Pravastatin Attenuates Acute Radiation-Induced Enteropathy and Improves Epithelial Cell Function

Background and Aim: Radiation-induced enteropathy is frequently observed after radiation therapy for abdominal and pelvic cancer or occurs secondary to accidental radiation exposure. The acute effects of irradiation on the intestine might be attributed to inhibition of mitosis in the crypts, as the loss of proliferative functions impairs development of the small intestinal epithelium and its barrier function. Especially, oxidative damage to intestinal epithelial cells is a key event in the initiation and progression of radiation-induced enteropathy. Pravastatin is widely used clinically to lower serum cholesterol levels and has been reported to have anti-inflammatory effects on endothelial cells. Here, we investigated the therapeutic effects of pravastatin on damaged epithelial cells after radiation-induced enteritis using in vitro and in vivo systems.Materials and Methods: To evaluate the effects of pravastatin on intestinal epithelial cells, we analyzed proliferation and senescence, oxidative damage, and inflammatory cytokine expression in an irradiated human intestinal epithelial cell line (InEpC). In addition, to investigate the therapeutic effects of pravastatin in mice, we performed histological analysis, bacterial translocation assays, and intestinal permeability assays, and also assessed inflammatory cytokine expression, using a radiation-induced enteropathy model.Results: Histological damage such as shortening of villi length and impaired intestinal crypt function was observed in whole abdominal-irradiated mice. However, damage was attenuated in pravastatin-treated animals, in which normalization of intestinal epithelial cell differentiation was also observed. Using in vitro and in vivo systems, we also showed that pravastatin improves the proliferative properties of intestinal epithelial cells and decreases radiation-induced oxidative damage to the intestine. In addition, pravastatin inhibited levels of epithelial-derived inflammatory cytokines including IL-6, IL-1β, and TNF-α in irradiated InEpC cells. We also determined that pravastatin could rescue intestinal barrier dysfunction via anti-inflammatory effects using the mouse model.Conclusion: Pravastatin has a therapeutic effect on intestinal lesions and attenuates radiation-induced epithelial damage by suppressing oxidative stress and the inflammatory response

Neurotrophic interactions between neurons and astrocytes following AAV1-Rheb(S16H) transduction in the hippocampus in vivo

Background and Purpose: We recently reported that AAV1-Rheb(S16H) transduction could protect hippocampal neurons through the induction of brain-derived neurotrophic factor (BDNF) in the rat hippocampus in vivo. It is still unclear how neuronal BDNF produced by AAV1-Rheb(S16H) transduction induces neuroprotective effects in the hippocampus and whether its up-regulation contributes to the enhance of a neuroprotective system in the adult brain. Experimental Approach: To determine the presence of a neuroprotective system in the hippocampus of patients with Alzheimer's disease (AD), we examined the levels of glial fibrillary acidic protein, BDNF and ciliary neurotrophic factor (CNTF) and their receptors, tropomyocin receptor kinase B (TrkB) and CNTF receptor α(CNTFRα), in the hippocampus of AD patients. We also determined whether AAV1-Rheb(S16H) transduction stimulates astroglial activation and whether reactive astrocytes contribute to neuroprotection in models of hippocampal neurotoxicity in vivo and in vitro. Key Results: AD patients may have a potential neuroprotective system, demonstrated by increased levels of full-length TrkB and CNTFRα in the hippocampus. Further AAV1-Rheb(S16H) transduction induced sustained increases in the levels of full-length TrkB and CNTFRα in reactive astrocytes and hippocampal neurons. Moreover, neuronal BDNF produced by Rheb(S16H) transduction of hippocampal neurons induced reactive astrocytes, resulting in CNTF production through the activation of astrocytic TrkB and the up-regulation of neuronal BDNF and astrocytic CNTF which had synergistic effects on the survival of hippocampal neurons in vivo. Conclusions and Implications: The results demonstrated that Rheb(S16H) transduction of hippocampal neurons could strengthen the neuroprotective system and this intensified system may have a therapeutic value against neurodegeneration in the adult brain. © 2019 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society1

Beta-Amyloid Instigates Dysfunction of Mitochondria in Cardiac Cells

Alzheimer’s disease (AD) includes the formation of extracellular deposits comprising aggregated β-amyloid (Aβ) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aβ in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aβ1-40 and Aβ1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aβ1-40 and Aβ1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aβ1-42 had more adverse effects than Aβ1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aβ1-42. Mitochondrial dysfunction induced by Aβ was associated with apoptosis of the cells. The effects of Aβ1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aβ1-40 and Aβ1-42 significantly increased Ca2+ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aβ on cell survival and mitochondria function in cardiac cells

Inhibition of JNK aggravates the recovery of rat hearts after global ischemia: the role of mitochondrial JNK.

c-Jun N-terminal kinase (JNK), a stress-activated MAPK, is activated during cardiac ischemia-reperfusion (IR). The role of JNK inhibitors in cardioprotection against IR still remains controversial, in part, due to spill-over effects of non-specific inhibitors. In the present study, we sought to examine whether inhibition of JNK by SU3327, a specific JNK inhibitor that inhibits upstream JNK signaling rather than the kinase activity of JNK, improves cardiac function and reduces heart damage during IR. Hearts of male Sprague-Dawley rats perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion in the presence or absence of SU3327. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion, mitochondria were isolated and used to measure respiration rates and mitochondrial permeability transition pore opening. Protein analysis of mitochondria predictably revealed that SU3327 inhibited JNK phosphorylation. Although SU3327 significantly reduced cell damage during the first minutes of reperfusion, it did not improve cardiac function and, furthermore, reduced the mitochondrial respiratory control index. Interestingly, SU3327 activated the other stress-related MAPK, p38, and greatly increased its translocation to mitochondria. Mitochondrial P-JNK and P-p38 were co-immunoprecipitated with complex III of the electron transfer chain. Thus, JNK plays an essential role in cardiac signaling under both physiological and pathological conditions. Its inhibition by SU3327 during IR aggravates cardiac function. The detrimental effects of JNK inhibition are associated with reciprocal p38 activation and mitochondrial dysfunction

Co-immunoprecipitation of mitochondrial proteins with P-JNK and P-p38.

<p>Representative immunoblots showing interaction between P-JNK, P-p38, the MPTP components ANT, VDAC, CyP-D, and components of mitochondrial oxidative phosphorylation. (<b><i>A</i></b>) Cardiac mitochondria from each group were immunoprecipitated (IP) with P-JNK and P-p38. The complexes were subjected to SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. Bands that underwent densitometry analysis are indicated by arrows (a,b). (<b><i>B</i></b>) Densitometric data for UQCRC2 (a component of mitochondrial complex III), were normalized to P-JNK. CS, non-ischemic hearts perfused for 60 min with 10 µM SU3327 added at 20 min after beginning of perfusion (n = 4). *, #, &: significantly different from the other indicators (<i>P</i><0.05). (<b><i>C</i></b>) Densitometric data for UQCRC2 (a component of mitochondrial complex III), normalized to P-p38. n = 3 per group.</p

Heart function.

<p>(<b><i>A</i></b>) Left ventricular (LV) developed pressure (LVDP) calculated as the difference between LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP). Data are expressed as a percentage of pre-ischemic values. *<i>P</i><0.0001 C <i>vs</i> other groups, ^#<i>P</i><0.05 IR <i>vs</i> IRS and IRSP. (<b><i>B</i></b>) LVEDP at the end of reperfusion (for IR, IRS, IRSP and IRSR) or perfusion (C). (<b><i>C</i></b>) The rate-pressure product (RPP) calculated as RPP = LVDP×HR. RPP recovery during the reperfusion period is shown as percent of pre-ischemia values. (<b><i>D</i></b>) Lactate dehydrogenase (LDH) activity in the coronary effluent. The dotted line represents 25 min of ischemia for IR, IRS, IRSP, and IRSR groups. Time in parentheses represents perfusion time for the C group (without IR). LDH activity is shown as decrement of absorbance at 340 nm per min for liter of perfusate per gram heart. *<i>P</i><0.001 C <i>vs</i> other groups, #, &: significantly different from the other indicators (<i>P</i><0.05). n = 5 for C, n = 8 for IR, n = 8 for IRS, n = 7 for IRSP, and n = 6 for IRSR groups.</p

Effect of SU3327 on H9C2 cells exposed to H₂O₂.

<p>Cardiomyocytes were pretreated with JNK inhibitor SU3327 (SU), then treated with 75 µM H₂O₂ (<b><i>A,B,C</i></b>). Cardiomyocytes were treated with SU only (<b><i>D,E,F</i></b>). Cell viability (<b><i>A,B</i></b>), total ROS (<b><i>B,E</i></b>), mitochondrial membrane potential (<b><i>C,F</i></b>). *, #, &, @: significantly different from the other indicators (<i>P</i><0.05). n = 6 per group.</p

Mitochondrial respiratory control index, MPTP opening and carbonylation of mitochondrial proteins.

<p>(<b><i>A</i></b>) Mitochondrial respiratory control index (RCI) at complex I measured in the presence of 2.5 mM 2-oxoglutarate and 1 mM L-malate substrates. (<b><i>B</i></b>) RCI at complex II measured in the presence of a 2.5 mM succinate substrate. (<b><i>C</i></b>) RCI at complex IV measured in the presence of 10 mM ascorbate and 0.3 mM TMPD. *, #, &, @, +: significantly different from the other indicators (<i>P</i><0.05). (<b><i>D</i></b>) Increment of the rate of mitochondrial swelling by addition of 100 mM CaCl₂. *: significantly different from group C (<i>P</i><0.05), (<b><i>E</i></b>) Carbonylation levels of mitochondrial proteins. The data were represented as the ratio of intensities from DNPH-treated samples to non-treated samples compared to control for each group. *, #, &, @: significantly different from the other indicators (<i>P</i><0.05). n = 5 for C, n = 8 for IR, n = 8 for IRS, n = 7 for IRSP, and n = 6 for IRSR groups.</p