Evidence for an intra-tumoral microbiome in pituitary neuroendocrine tumors with different clinical phenotypes.

PURPOSE: Bacteria have been observed in the tumor environment for decades and have been demonstrated to play important roles in the pathogenesis and development of several different tumors. So far there is a clear lack of specific studies relating to the presence of bacteria in pituitary neuroendocrine tumors (PitNETs). METHODS: In this study, we performed five region-based amplification and bacterial 16 S rRNA sequencing to identify the microbiome of PitNET tissues across four clinical phenotypes. Multiple filter procedures were performed to inhibit the risk of contamination with bacteria and bacterial DNA. Histological analysis was also conducted to validate the localization of bacteria in the intra-tumoral region. RESULTS: We identified common and diverse bacterial types across the four clinical phenotypes of PitNET. We also predicted the potential functions of these bacteria in tumor phenotypes and found that these functions were reported in certain previous mechanistic studies. Our data indicate that the pathogenesis and development of tumors may correlate with the behavior of intra-tumoral bacteria. Histological results, including lipopolysaccharide (LPS) staining and fluorescence in situ hybridization (FISH) for bacterial 16 S rRNA clearly demonstrated the localization of bacteria in the intra-tumoral region. Staining for Iba-1 suggested that the proportion of microglia was more abundant in FISH-positive regions than in FISH-negative regions. Furthermore, in FISH-positive regions, the microglia exhibited a longitudinally branched morphology that was different to the compact morphology observed in FISH-negative regions. CONCLUSION: In summary, we provide an evidence for the existence of intra-tumoral bacteria in PitNET.

PINK1 overexpression prevents forskolin-induced tau hyperphosphorylation and oxidative stress in a rat model of Alzheimer's disease.

PTEN-induced putative kinase 1 (PINK1)/parkin pathway mediates mitophagy, which is a specialized form of autophagy. Evidence shows that PINK1 can exert protective effects against stress-induced neuronal cell death. In the present study we investigated the effects of PINK1 overexpression on tau hyperphosphorylation, mitochondrial dysfunction and oxidative stress in a specific rat model of tau hyperphosphorylation. We showed that intracerebroventricular (ICV) microinjection of forskolin (FSK, 80 µmol) induced tau hyperphosphorylation in the rat brain and resulted in significant spatial working memory impairments in Y-maze test, accompanied by synaptic dysfunction (reduced expression of synaptic proteins synaptophysin and postsynaptic density protein 95), and neuronal loss in the hippocampus. Adeno-associated virus (AAV)-mediated overexpression of PINK1 prevented ICV-FSK-induced cognition defect and pathological alterations in the hippocampus, whereas PINK1-knockout significantly exacerbated ICV-FSK-induced deteriorated effects. Furthermore, we revealed that AAV-PINK1-mediated overexpression of PINK1 alleviated ICV-FSK-induced tau hyperphosphorylation by restoring the activity of PI3K/Akt/GSK3ß signaling. PINK1 overexpression reversed the abnormal changes in mitochondrial dynamics, defective mitophagy, and decreased ATP levels in the hippocampus. Moreover, PINK1 overexpression activated Nrf2 signaling, thereby increasing the expression of antioxidant proteins and reducing oxidative damage. These results suggest that PINK1 deficiency exacerbates FSK-induced tau pathology, synaptic damage, mitochondrial dysfunction, and antioxidant system defects, which were reversed by PINK1 overexpression. Our data support a critical role of PINK1-mediated mitophagy in controlling mitochondrial quality, tau hyperphosphorylation, and oxidative stress in a rat model of Alzheimer's disease.

Xanthoceraside attenuates amyloid ß peptide1-42-induced memory impairments by reducing neuroinflammatory responses in mice.

Xanthoceraside, a novel triterpenoid saponin extracted from the husks of Xanthoceras sorbifolia Bunge, has neuroprotective effects in vivo and anti-inflammatory properties in vitro. However, the exact mechanism of xanthoceraside on anti-amyloid beta (Aß)-induced neuroinflammatory responses has not been elucidated. Therefore, we used intracerebroventricular injection of amyloid 1-42 (Aß1-42) to establish a mouse model to test the effects of xanthoceraside on Aß-induced cognitive impairments and the TLR2/NF-κB and MAPK pathways. The mice received xanthoceraside (0.02, 0.08 or 0.32mg/kg) or vehicle from the day of Aß1-42 injection. The Morris water maze test was performed 4 days after Aß1-42 injection. The levels of inflammatory cytokines (interleukin (IL)-6 and IL-4) were measured by enzyme-linked immunosorbent assay (ELISA). The expression levels of glial fibrillary acidic protein (GFAP) and cluster of differentiation 11b (CD11b) in the hippocampus were determined with an immunohistochemistry assay. Inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) were analysed by Western blotting; iNOS, COX-2 and Toll-like receptor 2 (TLR2) mRNA expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR). Here, we observed that xanthoceraside at doses of 0.08 and 0.32mg/kg significantly improved learning and memory impairments and significantly inhibited GFAP and CD11b overexpression induced by Aß1-42 in mice. ELISA results revealed that xanthoceraside suppressed IL-6 release and increased IL-4 levels. Western blotting results showed that xanthoceraside reduced iNOS and COX-2 protein levels in hippocampus; xanthoceraside also inhibited translocation of NF-κB p50 and p65 into the nucleus and phosphorylation of extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38. RT-PCR confirmed that xanthoceraside decreased iNOS, COX-2 and TLR2 mRNA levels. These results suggest that xanthoceraside inhibition of the TLR2 pathway and down-regulation of MAPK and NF-κB activities may be related to the improvement in learning and memory impairments.

Xanthoceraside attenuates tau hyperphosphorylation and cognitive deficits in intracerebroventricular-streptozotocin injected rats.

RATIONALE: Xanthoceraside, a novel triterpenoid saponin extracted from the fruit husks of Xanthoceras sorbifolia Bunge, reverses cognitive deficits in intracerebroventricular injection of Aß25-35 or Aß1-42 mice. However, whether xanthoceraside has a positive effect on hyperphosphorylated tau protein remains unclear. OBJECTIVES: We investigated the effects of xanthoceraside on behavioural impairments induced by intracerebroventricular injection of streptozotocin (STZ) in rats and its potential mechanisms. MATERIALS AND METHODS: The rats were administered with xanthoceraside (0.06, 0.12 or 0.24 mg/kg) or vehicle once daily after STZ intracerebroventricular injections. The Y-maze test and novel object recognition test were performed 21 and 22 days after the second STZ injection, respectively. The levels of hyperphosphorylated tau, phosphatidylinositol-3-kinase (PI3K)/serine/threonine protein kinase B (Akt), glycogen synthase kinase-3ß (GSK-3ß), protein phosphatase 1 (PP-1) and protein phosphatase 2A (PP-2A) were also tested by Western blot. RESULTS: Xanthoceraside treatment significantly attenuated learning and memory impairments and reduced the level of STZ-induced hyperphosphorylated tau protein. Xanthoceraside also enhanced PP-2A and PP-1 expressions, increased PI3K (p85) and Akt (Ser473) phosphorylation and decreased GSK-3ß (tyr216) phosphorylation. CONCLUSIONS: Xanthoceraside has protective effect against learning and memory impairments and inhibits tau hyperphosphorylation in the hippocampus, possibly through the inhibition of the PI3K/Akt-dependent GSK-3ß signalling pathway and an enhancement of phosphatases activity.

Protective effect of xanthoceraside against ß-amyloid-induced neurotoxicity in neuroblastoma SH-SY5Y cells.

ß-Amyloid (Aß)-induced neurotoxicity is a major pathological mechanism of Alzheimer's disease (AD). Xanthoceraside, a triterpene extracted from the husk of Xanthoceras sorbifolia Bunge, has been shown to have therapeutic effects on learning and memory impairment induced by Aß intracerebroventricular infusion in mice. In this study, we investigated the effect of xanthoceraside on the neurotoxicity of Aß25-35 in SH-SY5Y cells. Cell viability was measured by MTT (3-(3,4-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. Cell apoptosis, reactive oxygen species (ROS) generation, and mitochondrion membrane potential (MMP) were measured using Annexin V/propidium iodide, 2,7-dichlorofluorescein diacetate, and rhodamine 123 with flow cytometry, respectively. Intracellular calcium level was determined with Fura-2/AM. Caspase-3 activity in cell lysates was measured using the spectrophotometric method. Results indicated that pretreatment with xanthoceraside (0.01 and 0.1 µM) obviously increased the viability of SH-SY5Y cells injured by Aß25-35 in a dose-dependent manner. Aß25-35-induced early apoptosis, ROS overproduction, MMP dissipation, intracellular calcium overload, and increase in caspase-3 activity were markedly reversed by xanthoceraside. These findings suggested that xanthoceraside might be useful in the prevention and treatment of AD.

Xanthoceraside inhibits pro-inflammatory cytokine expression in Aß25-35/IFN-γ-stimulated microglia through the TLR2 receptor, MyD88, nuclear factor-κB, and mitogen-activated protein kinase signaling pathways.

An accumulating body of evidence suggests that Alzheimer's disease (AD) is associated with microglia-mediated neuroinflammation and pro-inflammatory cytokine expression. Therefore, the suppression of neuroinflammation and pro-inflammatory cytokine might theoretically slow down the progression of AD. Xanthoceraside, a novel triterpenoid saponin extracted from the husks of Xanthoceras sorbifolia Bunge, has potent antiinflammatory and neuroprotective effects. However, the molecular mechanism underlying its anti-inflammatory action remains unclear. In the present study, we attempted to determine the effects of xanthoceraside on the production of pro-inflammatory mediators in amyloid ß25-35 (Aß25-35)/interferon-γ (IFN-γ)-stimulated microglia. Our results indicated that xanthoceraside (0.01 and 0.1 µM) significantly inhibited the release of nitric oxide (NO) and pro-inflammatory cytokines interleukin-1ß and tumor necrosis factor-α in a concentration-dependent manner. Reverse transcriptase-polymerase chain reaction and western blotting analyses showed that xanthoceraside decreased the Aß25-35/IFN-γ-induced production of cyclooxygenase-2 and inducible NO synthase. These effects were accompanied by inhibited activities of nuclear factor-κB and mitogen-activated protein kinase through Toll-like receptor 2 in a myeloid differentiation protein 88-dependent manner. Our results provide support for the therapeutic potential of xanthoceraside in AD.

[Assessment of the changes in autonomic nervous function during head up tilt test in syncopal patients using spectral analysis of heart rate variability].

OBJECTIVE: To study the pathological mechanism of vasovagal syncope, spectral analysis of heart rate variability was used to evaluate the changes of autonomic function during head up tilt test in patients with unexplained syncope. METHODS: 27 patients with recurrent episodes of unexplained syncope underwent 70 degrees head up tilt test. Spectral analysis was used to assess the changes in autonomic function before tilt testing, immediately after tilting, just before the occurrence of syncope or at the end of the test, during the syncope period or at the end of the test and after testing in supine rest. At the same time, haemodynamic changes were recorded. Spectral power in very low frequency (VLF, 0.003 - 0.04 Hz), low frequency (LF, 0.04 - 0.15 Hz) and high frequency (HF, 0.15 - 2.00 Hz) were computed with Fast Fourier Transform analysis, and LF and HF were normalized: LF norm = 100 x LF/(TP-VLF) and HF norm = 100 x HF/(TP-VLF). RESULTS: 12 patients (mean age 40 +/- 10 years) showed a negative response and 15 patients (mean age 37 +/- 9 years) showed a positive response. Both systolic and diastolic blood pressure decreased in all the patients [(118.00 +/- 10.42-->81.00 +/- 12.36) mm Hg, P < 0.01 and (76.00 +/- 8.40-->52.00 +/- 10.95) mm Hg, P < 0.01] and heart rate decreased in 8 patients (53%). No significant difference in all the indices of spectral analysis was observed in supine position between the subjects with positive and negative test results. LF norm in both the groups did not alter during the entire tilt procedure. The decreased HF norm and increased LF/HF persisted throughout head up tilt test in the negative patients. In the positive patients, similar patterns of changes were observed before the occurrence of positive symptoms, and during the occurrence of the symptoms. HF norm abruptly rose (10.47 +/- 4.04-->32.95 +/- 10.48) and obviously exceeded that before tilt testing (23.44 +/- 4.20-->32.95 +/- 10.48, P < 0.05) and LF/HF dropped (3.28 +/- 0.39-->1.07 +/- 0.31, P < 0.01). At supine rest just after test, all the indices in both groups came back. CONCLUSIONS: In the supine position, autonomic function is similar between positive and negative subjects. Positive patients have a different pattern of response to the tilting test. The pathological mechanism leading to vasovagal syncope appears to be related with the abrupt and excessive increase of vagal activity.