Guo-Xing Zhang

Apoptosis and autophagy contribute to gender difference in cardiac ischemia–reperfusion induced injury in rats

AIMS Gender difference in cardiac ischemia-reperfusion (IR) induced injury has been reported in animal models. However, a large-scale clinical trial found an increase in cardiovascular incidents in women with hormone replacement therapy. The present study is aimed to explore possible mechanisms of gender difference in cardiac IR induced injury. MAIN METHODS Male and female Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion. The infarct size and apoptotic cell number at 24h after reperfusion were significantly lower in female rats than in male rats. KEY FINDINGS Male rats expressed higher anti-apoptotic protein Bcl2 levels compared with female rats under physiological conditions. However, levels of Bcl2 were reduced significantly after IR in male rats but not in, female rats. Levels of pro-apoptotic protein, Bax and phospho-p38, showed similar under physiological conditions. In response to IR expression of Bax was markedly reduced in female rats but not in male rats, and expression of phospho-p38 was significantly increased in male rats but not in female rats. In addition, female rats showed marked increase of autophagy marker, ratio of LC3B to LC3A, while male rats significantly decreased the ratio in response to IR. SIGNIFICANCE Gender difference in IR injury is due to the different regulation of anti-apoptotic protein, pro-apoptotic protein and autophagy protein levels in male rats and levels in female rats. Our results provide better understanding of sex differences in cardiac IR injury.

Contribution of the renin–angiotensin system in chronic foot-shock induced hypertension in rats

AIMS Chronic foot shock has been demonstrated to induce hypertension. The present study was designed to explore whether the renin-angiotensin system (RAS) plays a role in this process and the possible mechanisms involved in chronic-foot-shock-induced hypertension. MAIN METHODS Male Sprague-Dawley rats were subjected to a two-week foot shock with or without an angiotensin II (Ang II) type 1 receptor blocker (ARB, candesartan) or an angiotensin I converting enzyme inhibitor (ACEI, captopril). The expression of RAS components in the central nervous and circulatory systems was examined. Antioxidant levels in the plasma were monitored. KEY FINDINGS Two-week foot shock significantly increased systolic blood pressure (SBP). Angiotensinogen, angiotensin I converting enzyme (ACE)-1, ACE-2, angiotensin type 1a and type 1b receptors, and vasopressin (VAP) mRNA expression in the cerebral cortex and hypothalamus were increased along with the concentration of renin and Ang II in the plasma; these changes were accompanied by decreased glutathione peroxidase activity and increased lipid peroxidation levels and plasma corticosterone concentrations. Both candesartan and captopril suppressed not only the increases in SBP but also the increases in VAP expression in the hypothalamus and RAS components in the central nervous system and the circulatory system. The decreases in antioxidant levels and the increases in lipid peroxidation and corticosterone levels were also partially reversed by candesartan or captopril treatment. SIGNIFICANCE Chronic foot shock increases expression of the main RAS components, which play an important role in the development of high blood pressure through increased VAP levels, oxidative stress levels and stress hormone levels.

The pathway of subarachnoid CSF moving into the spinal parenchyma and the role of astrocytic aquaporin-4 in this process

Aims: It has been proved that cerebrospinal fluid (CSF) in the subarachnoid space could reenter the brain parenchyma via the perivascular space. The present study was designed to explore the pathway of subarachnoid CSF flux into the spinal cord and the potential role of aquaporin‐4 (AQP4) in this process. Main methods: Fluorescently tagged cadaverine, for the first time, was used to study CSF movement in mice. Following intracisternal infusion of CSF tracers, the cervical spinal cord was sliced and prepared for fluorescence imaging. Some sections were subject with immunostaining in order to observe tracer distribution and AQP4 expression. Key findings: Fluorescently tagged cadaverine rapidly entered the spinal cord. Tracer influx into the spinal parenchyma was time dependent. At 10 min post‐infusion, cadaverine was largely distributed in the superficial tissue adjacent to the pial surface. At 70 min post‐infusion, cadaverine was distributed in the whole cord and especially concentrated in the gray matter. Furthermore, fluorescent tracer could enter the spinal parenchyma either along the perivascular space or across the pial surface. AQP4 was observed highly expressed in the astrocytic endfeet surrounding blood vessels and the pial surface. Blocking AQP4 by its specific inhibitor TGN‐020 strikingly reduced the inflow of CSF tracers into the spinal cord. Significance: Subarachnoid CSF could flow into the spinal cord along the perivascular space or across the pial surface, in which AQP4 is involved. Our observation provides a basis for the study on CSF movement in the spinal cord when some neurological diseases occur.

Protective effects of tanshinone IIA sodium sulfonate on ischemia-reperfusion-induced myocardial injury in rats

Objective(s): This study investigated the protective effect of tanshinone IIA sodium sulfonate (TSS) on ischemia-reperfusion (I/R) induced cardiac injury, and the underlying mechanism of action. Materials and Methods: Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by 24 hours’ reperfusion. Half an hour before the left coronary artery ligation, rats were pretreated with TSS in three different dosages (15, 30, 70 mg/kg, IP). Twenty-four hours later, cardiac function was measured and the ratio of infarct size to area at risk (AAR) was calculated. Western blotting examined the expression of the inflammatory mediator high-mobility group box1 (HMGB-1), anti-apoptotic protein Bcl-2, pro-apoptotic mediators such as Bax and Caspase-3, markers of autophagy such as ratio of LC3B/LC3A and Beclin-1 expression. Results: Our results showed that TSS dose-dependently improves cardiac function, accompanied with decrease of HMGB1 level, increase of LC3B/LC3A ratio and increase of Beclin-1 expression. TSS treatment down-regulates Bax and Caspase-3 expression, while up-regulating Bcl-2 levels. Conclusion: TSS ameliorates I/R induced myocardial injury and improves cardiac function via reducing inflammation and apoptosis, while enhancing autophagy.

Role of the renin-angiotensin system, renal sympathetic nerve system, and oxidative stress in chronic foot shock-induced hypertension in rats.

Objective: The renin-angiotensin system (RAS) and renal sympathetic nerve system (RSNS) are involved in the development of hypertension. The present study is designed to explore the possible roles of the RAS and the RSNS in foot shock-induced hypertension. Methods: Male Sprague-Dawley rats were divided into six groups: control, foot shock, RSNS denervation, denervation plus foot shock, Captopril (angiotensin I converting enzyme inhibitor, ACE inhibitor) plus foot shock, and Tempol (superoxide dismutase mimetic) plus foot shock. Rats received foot shock for 14 days. We measured the quantity of thiobarbituric acid reactive substances (TBARS), corticosterone, renin, and angiotensin II (Ang II) in plasma, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and renal noradrenaline content. RAS component mRNA and protein levels were quantified in the cerebral cortex and hypothalamus. Results: The two week foot shock treatment significantly increased systolic blood pressure, which was accompanied by an increase in angiotensinogen, renin, ACE1, and AT1a mRNA and protein expression in the cerebral cortex and hypothalamus, an increase of the plasma concentrations of renin, Ang II, corticosterone, and TBARS, as well as a decrease in plasma SOD and GSH-Px activities. Systolic blood pressure increase was suppressed by denervation of the RSNS or treatment with Captopril or Tempol. Interestingly, denervation or Tempol treatment both decreased main RAS components not only in the circulatory system, but also in the central nervous system. In addition, decreased antioxidant levels and increased TBARS and corticosterone levels were also partially restored by denervation or treatment with Tempol or Captopril. Conclusions: RAS, RSNS and oxidative stress reciprocally potentiate to play important roles in the development of foot shock-induced hypertension.

Effects of Extracts from Tiaozhi Granule and Its Components on Expression of Scavenger Receptor Class B Type I.

Sera from the rats with different drug treatments (atorvastatin, Tiaozhi granule, or its extracts) were collected. LO-2 cells or HepG2 cells were pretreated with different sera as the following groups randomly: (1) blank control group, (2) positive control group (atorvastatin group), (3) Tiaozhi granule water extract groups, (4) Tiaozhi granule alcohol extract groups, and (5) alcohol extracts for each component: Pollen Typhae Angustifoliae, Curcuma longa L., and Rhizoma Alismatis. LO-2 cells were cotransfected with plasmid carrying SR-BI and pRL-TK promoter genes. Promoter activity was measured by the luciferase reporter gene assay. The mRNA and protein expressions of SR-BI were examined using real-time PCR and western blot analyses. Our results show that promoter activity and mRNA and protein expression levels of the SR-BI were significantly upregulated by Tiaozhi granules alcohol or water extracts in a dose-dependent manner. Pollen Typhae Angustifoliae alcohol extract with a high dosage could also increase SR-BI activity and expression, but not the extracts from Curcuma longa L. and Rhizoma Alismatis. Both Tiaozhi granule alcohol and water extracts can upregulate SR-BI gene expression. Among the components, Pollen Typhae Angustifoliae are important for the regulatory effect coordinating with Curcuma longa L. and Rhizoma Alismatis.

Characterizing the glymphatic influx by utilizing intracisternal infusion of fluorescently conjugated cadaverine

Aims: Accumulating evidence supports that cerebrospinal fluid (CSF) in the subarachnoid space (SAS) could reenter the brain parenchyma via the glymphatic influx. The present study was designed to characterize the detailed pathway of subarachnoid CSF influx by using a novel CSF tracer. Main methods: Fluorescently conjugated cadaverine (A488‐ca), for the first time, was employed to investigate CSF movement in the brain. Following intracisternal infusion of CSF tracers, mice brain was sliced and prepared for fluorescence imaging. Some brain sections were immunostained in order to observe tracer distribution and cellular uptake. Key findings: A488‐ca moved into the brain parenchyma rapidly, and the influx was time and region dependent. A488‐ca entered the mice brain more readily and spread more widely than another commonly used CSF tracer‐fluorescently conjugated ovalbumin (OA‐45). Furthermore, A488‐ca could enter the brain parenchyma either along the paravascular space or across the pial surface. Suppression of glymphatic transport by administration with acetazolamide strikingly reduced the influx of A488‐ca. More importantly, relative to OA‐45 largely remained in the extracellular space, A488‐ca exhibited obvious cellular uptake by astrocytes surrounding the blood vessels and neurons in the cerebral cortex. Significance: Subarachnoid CSF could flow into the brain parenchyma via the glymphatic influx, in which the transcellular pathway was faithfully traced by intracisternal infusion with fluorescently conjugated cadaverine. These observations extend our comprehension on the glymphatic influx pathway.

Effects of Tiaozhi Granule on Regulation of Autophagy Levels in HUVECs

Sera from the rats with Tiaozhi granule treatment were collected. Human umbilical vein endothelial cells (HUVECs) were incubated with different dosage of sera with Tiaozhi granule for 48 hours. Rapamycin or angiotensin II was applied to activate autophagy in HUVECs with or without different dosages of sera of Tiaozhi granule. The mRNA expressions of Atg5, Atg7, Beclin-1, and mammal target of rapamycin (mTOR) were detected by real-time PCR. Autophagic flux markers (protein expression of LC3, Beclin-1, and p62) were examined by western blot analyses. The number of autophagosomes was visualized by immunofluorescence analysis with LC3-II labelling. Results showed that Tiaozhi granule sera increase cell autophagic levels by increase of mRNA of Atg5, Atg7, Beclin-1, and mTOR and increase of autophagic flux and also number of autophagosomes. However, in response to rapamycin or Ang II stimulation, activated autophagic levels were alleviated by Tiaozhi granule sera by reduction of mRNA of Atg5, Atg7, Beclin-1, mTOR, autophagic flux, and also number of autophagosomes. Our present data demonstrate that Tiaozhi granule plays a dual role in response to different cell conditions, which is to increase cell autophagy under physiological condition and to suppress cell excessive autophagy under pathological condition.

Selective peroxisome proliferator-activated receptor-α modulator K-877 regulates the expression of ATP-binding cassette transporter A1 in pancreatic beta cells

Abstract ATP‐binding cassette transporter A1 (ABCA1) protein is a pivotal regulator of cholesterol and phospholipid efflux from cells to high‐density lipoprotein (HDL) particles. Pancreatic ABCA1 functions in beta cell cholesterol homeostasis and affects insulin secretion. We investigated the effect of pemafibrate (K‐877), a novel selective PPAR&agr; modulator (SPPARM&agr;), on pancreatic ABCA1 expression. In vivo experiment, mice were divided into four treatment groups, namely, normal food plus placebo, high fat diet (HFD) plus placebo, normal food plus K‐877 (0.3 mg/kg/day), or HFD plus K‐877 (0.3 mg/kg/day), and treated for eight weeks. The results in vitro experiment indicate that K‐877 treatment increased levels of ABCA1 mRNA, as well as protein, subsequently reduced the cellular cholesterol content in INS‐1 cells. PPAR&agr; specific antagonist GW6471 attenuate K‐877 induced ABCA1 expression in INS‐1 cells. ABCA1 promoter activity increased with K‐877 treatment at concentration 1 &mgr;M and 10 &mgr;M. Glucose‐stimulated insulin secretion was ameliorated by K‐877 treatment in INS‐1 cells and isolated mouse islets. Although the expression of ABCA1 was reduced in mice with HFD treatment, both ABCA1 protein and mRNA levels were increased in mice with K‐877 treatment. K‐877 treatment improved glucose intolerance induced by HFD in mice. These findings raise the possibility that K‐877 may affect insulin secretion by controlling ABCA1 expression in pancreatic beta cells.