Tacrolimus reverses pemphigus vulgaris serum-induced depletion of desmoglein in HaCaT cells via inhibition of heat shock protein 27 phosphorylation.

BACKGROUND: Glucocorticoids are the first-line treatment for Pemphigus vulgaris (PV), but its serious side effects can be life-threatening for PV patients. Tacrolimus (FK506) has been reported to have an adjuvant treatment effect against PV. However, the mechanism underlying the inhibitory effect of FK506 on PV-IgG-induced acantholysis is unclear. OBJECTIVE: The objective of this study was to explore the effect of FK506 on desmoglein (Dsg) expression and cell adhesion in an immortalized human keratinocyte cell line (HaCaT cells) stimulated with PV sera. METHODS: A cell culture model of PV was established by stimulating HaCaT cells with 5% PV sera with or without FK506 and clobetasol propionate (CP) treatment. The effects of PV sera on intercellular junctions and protein levels of p38 mitogen-activated protein kinase (p38MAPK), heat shock protein 27 (HSP27), and Dsg were assayed using western blot analysis, immunofluorescence staining, and a keratinocyte dissociation assay. RESULTS: PV sera-induced downregulation of Dsg3 was observed in HaCaT cells and was blocked by FK506 and/or CP. Immunofluorescence staining revealed that linear deposits of Dsg3 on the surface of HaCaT cells in the PV sera group disappeared and were replaced by granular and agglomerated fluorescent particles on the cell surface; however, this effect was reversed by FK506 and/or CP treatment. Furthermore, cell dissociation assays showed that FK506 alone or in combination with CP increased cell adhesion in HaCaT cells and ameliorated loss of cell adhesion induced by PV sera. Additionally, FK506 noticeably decreased the PV serum-induced phosphorylation of HSP 27, but had no effect on p38MAPK phosphorylation. CONCLUSION: FK506 reverses PV-IgG induced-Dsg depletion and desmosomal dissociation in HaCaT cells, and this effect may be obtained by inhibiting HSP27 phosphorylation.

Electroacupuncture inhibits PDK1/Akt/HCN4 pathway to improve neurogenic urinary retention in rats. / 电针通过抑制PDK1/Akt/HCN4é€šè·¯æ”¹å–„å¤§é¼ ç¥žç»æºæ€§å°¿æ½´ç•™.

OBJECTIVES: To observe the therapeutic effect of electroacupuncture (EA) on neurogenic urinary retention rats, so as to explore the underlying mechanism of EA in treating neurogenic urinary retention by focusing on 3-phosphoinositide-dependent protein kinase 1 (PDK1)/protein kinase B (Akt)/hyperpolarization activated cyclic nucleotide-gated cation channel 4 (HCN4) pathway. METHODS: Female SD rats were randomly divided into sham operation, model, EA, PDK1 inhibitor, HCN4 blocker and EA + HCN4 blocker groups, with 20 rats in each group. The model of sacral spinal cord injury was established by modified Hassan Shaker spinal cord transection method. EA (2 Hz/15 Hz, 0.5 mA) was applied to "Zhongji" (CV3) and "Zhongliao" (BL33) for 20 min, once daily for 10 days. Rats of the PDK1 inhibitor group received intraperitoneal injection of OSU-03012 (20 mg/kg), and rats of the HCN4 blocker group received intraperitoneal injection of ivabradine (10 mg/kg), both once every other day for 10 days. The urodynamic indexes of rats were detected by multi-channel physiological recorder；muscle strip test was used to detect detrusor excitability；the morphological changes of bladder were observed by HE staining. Immunofluorescence double staining was used to detect the co-expression of HCN4 and C-Kit, a specific marker of interstitial cells of Cajal in bladder. Western blot was used to detect the expression of PDK1/Akt/HCN4 pathway proteins in bladder tissue and heat shock protein 27 (HSP27), a protein related to bladder contraction function. RESULTS: Compared with the sham operation group, the rats in the model group showed urinary dysfunction, decreased leak point pressure, isolated detrusor spontaneous contraction frequency, fluorescence intensity of C-Kit positive cells, HCN4+/C-Kit+ co-expression, HCN4 and p-HSP27/HSP27 protein expression in bladder tissue (P<0.05), and increased maximum bladder capacity and comp-liance, minimum tension during contraction of isolated detrusor, PDK1 and p-Akt/Akt protein expression in bladder tissue (P<0.05). Meanwhile, the above index were all reversed after EA and PDK1 inhibitor intervention (P<0.05). In comparison with the EA group, the rats had severe urinary dysfunction, the urine leakage point pressure, spontaneous contraction frequency, fluorescence intensity of C-Kit positive cells, the co-expression of HCN4+/C-Kit+, and the protein expression of HCN4 and p-HSP27/HSP27 were decreased (P<0.05), the maximum bladder capacity and compliance, the minimum tension during contraction of isolated detrusor, and the protein expression of PDK1 and p-Akt/Akt in bladder tissue were increased (P<0.05) in both HCN4 blocker and EA+HCN4 blocker groups. HE staining showed exfoliated bladder epithelium and disordered layers, vacuolization of bladder wall cells, with infiltration of neutrophils in mucosal and muscular layers in the model group, which were relatively milder in the EA and PDK1 inhibitor groups, but worse in the HCN4 blocker and EA + HCN4 blocker groups. CONCLUSIONS: EA can improve the urinary dysfunction in rats with neurogenic urinary retention, which may be related to its effect in inhibiting the activation of PDK1/Akt pathway, promo-ting HCN4-mediated detrusor excitatory contraction and urinary electrical signal activation.

Mangiferin prevents hepatocyte epithelial-mesenchymal transition in liver fibrosis via targeting HSP27-mediated JAK2/STAT3 and TGF-ß1/Smad pathway.

Hepatocytes has been confirmed to undergo EMT and can be converted into myofibroblasts during hepatic fibrogenesis. However, the mechanism of hepatocyte EMT regulation in hepatic fibrosis, particularly through HSP27 (human homologue of rodent HSP25), remains unclear. Mangiferin (MAN), a compound extracted from Mangifera indica L, has been reported to attenuate liver injury. This study aimed to investigate the mechanisms underlying HSP27 inhibition and the anti-fibrotic effect of MAN in liver fibrosis. Our results revealed that the expression of HSP27 was remarkably increased in the liver tissues of patients with liver cirrhosis and CCl4 -induced fibrotic rats. However, HSP27 shRNA treatment significantly alleviated fibrosis. Furthermore, MAN was found to inhibit CCl4 - and TGF-ß1-induced liver fibrosis and reduced hepatocyte EMT. More importantly, MAN decreased HSP27 expression to suppress the JAK2/STAT3 pathway, and subsequently blocked TGF-ß1/Smad signaling, which were consistent with its protection against CCl4 -induced EMT and liver fibrosis. Together, these results suggest that HSP27 may play a crucial role in hepatocyte EMT and liver fibrosis by activating JAK2/STAT3 signaling and TGF-ß1/Smad pathway. The suppression of HSP27 expression by MAN may be a novel strategy for attenuating the hepatocyte EMT in liver fibrosis.

The nephroprotective properties of taurine-amikacin treatment in rats are mediated through HSP25 and TLR-4 regulation.

Amikacin (AMK) is one of the most effective aminoglycoside antibiotics. However, nephrotoxicity is a major deleterious and dose-limiting side effect associated with its clinical use especially in high dose AMK-treated patients. The present study assessed the ability of taurine (TAU) to alleviate or prevent AMK-induced nephrotoxicity if co-administrated with AMK focusing on inflammation, apoptosis, and fibrosis. Male Sprague Dawley rats were assigned to six equal groups. Group 1: rats received saline (normal control), group 2: normal rats received 50 mg kg-1 TAU intraperitoneally (i.p.). Groups 3 and 4: received AMK (25 or 50 mg kg-1; i.p.). Groups 5 and 6: received TAU (50 mg kg-1; i.p.) concurrently with AMK (25 or 50 mg kg-1; i.p.) for 3 weeks. AMK-induced nephrotoxicity is evidenced by elevated levels of serum creatinine (CRE), blood urea nitrogen (BUN), and uric acid (UA). Histopathological investigations provoked damaging changes in the renal tissues. Heat shock proteins (HSP)25 and Toll-like receptor-4 (TLR-4) elevated levels were involved in the induction of inflammatory reactions and focal fibrosis. The improved activation of TLR-4 may stimulate monocytes to upgrade Interleukin (IL)-18 production rather than IL-10. TAU proved therapeutic effectiveness against AMK-induced renal toxicity through downregulation of HSP25, TLR-4, caspase-3, and IL-18 with up-regulation of IL-10 levels.

Neuroprotective Effects of Asparagus officinalis Stem Extract in Transgenic Mice Overexpressing Amyloid Precursor Protein.

To mimic Alzheimer's disease, transgenic mice overexpressing the amyloid precursor protein (APP) were used in this study. We hypothesize that the neuroprotective effects of ETAS®50, a standardized extract of Asparagus officinalis stem produced by Amino Up Co., Ltd. (Sapporo, Japan), are linked to the inhibition of the apoptosis cascade through an enhancement of the stress-response proteins: heat shock proteins (HSPs). APP-overexpressing mice (double-transgenic APP and PS1 mouse strains with a 129s6 background), ages 6-8 weeks old, and weighing 20-24 grams were successfully bred in our laboratory. The animals were divided into 5 groups. APP-overexpressing mice and wild-type (WT) mice were pretreated with ETAS®50 powder (50% elemental ETAS and 50% destrin) at 200 mg/kg and 1000 mg/kg body weight. Saline, the vehicle for ETAS®50, was administered in APP-overexpressing mice and WT mice. ETAS®50 and saline were administered by gavage daily for 1 month. Cognitive assessments, using the Morris Water Maze, demonstrated that memory was recovered following ETAS®50 treatment as compared to nontreated APP mice. At euthanization, the brain was removed and HSPs, amyloid ß, tau proteins, and caspase-3 were evaluated through immunofluorescence staining with the appropriate antibodies. Our data indicate that APP mice have cognitive impairment along with elevated amyloid ß, tau proteins, and caspase-3. ETAS®50 restored cognitive function in these transgenic mice, increased both HSP70 and HSP27, and attenuated pathogenic level of amyloid ß, tau proteins, and caspsase-3 leading to neuroprotection. Our results were confirmed with a significant increase in HSP70 gene expression in the hippocampus.

MicroRNA216b mediated downregulation of HSP27/STAT3/AKT signaling is critically involved in lambertianic acid induced apoptosis in human cervical cancers.

Since heat shock protein (HSP27) is a prognostic marker in cervical cancer, in the present study, the apoptotic mechanism of lambertianic acid (LA) was investigated in human cervical cancers in association with HSP27/STAT3/AKT signaling axis. LA exerted significant cytotoxicity, induced sub-G1 population, and increased the cleavage of Poly (ADP-ribose) polymerase (PARP) and cysteine aspartyl-specific protease 3 (caspase3) in HeLa and Caski cancer cells. Consistently, LA downregulated anti-apopotic genes such as B-cell lymphoma 2 (Bcl-2) and inhibitors of apoptosis proteins (c-IAP) in HeLa and Caski cells. Furthermore, LA-inhibited phosphorylation of HSP27, signal transducer, and activator of transcription 3 (STAT3) and Protein kinase B (AKT) through disturbing the binding of HSP27 with STAT3 or AKT in HeLa cells. Notably, LA upregulated the level of miR216b in HeLa and Caski cells. Consistently, miR216b mimic suppressed phosphorylation of HSP27 and reduced the expression of pro-PARP, while miR216b inhibitor reversed the ability of LA to attenuate phosphorylation of AKT, HSP27, and STAT3 and to reduce the expression of pro-PARP in HeLa cells. Overall, our findings suggest that miRNA216b mediated inhibition of HSP27/STAT3/ AKT signaling axis is critically involved in LA-induced apoptosis in cervical cancers.

Adverse Effects in Skeletal Muscle Following the Medicinal Use of Nicotiana glauca.

Nicotiana glauca is a cosmopolitan shrub, used in medicine to treat swellings, wounds, sores and cancer. However, its users lack of knowledge of the adverse effects. We seek to evaluate the effects of lipid extracts from N. glauca on myoblasts, identifying the compounds which cause undesirable effects. Myoblasts are important in muscle homeostasis, thus a high death rate of them cause myopathies. We performed an ethanolic extraction from leaves of N. glauca and the extract was successively partitioned with hexane, chloroform and ethyl acetate. The effects of extracts in C2C12 cells were analysed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), Mitotracker and 4',6-diamidino-2-phenylindole (DAPI) staining, Western blotting, real-time PCR and immunofluorescence assays. Caspase activity was studied. The fraction with the highest apoptotic effects was analysed by chromatography, NMR and GC-MS spectrometry were used to identify the apoptotic agent, after which its biological activity was evaluated. The extracts from N. glauca induced apoptosis in C2C12 cells involving caspase-3/7. We found that the extracts trigger a defence response in muscle through Akt and heat shock protein 27 (HSP27). We identified an apoptotic agent as palmitic acid. These data suggest that the use of N. glauca in hormone replacement therapy, or in other therapies affects skeletal muscle homeostasis, worsening the negative effects of the menopause. Thus, the relevance of this work lies in the fact that it is the first time that a report about the molecular mechanism responsible for the side effects of medicinal use of N. glauca, has been shown. Moreover the compound responsible for these effects has been identified.

Novel compound VB-037 inhibits Aß aggregation and promotes neurite outgrowth through enhancement of HSP27 and reduction of P38 and JNK-mediated inflammation in cell models for Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is involved in the aggregation of misfolded amyloid ß (Aß), which upregulates the activity of acetylcholinesterase (AChE), increases the production of reactive oxygen species (ROS), enhances neuroinflammation, and eventually leads to neuronal death. Therefore, compounds targeting these mechanisms may be candidates for multitarget drugs in AD treatment. We found that two quinoline derivatives, VB-030 and VB-037, markedly reduced Aß aggregation and ROS levels in the thioflavin T biochemical assay and Tet-On Aß-green fluorescent protein (GFP) 293 AD cell model. These compounds further improved neurite outgrowth, reduced AChE activity and upregulated the molecular chaperone heat shock protein family B [small] member 1 (HSP27), whereas knockdown of HSP27 counteracted the compounds' neuroprotective effects on the Tet-On Aß-GFP SH-SY5Y AD neuronal model. Furthermore, VB-037 attenuated lipopolysaccharide (LPS)/interferon (IFN)-γ-induced activation of BV-2 microglial cells. In addition, VB-037 demonstrated its potential to diminish LPS/IFN-γ-induced upregulation of caspase 1 activity, expression of interleukin (IL)-1ß, and active phosphorylation of mitogen-activated protein kinase 14 (P38), mitogen-activated protein kinase 8 (JNK), and Jun proto-oncogene, AP-1 transcription factor subunit (JUN) signalings, as well as improve cell viability in the Tet-On Aß-GFP SH-SY5Y AD neuronal model. Our findings strongly indicate the potential of VB-037 for modifying AD progression by targeting multiple mechanisms, thereby offering a new drug development avenue for AD treatment.

Curcumin Reverses 5-Fluorouracil Resistance by Promoting Human Colon Cancer HCT-8/5-FU Cell Apoptosis and Down-regulating Heat Shock Protein 27 and P-Glycoprotein.

OBJECTIVE: To investigate the potential mechanisms that curcumin reverses 5-fluorouracil (5-FU) multidrug resistance (MDR). METHODS: Cell growth and the inhibitory rate of curcumin (2-25 µg/mL) and/or 5-FU (0.05-1000 µg/mL) on human colon cancer HCT-8 and HCT-8/5-FU (5-FU-resistant cell line) were determined using cell counting kit-8 (CCK-8) assay. Apoptosis and cell cycle after 5-FU and/or curcumin treatment were detected by flow cytometry (FCM) and transmission electron microscopy (TEM). The expression of the multidrug resistance related factors p-glycoprotein (P-gp) and heat shock protein 27 (HSP-27) genes and proteins were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. RESULTS: The inhibitory rate of curcumin or 5-FU on HCT-8 and HCT-8/5-FU cells proliferation at exponential phase were in a dosedependent manner, HCT-8 cell line was more sensitive to curcumin or 5-FU when compared the inhibitory rate of HCT-8/5-FU. The 50% inhibitory concentration (IC50) of combination 5-FU and curcumin (4.0 µg/mL) in HCT-8/5-FU was calculated as 179.26 µg/mL, with reversal fold of 1.85. Another IC50 of combination 5-FU and curcumin (5.5 µg/mL) in HCT-8/5-FU was calculated as 89.25 µg/mL, with reversal fold of 3.71. Synergistic effect of 5-FU and curcumin on HCT-8 and HCT-8/5-FU cells were found. The cell cycle analysis performed by FCM showed that HCT-8 and HCT-8/5-FU cells mostly accumulated at G0/G1 phase, which suggested a synergistic effect of curcumin and 5-FU to induce apoptosis. FCM analysis found that the percentage of apoptosis of cells treated with curcumin, 5-FU and their combination were significantly increased compared to the control group (P<0.05), and the percentage of apoptosis of the combination groups were slightly higher than other groups (P<0.05). The mRNA levels of P-gp (0.28±0.02) and HSP-27 (0.28±0.09) in HCT-8/5-FU cells treated with combination drugs were lower than cells treated with 5-FU alone (P-gp, 0.48±0.07, P=0.009; HSP-27, 0.57±0.10, P=0.007). The protein levels of P-gp (0.25±0.06) and HSP-27 (0.09±0.02) in HCT-8/5-FU cells treated with combination drugs were decreased when compared to 5-FU alone (P-gp, 0.46±0.02, P=0.005; HSP-27, 0.43±0.01, P=0.000). CONCLUSIONS: Curcumin can inhibit the proliferation of human colon cancer cells. Curcumin has the ability of reversal effects on the multidrug resistance of human colon cancer cells lines HCT-8/5-FU. Down-regulation of P-gp and HSP-27 may be the mechanism of curcumin reversing the drug resistance of HCT-8/5-FU to 5-FU.

Dependence of HSP27 cellular level on protein kinase CK2 discloses novel therapeutic strategies.

BACKGROUND: HSP27 plays a role in various diseases, including neurodegenerative diseases, ischemia, and atherosclerosis. It is particularly important in the regulation of the development, progression and metastasis of cancer as well as cell apoptosis and drug resistance. However, the absence of an ATP binding domain, that is, instead, present in other HSPs such as HSP90 and HSP70, hampers the development of small molecules as inhibitors of HSP27. METHODS: Knockout cell lines generated by Crispr/Cas9 gene editing tool, specific kinase inhibitors and siRNA transfections were exploited to demonstrate that the expression of HSP27 is dependent on the integrity/activity of protein kinase CK2 holoenzyme. The interaction between these proteins has been confirmed by co-immunoprecipitation, confocal immunofluorescence microscopy, and by density gradient separation of protein complexes. Finally, using a proliferation assay this study demonstrates the potential efficacy of a combinatory therapy of heath shock and CK2 inhibitors in cancer treatment. RESULTS: Our data demonstrate that CK2 is able to regulate HSP27 turnover by affecting the expression of its ubiquitin ligase SMURF2 (Smad ubiquitination regulatory factor 2). Moreover, for the first time we show an increased sensitivity of CK2-inhibited tumour cells to hyperthermia treatment. CONCLUSION: Being HSP27 involved in several pathological conditions, including protein conformational diseases (i.e Cystic Fibrosis) and cancer, the need of drugs to modulate its activity is growing and CK2-targeting could represent a new strategy to reduce cellular HSP27 level. GENERAL SIGNIFICANCE: This study identifies CK2 as a molecular target to control HSP27 cellular expression.

Taurine Supplementation Alleviates Puromycin Aminonucleoside Damage by Modulating Endoplasmic Reticulum Stress and Mitochondrial-Related Apoptosis in Rat Kidney.

Taurine (TAU) is a sulfur-containing beta amino acid that is not involved in protein composition and anabolism, conditionally essential in mammals provided through diet. Growing evidence supports a protective role of TAU supply in osmoregulation, calcium flux, and reduction of inflammation and oxidant damage in renal diseases like diabetes. Endoplasmic reticulum (ER) stress, due to abnormal proteostasis, is a contributor to nephrotic syndrome and related renal damage. Here, we investigated the effect of dietary TAU (1.5% in drinking water for 15 days) in an established rat model that mimics human minimal change nephrosis, consisting of a single puromycin aminonucleoside (PAN) injection (intraperitoneally 15 mg/100 g body weight), with sacrifice after eight days. TAU limited proteinuria and podocytes foot processes effacement, and balanced slit diaphragm nephrin and glomerular claudin 1 expressions. In cortical proximal tubules, TAU improved lysosomal density, ER perimeter, restored proper ER-mitochondria tethering and mitochondrial cristae, and decreased inflammation. Remarkably, TAU downregulated glomerular ER stress markers (GRP78, GRP94), pro-apoptotic C/EBP homologous protein, activated caspase 3, tubular caspase1, and mitochondrial chaperone GRP75, but maintained anti-apoptotic HSP25. In conclusion, TAU, by targeting upstream ER stress separate from mitochondria dysfunctions at crucial renal sites, might be a promising dietary supplement in the treatment of the drug-resistant nephrotic syndrome.

Combined Extracts of Artemisia and Green Tea, Mitigated Alcoholic Gastritis Via Enhanced Heat-shock Protein 27.

Background/Aims: Several lines of evidence from epidemiologic and laboratory studies have shown that the consumption of Artemisia or green tea extracts (MPGT) is inversely associated with the risk of alcohol-induced damage and other chronic diseases. Supported by previous studies showing that the combined extract of Artemisia and green tea, MPGT, exerted significantly either antioxidative or anti-inflammatory actions against Helicobacter pylori-associated gastric diseases, it was hypothesized that MPGT can offer protection against alcoholic gastritis. Methods: Ethanol was administered to induce gastric damage in Wistar rats, which had been pretreated with various doses of MPGT, to measure the rescuing action of a MPGT pretreatment against ethanol-induced gastric damage. In addition, the molecular mechanisms for the preventive effects were examined. Results: The MPGT pretreatment (100, 300, and 500 mg/kg) alleviated the ethanol-induced gastric damage, which was evidenced by the significant decrease in calcium-dependent phospholipase A2, MAPKs, and NF-κB levels compared to ethanol alone. Furthermore, the MPGT pretreatment preserved 15-prostaglandin dehydrogenase, whereas cyclooxygenase-2 was decreased significantly. All of these biochemical changes led to the significant alleviation of alcohol-associated gastric mucosal damage. Ethanol significantly increased the TUNEL positivity in the stomach, but MPGT decreased the apoptotic index significantly, which was associated with significantly lower pathological scores of ethanol-induced mucosal ulcerations. The significant protective changes observed alcoholic gastritis with MPGT were related to the increased expression of cytoprotective genes, such as heat-shock protein (HSP)27, HSP60, and PDGF. Conclusions: The efficient anti-inflammatory, anti-apoptotic, and regenerative actions of MPGT make it a potential nutrient phytoceutical to rescue the stomach from alcoholic gastritis.

[Effects of Electroacupuncture and Moxibustion Pretreatment on Expressions of HSP 27, HSP 70, HSP 90 at Different Time-points in Rabbits with Myocardial Ischemia-reperfusion Injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) and moxibustion (Moxi) pretreatment on expression of myocardial heat shock protein (HSP) in acute myocardial ischemia-reperfusion injury (MIRI) rabbits. METHODS: A total of 72 New Zealand rabbits were randomly divided into 4 groups:sham operation, MIRI model, EA pretreatment and Moxi pretreatment (n=18 rabbits in each group) which were further divided into 0, 24 and 48 h (time-point) subgroups (n=6 in each). The MIRI model was established by occlusion of the anterior descending branch (ADB) of the left coronary artery for 40 min and reperfusion for 60 min. EA and Moxi stimulation was respectively applied to bilateral "Neiguan"(PC 6) for 20 min, once daily for 5 days before ADB occlusion. The expressions of myocardial HSP 27, HSP 70 and HSP 90 were detected by immunohistochemistry. The pathological and ultrastructural changes of left ventricular ischemia tissue were observed under light and transmission electronic microscope (TEM), respectively. RESULTS: Outcomes of H.E. staining and ultrastructure showed that MIRI-induced changes of disordered arrangement of cardiomyocytes, vague myocardial transverse striation, inflammatory infiltration, cardiac myofibre necrosis and fibrolysis (light microscope), and myofiber atrophy, vague and disorder in the arrangement of myofiber, myofilament necrosis, interstitial edema, mitochondrial swelling, microvessel expansion, etc. (TEM) were relatively milder in both EA and Moxi pretreatment groups (48 h). In comparison with the sham group, the expression levels of myocardial HSP 27, HSP 70 and HSP 90 had no significant changes after MIRI at the 3 time-points (P>0.05). In the pretreatment groups, the expression levels of HSP 27 at 24 and 48 h in both EA and Moxi groups, HSP 70 at 48 h in both groups, HSP 70 at 0 and 24 h in the Moxi group were significantly up-regulated compared with the model group (P<0.05, P<0.01). No significant changes were found in the expression of HSP 90 at the 3 time-points in the EA and Moxi pretreatment groups (P>0.05). No significant differences were found between EA and Moxi in up-regulating expressions of myocardial HSP 27, HSP 70 and HSP 90 proteins at the 3 time-points (P>0.05) except HSP 70 at 24 h (Moxi being stronger relative to EA, P<0.05). CONCLUSIONS: EA and Moxi pretreatment has a protective effect on ischemic myocardium in MIRI rabbits, which Feb be associated with their actions in up-regulating myocardial HSP 27 and HSP 70 expression.

Zhenbao Pill reduces the percentage of Treg cells by inducing HSP27 expression.

CONTEXT: Zhenbao pill containing Nacre, Safflower, Musk and Cornu Bubahas has been proved to have a good therapeutic effect on the repair of spinal cord injury (SCI). However, its complex mechanism of repairing SCI is not yet known. OBJECTIVE: This study attempts to investigate the role of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in the mechanism of the action of Zhenbao pill, and further explore the relationship between Tregs and HSP27 expression in repair mechanism. MATERIALS AND METHODS: Treatment of human peripheral blood mononuclear cells (PBMCs) with different concentrations (0, 0.5, 2.5, 5, 10mg/mL) of Zhenbao pill, flow cytometry was used to detect the expression of the specific factors CD4+CD25+Foxp3+ in Tregs, and detection of Tregs related regulatory factor TGF-ß content was performed with ELISA assay. The relationship between miR-214 and HSP27 was assessed by Luciferase assay, and the level of miR-214 was detected by qPCR. The expression of HSP27 was examined with qPCR and western blotting. RNA interference technology and gene recombination were used to inhibit and up-regulate the expression of HSP27. RESULTS: Zhenbao pill with 11.61mg/mL of IC50 for Tregs can significantly inhibit the differentiation into Tregs in human PBMCs and up-regulate by more than 1-fold of HSP27 expression. Essentially, it enhanced the expression of HSP27 by inhibiting miR-214 expression (50%). Inhibition of HSP27 expression, followed by the differentiation into Tregs, was promoted in human PBMCs. When the HSP27 expression was up-regulated, the differentiation into Tregs was decreased by 30%. It indicated that the expression of HSP27 regulated the differentiation into Tregs. Inhibition of HSP27 expression and Zhenbao pill treatment, the differentiation into Tregs was decreased but remained at a higher level than that of the group was only treated with pill. Under the action of Zhenbao pill, the expression of HSP27 was not completely interfered, and its expression level was still increased. CONCLUSIONS: In the process of repairing the SCI, Zhenbao pill inhibits reduces numbers of Treg lymphocytes as well as TGF-ß levels by inducing HSP27 expression.

Spasmolytic Mechanism of Aqueous Licorice Extract on Oxytocin-Induced Uterine Contraction through Inhibiting the Phosphorylation of Heat Shock Protein 27.

Licorice derived from the roots and rhizomes of Glycyrrhiza uralensis Fisch. (Fabaceae), is one of the most widely-used traditional herbal medicines in China. It has been reported to possess significant analgesic activity for treating spastic pain. The aim of this study is to investigate the spasmolytic molecular mechanism of licorice on oxytocin-induced uterine contractions and predict the relevant bioactive constituents in the aqueous extract. The aqueous extraction from licorice inhibited the amplitude and frequency of uterine contraction in a concentration-dependent manner. A morphological examination showed that myometrial smooth muscle cells of oxytocin-stimulated group were oval-shaped and arranged irregularly, while those with a single centrally located nucleus of control and licorice-treated groups were fusiform and arranged orderly. The percentage of phosphorylation of HSP27 at Ser-15 residue increased up to 50.33% at 60 min after oxytocin stimulation. Furthermore, this increase was significantly suppressed by licorice treatment at the concentration of 0.2 and 0.4 mg/mL. Colocalization between HSP27 and α-SMA was observed in the myometrial tissues, especially along the actin bundles in the oxytocin-stimulated group. On the contrary, the colocalization was no longer shown after treatment with licorice. Additionally, employing ChemGPS-NP provided support for a preliminary assignment of liquiritigenin and isoliquiritigenin as protein kinase C (PKC) inhibitors in addition to liquiritigenin, isoliquiritigenin, liquiritin and isoliquiritin as MAPK-activated protein kinase 2 (MK2) inhibitors. These assigned compounds were docked with corresponding crystal structures of respective proteins with negative and low binding energy, which indicated a high affinity and tight binding capacity for the active site of the kinases. These results suggest that licorice exerts its spasmolytic effect through inhibiting the phosphorylation of HSP27 to alter the interaction between HSP27 and actin. Furthermore, our results provide support for the prediction that potential bioactive constituents from aqueous licorice extract inhibit the relevant up-stream kinases that phosphorylate HSP27.

Effects of sauna bathing on stress-related genes expression in athletes and non-athletes.

INTRODUCTION AND OBJECTIVE: Heat stress induces the expression of genes encoding heat-shock proteins and immune response mediators. The aim of this study was to determine the differences in the expression of genes encoding heat-shock proteins 70 kDa and27 kDa, interleukin 6, interleukin 10and C-reactive protein, between athletes and non-athletes after sauna bathing. MATERIALS AND METHOD: Athletes (n=9) and non-athletes (n=9) were exposed to a Finnish sauna twice during one session at a temperature of 98.2 °C and humidity of 10% ± 2%, with a 5 min break for cooling down under a shower. The groups did not differ in terms of age, height or body mass. Blood samples were taken before and after sauna exposure in order to assess gene expression, using reverse transcription polymerase chain reaction. RESULTS: Differences were observed in leukocyte mRNA levels of tested genes between athletes and non-athletes. In the non-athlete group, all the tested genes were expressed at higher levels as a response to the same heat challenge. CONCLUSION: It appears that expression of stress-related genes induced by heat stress is dependent on the level of physical activity.

Heat shock protein 27 is a potential indicator for response to YangZheng XiaoJi and chemotherapy agents in cancer cells.

Heat shock protein 27 (HSP27) is a member of the heat shock protein family which has been linked to tumour progression and, most interestingly, to chemotherapy resistance in cancer patients. The present study examined the potential interplay between HSP27 and YangZheng XiaoJi, a traditional Chinese medicine used in cancer treatment. A range of cell lines from different tumour types including pancreatic, lung, gastric, colorectal, breast, prostate and ovarian cancer (both wild-type and resistant) were used. Levels and activation of HSP27 and its potential associated signalling pathways were evaluated by protein array and western blotting. Knockdown of HSP27 in cancer cells was achieved using siRNA. Localisation and co-localisation of HSP27 and other proteins were carried out by immunofluorescence. Cell growth and migration were evaluated in their response to a range of chemotherapeutic agents. The present study first identified, by way of protein array, that YangZheng XiaoJi was able to inhibit the phosphorylation of HSP27 protein in cancer cells. We further demonstrated that HSP27, which is co-localised with caspase-9, can be blocked from localising in focal adhesions and co-localising with caspase-9 by YangZheng XiaoJi. The study also demonstrated that YangZheng XiaoJi was able to sensitise cancer cells including those cells that were resistant to chemotherapy, to chemotherapeutic agents. Finally, knocking down HSP27 markedly reduced the migration of cancer cells and increased the sensitivity of cancer cells to the inhibitory effect on cellular migration by YangZheng XiaoJi. YangZheng XiaoJi can act as an agent in first sensitising cancer cells to chemotherapy and secondly to overcome, to some degree, chemoresistance when used in an appropriate fashion in patients who have active HSP27.

HSPB1 deficiency sensitizes melanoma cells to hyperthermia induced cell death.

Hyperthermia has shown clinical potency as a single agent or as adjuvant to other therapies in cancer treatment. However, thermotolerance induced by thermosensitive genes such as the heat shock proteins can limit the efficacy of hyperthermic treatment. In the present study, we identified HSPB1 (HSP27) is hyperthermically inducible or endogenously highly expressed in both murine and human melanoma cell lines. We used a siRNA strategy to reduce HSPB1 levels and showed increased intolerance to hyperthermia via reduced cell viability and/or proliferation of cells. In the investigation of underlying mechanisms, we found knock down of HSPB1 further increased the proportion of apoptotic cells in hyperthermic treated melanoma cells when compared with either single agent alone, and both agents leaded to cell cycle arrest at G0/G1 or G2/M phases. We concluded that hyperthermia combined with silencing of HSPB1 enhanced cell death and resulted in failure to thrive in melanoma cell lines, implying the potential clinical utility of hyperthermia in combination with HSPB1 inhibition in cancer treatment.

l-glutamine and l-alanine supplementation increase glutamine-glutathione axis and muscle HSP-27 in rats trained using a progressive high-intensity resistance exercise.

In this study we investigated the chronic effects of oral l-glutamine and l-alanine supplementation, either in their free or dipeptide form, on glutamine-glutathione (GLN-GSH) axis and cytoprotection mediated by HSP-27 in rats submitted to resistance exercise (RE). Forty Wistar rats were distributed into 5 groups: sedentary; trained (CTRL); and trained supplemented with l-alanyl-l-glutamine, l-glutamine and l-alanine in their free form (GLN+ALA), or free l-alanine (ALA). All trained animals were submitted to a 6-week ladder-climbing protocol. Supplementations were offered in a 4% drinking water solution for 21 days prior to euthanasia. Plasma glutamine, creatine kinase (CK), myoglobin (MYO), and erythrocyte concentration of reduced GSH and glutathione disulfide (GSSG) were measured. In tibialis anterior skeletal muscle, GLN-GSH axis, thiobarbituric acid reactive substances (TBARS), and the expression of heat shock factor 1 (HSF-1), 27-kDa heat shock protein (HSP-27), and glutamine synthetase were determined. In CRTL animals, high-intensity RE reduced muscle glutamine levels and increased GSSG/GSH rate and TBARS, as well as augmented plasma CK and MYO levels. Conversely, l-glutamine-supplemented animals showed an increase in plasma and muscle levels of glutamine, with a reduction in GSSG/GSH rate, TBARS, and CK. Free l-alanine administration increased plasma glutamine concentration and lowered muscle TBARS. HSF-1 and HSP-27 were high in all supplemented groups when compared with CTRL (p < 0.05). The results presented herein demonstrate that l-glutamine supplemented with l-alanine, in both a free or dipeptide form, improve the GLN-GSH axis and promote cytoprotective effects in rats submitted to high-intensity RE training.

Atorvastatin attenuates contrast-induced nephropathy by modulating inflammatory responses through the regulation of JNK/p38/Hsp27 expression.

This study aimed to investigate whether atorvastatin reduce the contrast-induced nephropathy inflammatory response and apoptosis of renal tubular epithelial cells and the relationship with MAPK signaling pathway. We utilized the iopamidol-induced contrast-induced nephropathy (CIN) rat model which was induced by a single dose of iopamidol (2.9 g iodine/kg) and a cell model in which human embryonic proximal tubular (HK2) cells were treated with iopamidol. The rats were divided into five groups: (1) control rats (CR); (2) atorvastatin (CA); (3) iopamidol (CM); (4) iopamidol and atorvastatin (20 mg/kg d) (CMA2); (5) iopamidol and atorvastatin (40 mg/kg d) (CMA4). On days 1, 2 and 6 after iopamidol injection, the urea nitrogen and cystatin C increased in CM compared with CR but decreased in CMA compared with CM. Inflammatory parameters and the percentage of apoptotic cells were increased in CM compared with CR and CA, but they were decreased in CMA compared with CM. We also found that atorvastatin ameliorated the renal tubular necrosis, apoptosis, and the deterioration of renal function in a dose dependent manner (P < 0.05). Furthermore, in vivo, both of SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) could decrease the expression of Bax and caspase-3, but increase Bcl-2 levels in HK2 cells treated with iopamidol. Our study demonstrates that high-dosage atorvastatin treatment attenuates both the inflammatory processes and apoptosis in contrast-induced acute kidney injury, and that the JNK/p38 MAPK pathway participates in the contrast-induced apoptosis of renal tubular cells. Finally, atorvastatin reduces CIN by suppression of apoptosis, which may be through inhibition of JNK/p38 MAPK pathways.