Jiao Xu

In vitro evaluation of aspirin-induced HspB1 against heat stress damage in chicken myocardial cells.

To understand the potential association of heat stress resistance with HspB1 induction by aspirin (ASA) in chicken myocardial cells, variations of HspB1 expression and heat stressed-induced damage of myocardial cells after ASA administration were studied in primary cultured myocardial cells. Cytopathological lesions as well as damage-related enzymes, such as creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), indicated the considerable protective ability of ASA pre-treatment against acute heat stress. Immunostaining assays showed that heat stress caused HspB1 to relocate into the nucleus, while ASA did not. ELISA analysis, revealed that HspB1 expression induced by ASA averaged 45.62-fold higher than that of the control. These results indicated that the acute heat-stressed injuries were accompanied by comparatively lower HspB1 expression caused by heat stress in vitro. ASA pre-treatment induced a level of HspB1 presumed to be sufficient to protect myocardial cells from acute heat stress in the extracorporal model, although more detailed mechanisms will require further investigation.

Hsp70 expression induced by Co-Enzyme Q10 protected chicken myocardial cells from damage and apoptosis under in vitro heat stress

&NA; The aim of this study was to investigate whether induction of Hsp70 expression by co‐enzyme Q10 (Q10) treatment protects chicken primary myocardial cells (CPMCs) from damage and apoptosis in response to heat stress for 5 hours. Analysis of the expression and distribution of Hsp70 and the levels of the damage‐related enzymes creatine kinase‐MB (CK‐MB) and lactate dehydrogenase (LDH), as well as pathological analysis showed that co‐enzyme Q10 alleviated the damage caused to CPMCs during heat stress. Further, analysis of cell apoptosis and the expression of cleaved caspase‐3 indicated that co‐enzyme Q10 did have an anti‐apoptotic role during heat stress. Western blot analysis showed that pretreatment with co‐enzyme Q10 led to a significant increase in the expression of Hsp70 during heat stress. Immunostaining assays confirmed the results of western blot analysis and also showed that co‐enzyme Q10 could accelerate the translocation of Hsp70 into the nucleus during heat stress, but this was not observed in the group that was treated with only co‐enzyme Q10. These findings seem to indicate that co‐enzyme Q10 protected CPMCs from heat stress via the induction of Hsp70. To investigate this, 200 &mgr;M quercetin, an Hsp70 inhibitor, was used to inhibit the expression of Hsp70 2 h before heat stress. Quercetin pre‐treatment was observed to suppress the expression of Hsp70 as well the protective function of co‐enzyme Q10 at 5 h of heat stress. This finding confirms that Q10 brought about its effects via Hsp70 expression, but the mechanism underlying this needs further investigation.

Co-enzyme Q10 and acetyl salicylic acid enhance Hsp70 expression in primary chicken myocardial cells to protect the cells during heat stress

We investigated the effects of co-enzyme Q10 (Q10) and acetyl salicylic acid (ASA) on expression of Hsp70 in the protection of primary chicken myocardial cells during heat stress. Western blot analysis showed that Q10 and ASA accelerated the induction of Hsp70 when chicken myocardial cells were exposed to hyperthermia. In the absence of heat stress, however, neither Q10 nor ASA are able to upregulate Hsp70 expression. Analysis of enzymes that respond to cellular damage and pathological examination revealed that ectopic expression of ASA and Q10 alleviate cellular damage during heat stress. Quantification of heat shock factors (HSF) indicated that treatment of ASA increased the expression of HSF-1 and HSF-3 during heat stress. Treatment with Q10 resulted in the elevation of HSF-1 expression. Expression of HSF-2 and HSF-4 was not affected by ASA or Q10. Subcellular distribution analysis of HSF-1 and HSF-3 showed that in response to heat stress ASA promoted nuclear translocation of HSF-1 and HSF-3, while Q10 promoted only HSF-1 nuclear translocation. Chromatin immunoprecipitation (ChIP) analysis indicated that HSF-1 occupies the Hsp70 promoter in chicken primary myocardial cells during heat stress and under normal conditions, while HSF-3 occupies the Hsp70 promoter only during heat stress. Real-time PCR analysis revealed that ASA induces HSF-1 and HSF-3 binding to Hsp70 HSE, while Q10 only induces HSF1 binding to Hsp70 HSE, in agreement with the impact of HSF1 and HSF3 silencing on Hsp70 expression. These data demonstrate that ASA and Q10 both induce the expression of Hsp70 to protect chicken primary myocardial cells during heat stress, but through distinct pathways.

Lenti-siRNA Hsp60 promote bax in mitochondria and induces apoptosis during heat stress

Hsp60 is a typical mitochondrial protein in eukaryotes, and is involved in facilitating the correction of misfolded protein back into the correct conformation. Previous, we identified aspirin-induced HSPs in response to heat stress [1]. To investigate whether Hsp60 can protect against death under heat stress, we used lenti-siRNA to knock down the expression of Hsp60. When exposed to heat stress, more apoptosis was observed with increasing exposure to heat stress, while necrosis was not affected. Furthermore, heat stress induced the loss of mitochondrial membrane potential (ΔΨm) and a significant increase of reactive oxygen species (ROS) produced in mitochondria as measured by TMRE and MitoSOXTM red. The loss of ΔΨm indicated a change in inner mitochondrial function. Real-time Quantitative PCR was used to investigate the mechanism by detecting mRNA expression profile of the inner mitochondrial membrane, including CypD, ANT, and PIC. Results showed no differences between lenti-siRNA Hsp60 and control. However, bax in the cytoplasm translocated to mitochondrial during heat stress and regulated the permeability of outer mitochondrial membrane. We hypothesize that Hsp60 can protect cardiac myocytes against apoptosis involving in outer mitochondrial membrane not the inner mitochondrial membrane under heat stress.

Co-enzyme Q10 upregulates Hsp70 and protects chicken primary myocardial cells under in vitro heat stress via PKC/MAPK

In this report, we investigate the protective mechanism of co-enzyme Q10 on chicken primary myocardial cells during heat stress. Morphological observations indicate that addition of co-enzyme Q10 protects myocardial cells from heat stress, reduces the damage of mitochondria and nucleus, and decreases the mean number of vacuolated mitochondria. We have previously shown that co-enzyme Q10 can protect myocardial cells by upregulating the expression of Hsp70. Therefore, signaling pathways involved in this process were explored. No changes of total MAPK protein (P38MAPK, JNK, ERK) expression in the experimental groups were detected, with the exception of total JNK1. Co-enzyme Q10 failed to increase the expression of JNK1 compared to the HS group which was treated with heat stress only. Addition of Q10 upregulated the expression of p-P38MAPK, p-JNK, and p-ERK1. Inhibitors of P38MAPK and JNK, SB203580 and SP600125, respectively, weakened the upregulation of Hsp70 by co-enzyme Q10, indicating that MAPK pathways participate in the Hsp70 upregulation by co-enzyme Q10. Co-enzyme Q10 upregulates the expression of p-MEK3/6 and p-MEK4, but not p-MEK7 during heat stress. Expression of p-PKCα and p-PKCβ1 was also elevated following the addition of co-enzyme Q10 during heat stress, and addition of PKC inhibitors decreased the expression of Hsp70 induced by co-enzyme Q10. This confirms that PKC is also associated with the upregulation of Hsp70. In HS+Q10 group, addition of SP600125 or SB203580 could increase cell apoptosis under heat stress. Our results suggest that co-enzyme Q10 upregulates the expression of Hsp70 during heat stress to protect chicken primary myocardial cells via the PKC-MEK3/4/6-P38MAPK/JNK pathways.

Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/β and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/β level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/β. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

Co-enzyme Q10 protects chicken hearts from in vivo heat stress via inducing HSF1 binding activity and Hsp70 expression

ABSTRACT In this report, we investigated the protective function of co‐enzyme Q10 on chicken hearts during in vivo heat stress (HS) and the relationship with Hsp70 expression. The concentration of co‐enzyme Q10 (Q10) in the serum indicated that Q10 exogenously added prior HS was fully absorbed by chickens and is maintained at high levels during HS. The level of heart and oxidative damage‐associated enzymes in the serum revealed that treatment with Q10 decreased the activity of CK‐MB, CK, and LDH compared with the HS group; moreover, oxidative injury was also alleviated by Q10 according to the level of SOD, MDA, and T‐AOC in the serum compared with HS group during heat stress. A pathological examination indicated that the chicken hearts suffered serious damage during HS, including hemorrhage, granular changes, karyopyknosis, and cardiac muscle fiber disorder; however, the extent of heart damage was reduced in HS + Q10 group. Our results indicated that the addition of Q10 could upregulate the expression of Hsp70 during HS compared with the HS group. Compared with the HS group, the addition of Q10 significantly increased the gene expression of hsf1 during HS and hsf3 at 5 h of HS. The expression of hsf2 and hsf4 was not influenced by HS. Q10 could only accelerate the trimerization of HSF1 as well binding activities to Hsp70 HSE according to native page and ChIP assays. These findings suggest that co‐enzyme Q10 can protect chicken hearts from in vivo HS by inducing HSF1 binding activity and Hsp70 expression.

Rosemary Reduces Heat Stress by Inducing CRYAB and HSP70 Expression in Broiler Chickens

Heat stress negatively affects poultry production and animal health. In response, animals invoke a heat stress response by inducing heat shock proteins (HSPs). Scientists are actively seeking natural products that can enhance the heat shock response. The present study aimed at assessing the effects of a purified rosemary extract comprising antioxidant compounds on the heat shock response and HSP expression profile in broiler chickens. The response of broilers to HS in the presence of purified rosemary extract was assessed using an in vivo myocardial cell model. Pathological lesions of heart tissue were examined microscopically. The levels and activities of enzymes associated with heart damage and oxidative damage were detected. Immunohistochemical staining was performed for HSPs in myocardial cells. The results showed that lactate dehydrogenase (LDH), creatine kinase (CK), and myocardial CK (CKMB) levels were reduced by the purified rosemary extract before and during heat stress. Heat stress alone increased CK and CKMB levels. The levels of oxidative damage-associated enzymes were compared between the rosemary + heat stress and heat stress-alone groups. The results indicated that in terms of these enzymes, the purified rosemary extract induced a more antioxidative state. Pathological examinations showed that heat stress caused myocardial fiber fracture, karyopyknosis, and degeneration. The addition of purified rosemary extract ameliorated these lesions to some degree, preserving more of the basic structure. Heat stress decreased the cellular levels of crystallin alpha B (CRYAB) and HSP70. The addition of the purified rosemary extract significantly increased the levels of CRYAB and HSP70 during heat stress (p < 0.0001). Immunohistochemistry showed that after rosemary treatment, CRYAB and HSP70 showed more intense staining compared with the no heat stress control group. In the rosemary + heat group, after 10 hours of heat stress, the staining intensity of these two proteins remained higher than in the heat stress group. Thus, purified rosemary extract could induce high levels of HSP70 and CRYAB in chicken hearts before and during heat stress. Purified rosemary extract could be used to alleviate heat stress in broiler chickens.

Heat stress-induced renal damage in poultry and the protective effects of HSP60 and HSP47

The present study investigates the effects of heat stress on the kidney in broilers, based on previous findings which showed that heat stress caused cardiac damage in broilers. Further, the possible renoprotective role of aspirin and the heat shock proteins HSP60 and HSP47 was also investigated. The enzyme levels of urea and uric acid, which are indicators of renal damage, and lactate dehydrogenase, an indicator of oxidative damage, were measured in chickens that were only exposed to heat stress, chickens that were pretreated with aspirin before heat stress, and chickens that were only treated with aspirin. Further, histological examination of renal tissue from the three groups was also performed. Finally, expression of HSP60 and HSP47 was also examined. In the heat stress group, the enzyme measurements were indicative of renal dysfunction and oxidative damage, and the histological findings were indicative of renal ischemia and damage. Aspirin seemed to have a protective effect against the renal damage caused by the stress, based on the enzyme measurements and histopathological findings in the aspirin-treated group. The findings also indicate that aspirin may induce HSP60 and HSP47 expression in renal cells. Finally, the expression patterns of HSP60 and HSP47 indicated that they may play a renoprotective role, as their expression was higher in the aspirin-treated groups. In conclusion, the present findings show that heat stress causes renal damage in poultry and that aspirin may play a protective role against this damage via pathways that involve HSP60 and HSP47.

CRYAB protects cardiomyocytes against heat stress by preventing caspase-mediated apoptosis and reducing F-actin aggregation

CRYAB is a small heat shock protein (sHSP) that has previously been shown to protect the heart against various cellular stresses; however, its precise function in myocardial cell injury caused by heat stress remains unclear. This study aimed to investigate the molecular mechanism by which CRYAB protects cardiomyocytes against heat stress. We constructed two H9C2 cell lines that stably express CRYAB protein to differing degrees: CRYAB-5 and CRYAB-7. Both CRYAB-5 and CRYAB-7 showed significantly reduced granular degeneration and vacuolar degeneration following heat stress compared to control cells. In addition, CRYAB overexpression in H9C2 cells relieved cell cycle proportion at the G0/G1 phase following heat stress compared to control cells. These protective effects were associated with the level of CRYAB protein expression. Our immunofluorescence analysis showed CRYAB could translocate from the cytoplasm to the nucleus under heat stress conditions, but that CRYAB co-localized with F-actin (which accumulates under stress conditions). Indeed, overexpression of CRYAB significantly reduced the aggregation of F-actin in H9C2 cells caused by heat stress. Furthermore, overexpressing CRYAB protein significantly reduced the apoptosis of cardiomyocytes induced by heat stress, likely by reducing the expression of cleaved-caspase 3. Collectively, our results show overexpression of CRYAB significantly increases the heat resistance of H9C2 cardiomyocytes, likely by reducing F-actin aggregation (thus stabilizing the cytoskeleton), regulating the cell cycle, and preventing caspase-mediated apoptosis.