regulation model of lignin biosynthesis that has been illustrated for developmental lignin accumulation.

ETHYLENE RESPONSE FACTOR39-MYB8 complex regulates low-temperature-induced lignification of loquat fruit.

Flesh lignification is a specific chilling response that causes deterioration in the quality of stored red-fleshed loquat fruit (Eribotrya japonica) and is one aspect of wider chilling injury. APETALA2/ETHLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors are important regulators of plant low-temperature responses and lignin biosynthesis. In this study, the expression and action of 27 AP2/ERF genes from the red-fleshed loquat cultivar 'Luoyangqing' were investigated in order to identify transcription factors regulating low-temperature-induced lignification. EjERF27, EjERF30, EjERF36, and EjERF39 were significantly induced by storage at 0 °C but inhibited by a low-temperature conditioning treatment (pre-storage at 5 °C for 6 days before storage at 0 °C, which reduces low-temperature-induced lignification), and their transcript levels positively correlated with flesh lignification. A dual-luciferase assay indicated that EjERF39 could transactivate the promoter of the lignin biosynthetic gene Ej4CL1, and an electrophoretic mobility shift assay confirmed that EjERF39 recognizes the DRE element in the promoter region of Ej4CL1. Furthermore, the combination of EjERF39 and the previously characterized EjMYB8 synergistically transactivated the Ej4CL1 promoter, and both transcription factors showed expression patterns correlated with lignification in postharvest treatments and red-fleshed 'Luoyangqing' and white-fleshed 'Ninghaibai' cultivars with different lignification responses. Bimolecular fluorescence complementation and luciferase complementation imaging assays confirmed direct protein-protein interaction between EjERF39 and EjMYB8. These data indicate that EjERF39 is a novel cold-responsive transcriptional activator of Ej4CL1 that forms a synergistic activator complex with EjMYB8 and contributes to loquat fruit lignification at low temperatures.

Natural Occurrence, Bioactivity and Biosynthesis of Elaiophylin Analogues.

Elaiophylins belong to a special family of 16-membered macrodiolides with C2-symmetry. They have exhibited remarkable biological activities, such as antimicrobial, anthelmintic, anticancer, immunosuppressive, anti-inflammatory, antiviral, and α-glucosidase inhibitory activities. A member of elaiophylins, efomycin M, is as a specific small molecule inhibitor of selectin in preclinical trial for the treatment of psoriasis, ischemia-reperfusion, and allergy. The biosynthetic investigation of elaiophylins has uncovered a unique thioesterase, which is responsible for the formation of C2-symmetric diolide. We herein summarize the natural occurrence, bioactivity, and biosynthesis of elaiophylins covering the literatures from 1959 to 2019. Hopefully, this review will inspire further research interests of these compounds and encourage the discovery of new analogues by metabolic engineering or genome mining.

EjHAT1 Participates in Heat Alleviation of Loquat Fruit Lignification by Suppressing the Promoter Activity of Key Lignin Monomer Synthesis Gene EjCAD5.

Texture attributes such as firmness and lignification are important for fruit quality. Lignification has been widely studied in model plants and energy crops, but fruit lignification has rarely been investigated, despite having an adverse effect on fruit quality and consumer preference. Chilling-induced loquat fruit lignification that occurs after harvest can be alleviated by heat treatment (HT) applied prior to low temperature storage. Enzyme activity assay showed that HT treatment could retard the low temperature-induced increase in cinnamyl alcohol dehydrogenase (CAD) activity. Transcript analysis and substrate activity assays of recombinant CAD proteins highlighted the key role of EjCAD5 in chilling-induced lignin biosynthesis. A novel homeobox-leucine zipper protein ( EjHAT1) was identified as a negative regulator of EjCAD5. Therefore, the effect of HT treatment on lignification may be partially due to the suppression of the EjCAD5 promoter activity by EjHAT1.

Acupuncture and vitamin B12 injection for Bell's palsy: no high-quality evidence exists.

OBJECTIVE: To assess the efficacy of acupuncture combined with vitamin B12 acupoint injection versus acupuncture alone to reduce incomplete recovery in patients with Bell's palsy. DATA RETRIEVAL: A computer-based online retrieval of Medline, Web of Science, CNKI, CBM databases until April 2014 was performed for relevant trials, using the key words "Bell's palsy or idiopathic facial palsy or facial palsy" and "acupuncture or vitamin B12 or methylcobalamin". STUDY SELECTION: All randomized controlled trials that compared acupuncture with acupuncture combined with vitamin B12 in patients with Bell's palsy were included in the meta-analysis. The initial treatment lasted for at least 4 weeks. The outcomes of incomplete facial recovery were monitored. The scoring index varied and the definition of healing was consistent. The combined effect size was calculated by using relative risk (RR) with 95% confidence interval (CI) using the fixed effect model of Review Manager. MAIN OUTCOME MEASURES: Incomplete recovery rates were chosen as the primary outcome. RESULTS: Five studies involving 344 patients were included in the final analysis. Results showed that the incomplete recovery rate of Bell's palsy patients was 44.50% in the acupuncture combined with vitamin B12 group but 62.57% in the acupuncture alone group. The major acupoints were Taiyang (EX-HN5), Jiache (ST6), Dicang (ST4) and Sibai (ST2). The combined effect size showed that acupuncture combined with vitamin B12 was better than acupuncture alone for the treatment of Bell's palsy (RR = 0.71, 95%CI: 0.58-0.87; P = 0.001), this result held true when 8 patients lost to follow up in one study were included into the analyses (RR = 0.70, 95%CI: 0.58-0.86; P = 0.0005). In the subgroup analyses, the therapeutic effect in patients of the electroacupuncture subgroup was better than in the non-electroacupuncture subgroup (P = 0.024). There was no significant difference in the incomplete recovery rate by subgroup analysis on drug types and treatment period. Most of the included studies were moderate or low quality, and bias existed. CONCLUSION: In patients with Bell's palsy, acupuncture combined with vitamin B12 can reduce the risk of incomplete recovery compared with acupuncture alone in our meta-analysis. Because of study bias and methodological limitations, this conclusion is uncertain and the clinical application of acupuncture combined with vitamin B12 requires further exploration.

[Anaplastic lymphoma kinase fusion gene expression, clinical pathological characteristics and prognosis in 95 Chinese patients with non-small cell lung cancer].

OBJECTIVE: To examine the prevalence of anaplastic lymphoma kinase (ALK) fusion gene in Chinese patients with non-small cell lung cancer (NSCLC). METHODS: In this study, 95 patients with NSCLC and corresponding clinical information and formalin-fixed paraffin-embedded (FFPE) tissue blocks were included. Hematoxylin & eosin (HE) staining, conventional ALK immunochemistry (IHC) staining and intercalated antibody-enhanced polymer (iAEP) IHC staining, and dual-color split fluorescence in situ hybridization (FISH) for ALK fusion gene were performed. RESULTS: Eight ALK-positive cases were detected using anti-ALK immunohistochemistry with the iAEP method, and FISH analyses revealed 4 patients of them who harbored the ALK fusion gene (4.2%, 4/95), including 2 cases of female patients with solid signet-ring cell adenocarcinoma and 2 cases of male patients with adenosquamous carcinoma. The positive cases were all non-smokers without EGFR/KRAS mutations. Furthermore, the positive cases all survived, and the overall postsurgery survival time of 2 cases was more than 5 years. CONCLUSION: ALK IHC with the iAEP method is better than conventional ALK IHC, and the percentage of the positive cells is more important than that of the intensity. ALK translocations were infrequent in the entire NSCLC patient population (<5%) with better prognosis.

Klotho upregulation contributes to the neuroprotection of ligustilide in an Alzheimer's disease mouse model.

Klotho, an aging-suppressor gene, encodes a protein that potentially acts as a neuroprotective factor by modulating insulin-like growth factor 1 signaling and oxidative stress. In the present study, we investigated the potential role of Klotho in the therapeutic effect of ligustilide against Alzheimer's disease (AD)-like neuropathologies and memory impairment in aged senescence-accelerated mouse prone-8 (SAMP8) mice. Ligustilide treatment (10 and 40 mg/kg for 8 weeks, intragastrically) in 10-month-old SAMP8 mice reduced memory deficits, amyloid-ß(1)-42 accumulation, tau phosphorylation, and neuron loss, increased mitochondrial manganese-superoxide dismutase and catalase expression and activity, and decreased malondialdehyde, protein carbonyl, and 8-hydroxydesoxyguanosine levels in the brain. Ligustilide upregulated Klotho expression in the cerebral choroid plexus and serum, decreased Akt and Forkhead box class O1 phosphorylation. Moreover, ligustilide inhibited the insulin-like growth factor 1 pathway and induced Forkhead box class O1 activation in 293T cells along with Klotho upregulation. An inverse correlation was found between Klotho expression and the AD phenotype, suggesting that Klotho might be a novel therapeutic target for age-related AD, and Klotho upregulation might contribute to the neuroprotective effect of ligustilide against AD.

[Protective effects of Z-ligustilide against cytotoxicity induced by Abeta25-35 in neuron cells].

OBJECTIVE: To investigate the effects of Z-ligustilide (LIG) on Abeta25-35-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. METHODS: Cells were incubated with 0.1, 1.0, 2.5, 5.0 microg/mL LIG and then were exposed to 50 micromol/L Abeta25-35 to induce cytotoxicity in SH-SY5Y cell to establish the AD model in vitro. Cell viability was test by MTT method. Pro- and anti-apoptosis protein levels were investigated by Western blot. RESULTS: After exposure to Abeta25-35, the cell viability decreased significantly, and the expression of pro-apoptosis protein--Bax, cleaved caspase 3, cytochrome C, caspase 8 was up regulated while the anti-apoptosis protein Bcl-2 was down regulated. However, the treatment of LIG (0.1, 1.0, 2.5, 5.0 microg/mL) significantly restored these changes. CONCLUSION: LIG has the protective effect against cytotoxicity induced by in SH-SY5Y cells via inhibit the apoptosis induced by Abeta25-35.

Aqueous extract of Gleditsia sinensis Lam. fruits improves serum and liver lipid profiles and attenuates atherosclerosis in rabbits fed a high-fat diet.

ETHNOPHARMACOLOGICAL RELEVANCE: Gleditsia sinensis Lam. has been used in the traditional Chinese medicine as a chief ingredient of many polyherbal formulations for the treatment of obesity and thrombosis. AIM OF THE STUDY: To evaluate the effects of Gleditsia sinensis Lam. fruit aqueous extract (GAE) on hyperlipidemia and atherosclerosis in Japanese white rabbits on a high fat diet. MATERIALS AND METHODS: Rabbits were divided into four groups: the normal control with a normal diet, and high-fat diet-fed model group and GAE-treated groups supplemented with GAE (6 or 12 mg/kg/day, p.o.), respectively. The groups fed high-fat diets were given i.v. with bovine serum albumin (BSA) on the 4th week to induce atherosclerosis. The serum lipid profile, including triglycerides (TG), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C), was determined on the 0th, 4th, 8th and 14th week, respectively. And the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatine kinase (CK) were measured simultaneously. At the end of the experiment, the rabbits were sacrificed, and the atherosclerotic plaques as well as the histopathological changes of aorta and liver were assessed by oil-red or HE staining, respectively, and the aorta and liver lipid profiles were also assayed. RESULTS: Results showed that the prophylactic treatment with GAE could significantly decrease the lipid levels of serum, aorta and liver, attenuate aortic atherosclerosis and improve aortic remodeling without the significant liver and muscle toxicity. CONCLUSION: The present findings suggest that GAE can effectively attenuate the atherosclerotic at least through anti-hyperlipidemic activity and thus has the therapeutic potential in treating hyperlipidemia-related cardiovascular diseases.

Saponins isolated from the root of Panax notoginseng showed significant anti-diabetic effects in KK-Ay mice.

Panax notoginseng, a well-known and commonly used traditional Chinese herb, has been used in China for six hundred years. Panax notoginseng saponins (PNS) were extracted from the root of the plant. This is the first study on anti-hyperglycemic and anti-obese effects of PNS in genetic model mice. Additionally, the preventive effect on diabetic nephropathy was investigated. Animals received intraperitoneal injections of PNS 50 or 200 mg/kg daily. On day 12, 22 and 30, PNS-treated groups had significantly lower fasting blood glucose levels and smaller body weight incremental percentage. After a 12-day treatment, glucose tolerance of PNS groups were significantly improved; these indices in PNS-treated mice exhibited a dose-dependent improvement. Furthermore, on day 30, the serum insulin resistance index and triglyceride levels of PNS-treated groups decreased significantly, and the development of the mice glomerular lesions was prevented significantly. The results in this present paper indicate that PNS possesses anti-diabetes and anti-obese activities and may prove to be of clinical importance in improving the management of type 2 diabetes.