Increased Expression Of HSP70 Research Articles

The Long-term influences of Age-At-Injury on Neuroinflammation and neuronal Apoptosis following Traumatic Brain Injury in Pediatric and Adulthood Mice.

The study aims to investigate the long-term impacts of traumatic brain injury (TBI) on neuroinflammation and neuronal apoptosis in pediatric and adult mice, specifically focusing on how age-at-injury influences these processes. Controlled cortical impact (CCI) was used to induce TBI in pediatric (21-25 days old) and adult (8-12 weeks old) C57Bl/6 male mice. Neuroinflammation was evaluated through immunoreactivity for Iba-1 and GFAP, while apoptosis was assessed using markers such as Bax, Bcl- 2, and pro-caspase-3. Additionally, HSP70 expression was measured to understand the stress response. Following CCI, pediatric mice exhibited a significant reduction in NeuN expression(p < 0.001), significant increase in GFAP (p < 0.01) and AIF-1/Iba1 expression (p < 0.05) at 3 dpi compared to sham controls. In contrast, adult mice exhibited no significant change in AIF-1/Iba1 expression and a less pronounced increase in GFAP (p < 0.05) at 3 dpi compared to sham controls. Pediatric mice demonstrated a more significant increase in Bax/Bcl-2 ratio at 7 dpi (p < 0.01), While adult mice a little weak significant increase in Bax/Bcl-2 ratio at 7 dpi (p < 0.05). Both age groups showed a significant but transient increase in HSP70 levels at 7 dpi, which normalized by 90 dpi. Pediatric and adult mice exhibited significant time-dependent differences in neuroinflammation and apoptosis following TBI, with pediatric mice showing more intense early responses, highlighting age-specific vulnerabilities in post-injury outcomes. Both age groups showed a significant but transient increase in HSP70 expression, suggesting an acute stress response postinjury.

HSP70 promotes pancreatic cancer cell epithelial-mesenchymal transformation and growth via the NF-κB signaling pathway.

To study the effects of HSP70 on proliferation, migration, invasion, and epithelial-mesenchymal transformation (EMT) of pancreatic cancer cells and explore its underlying mechanisms. Pancreatic cancer cell models with either reduced HSP70 or increased HSP70 expression were established. RT-qPCR and Western blot assays were used to determine mRNA and protein levels of HSP70, IKK/IκBa/NF-κB signaling pathway-related genes, and EMT markers. The CCK-8 and cell cloning assays were used to evaluate cell proliferation and cloning abilities. Transwell and wound healing assays were used to assess the invasive and migratory properties of the cells. Effects of NF-κB signaling modulation were explored using an IKK inhibitor (BAY11-7082) and an IKK overexpression vector (pCMV-IKK). Electrophoretic mobility shift assay (EMSA) and luciferase reporter assays were conducted to analyze NF-κB's promoter binding and transcriptional activities. HSP70 knockdown inhibited p-p65 nuclear translocation and reduced the expression of p-p65, p-IKKα/β, p-IκBα, N-cadherin, Vimentin, and Twist. It also decreased NF-κB's promoter binding and transcriptional activities, increased E-cadherin levels, and suppressed pancreatic cancer cell proliferation, cloning, migration, and invasion. In contrast, HSP70 overexpression led to increased expression of p-p65, p-IKKα/β, p-IκBα, N-cadherin, Vimentin, and Twist, decreased E-cadherin levels, and enhanced cell proliferation, cloning, migration, and invasion capabilities. NF-κB signaling pathway modulation reversed EMT changes induced by altered HSP70 expression levels. rhHSP70 also increased p-IKKα/β and p-IκBα protein levels. HSP70 promotes the EMT and enhances pancreatic cancer cell proliferation, migration, and invasion by activating the NF-κB pathway.

High-Intensity Focused Ultrasound Increases Facial Adipogenesis in a Swine Model via Modulation of Adipose-Derived Stem Cell Cilia.

Decreased medial cheek fat volume during aging leads to loss of a youthful facial shape. Increasing facial volume by methods such as adipose-derived stem cell (ASC) injection can produce facial rejuvenation. High-intensity focused ultrasound (HIFU) can increase adipogenesis in subcutaneous fat by modulating cilia on ASCs, which is accompanied by increased HSP70 and decreased NF-κB expression. Thus, we evaluated the effect of HIFU on increasing facial adipogenesis in swine (n = 2) via modulation of ASC cilia. Expression of CD166, an ASC marker, differed by subcutaneous adipose tissue location. CD166 expression in the zygomatic arch (ZA) was significantly higher than that in the subcutaneous adipose tissue in the mandible or lateral temporal areas. HIFU was applied only on the right side of the face, which was compared with the left side, where HIFU was not applied, as a control. HIFU produced a significant increase in HSP70 expression, decreased expression of NF-κB and a cilia disassembly factor (AURKA), and increased expression of a cilia increasing factor (ARL13B) and PPARG and CEBPA, which are the main regulators of adipogenesis. All of these changes were most prominent at the ZA. Facial adipose tissue thickness was also increased by HIFU. Adipose tissue volume, evaluated by magnetic resonance imaging, was increased by HIFU, most prominently in the ZA. In conclusion, HIFU increased ASC marker expression, accompanied by increased HSP70 and decreased NF-κB expression. Additionally, changes in cilia disassembly and length and expression of adipogenesis were observed. These results suggest that HIFU could be used to increase facial volume by modulating adipogenesis.

Aqueous PM2.5 promotes lipid accumulation, classical macrophage polarisation and heat shock response

Fine particulate matter (PM2.5) is an air pollutant that enhances susceptibility to cardiovascular diseases. Macrophages are the first immune cells to encounter the inhaled particles and orchestrate an inflammatory response. Given their role in atherosclerosis development, we investigated whether aqueous PM2.5 could elicit atherogenic effects by polarising macrophages to a pro-oxidative and pro-inflammatory phenotype and enhancing foam cell formation. The RAW264.7 macrophage cell line was exposed to PM2.5 for 48 h, with PBS as the control. Aqueous PM2.5 induced apoptosis and reduced cell proliferation. In surviving cells, we observed morphological, phagocytic, oxidative, and inflammatory features (i.e. enhanced iNOS, Integrin-1β, IL-6 expression), indicative of classical macrophage activation. We also detected an increase in total and surface HSP70 levels, suggesting macrophage activation. Further, exposure of high-cholesterol diet-fed mice to PM2.5 resulted in aortic wall enlargement, indicating vascular lesions. Macrophages exposed to PM2.5 and non-modified low-density lipoprotein (LDL) showed exacerbated lipid accumulation. Given the non-oxidised LDL used and the evidence linking inflammation to disrupted cholesterol negative feedback, we hypothesise that PM2.5-induced inflammation in macrophages enhances their susceptibility to transforming into foam cells. Finally, our results indicate that exposure to aqueous PM2.5 promotes classical macrophage activation, marked by increased HSP70 expression and that it potentially contributes to atherosclerosis.

Correlation of SNR Value on DPOAE Examination with HSP70 Levels in Blood and HSP70 Expression in Cochlea of Noise Model Rattus norvegicus as an Indicator of Inner Ear Damage

Background: Cellular stress caused by noise-induced hearing loss can be observed through the damage of cochlear hair cells, mechanically and metabolically. SNR value on the DPOAE examination assesses the sensory function of cochlear outer hair cells. HSP70 as marker of cellular stress can be identified from its levels in blood and its expression in the cochlea.Methods: Three groups of rats were used for the noise intervention: Group 1 was the control group, Group 2 had a noise exposure of 100 dB, and Group 3 received a noise exposure of 110 dB. DPOAE examination was then conducted, blood samples from rats’ tail were collected after a noise treatment, followed by the measurement of HSP70 levels by ELISA readings. Rats were terminated afterwards and immunohistochemical examination of the cochlear organ of Corti was performed to calculate the expression of HSP70. The data obtained were analyzed using SPSS.Results: A decrease occurred in the values of SNR and an increase occurred in HSP70 levels in blood along with its expression in the cochlea of noise model Rattus norvegicus, and there is a correlation between the three.Conclusion: The decrease in SNR values, increase in HSP70 levels in blood, and increase in HSP70 expression in the cochlear organ of Corti of Rattus norvegicus, proves that noise-induced hearing loss triggers the production of HSP70 as a cytoprotective agent in preventing inner ear damage.Keywords: DPOAE; Rattus norvegicus; NIHL; Heat Shock Protein 70

Evaluation of Bis(2-ethylhexyl) phthalate toxicity on the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg9

Bis(2-ethylhexyl) phthalate, generally known as DEHP is a synthetic compound mainly used as a plasticizer to make polyvinyl chloride products flexible and soft. The present work aimed to study the toxicity of Bis(2-ethylhexyl) phthalate on the third instar larvae of transgenic Drosophila melanogaster(hsp70-lacZ) Bg9. The hsp70 gene is associated with the β-galactosidase in our present transgenic strain therefore, the more activity of β-galactosidase will indirectly correspond to hsp70 expression. The third instar larvae were allowed to feed on the diet for 24 h having 0.001, 0.005, 0.01, and 0.02 M of Bis(2-ethylhexyl) phthalate at the final concentration. After the exposure of 24hrs, the larvae were subjected to ONPG assay, X-gal staining, trypan blue exclusion test, oxidative stress markers assays, and comet assay. A dose-dependent increase in hsp70 expression, tissue damage, Glutathione-S-transferase (GST) activity, lipid peroxidation, monoamine oxidase, caspase-9 & 3, protein carbonyl content (PCC), DNA damage and decrease in the glutathione (GSH) content, delta-aminolevulinic acid dehydrogenase (ẟ-ALD-D) and acetylcholinesterase activity were observed in the larvae exposed to 0.005, 0.01, 0.02 M of Bis-(2-ethylhexyl) phthalate. The dose of 0.001 M of Bis(2-ethylhexyl) phthalate did not showed any toxic effects and hence can be considered as No Observed Adverse Effect Level (NOAEL) for Bis(2-ethylhexyl) phthalate. The study supports the use of Drosophila for the evaluation of possible toxic effects associated with synthetic compounds.

Structural changes and features of heat shock protein HSP70 and HSP90 expression in the pyramidal neurons of the hippocampus of rat in the experimental discirculatory encephalopathy modeling

AbstractBackgroundThe heat shock protein (Hsp) system acts as intracellular chaperone molecules responsible for maintaining normal intracellular homeostasis by regulating the correct coagulation of newly synthesized peptides, as well as the transport and degradation of mature proteins. In addition to their role as chaperones, proteins of the Hsp family stabilize the cell membrane and inhibit the caspase‐dependent apoptosis cascadeMethodHypertensive encephalopathy was modeled on 12‐month‐old rats by the method of exposure to negative longitudinal overloads in the caudo‐cranial vector with a 9 G force for 5 minutes with 12 hours interval for 28 days. The behavioral responses, cognitive and mnestic functions, the relative number of pyramidal neurons with hyperchromic cytoplasm and shrunken perikariya, and the level of expression of heat shock proteins 70 kDa (Hsp70) and 90 kDa (Hsp90) were assessedResultAfter 28 days of exposure to 9 G negative longitudinal overloads, the rats showed functional and morphological manifestations characteristic of hypertensive encephalopathy. There was a decrease in the basic reflexes, disorders of the cognitive sphere, as well as decrease in memory and orientation‐exploratory activity. Morphological examination of the hippocampus against the background of pronounced edema revealed a significant increase in the relative number of pyramidal neurons with hyperchromatosis and perikaryon shrinkage in all zones of the pyramidal layer (p &lt;0.01). These signs of reversible and irreversible damage were accompanied by a significant increase in Hsp70 expression, especially pronounced in the CA3 zone (p &lt;0.01). A significant increase in Hsp90 expression was found in the CA1 zone (p &lt;0.05)ConclusionDiscirculatory encephalopathy experimental modeling demonstrates signs of both reversible and irreversible alteration in all regions of the pyramidal layer of hippocampus. Differences in the ability of neurons of the pyramidal layer of different regions of the hippocampus to express Hsp70, Hsp90 were noted. In the CA1 and CA3 zones, compensatory reactions are more pronounced and are accompanied by a more pronounced expression of Hsp70, while in CA1, the expression of Hsp70 and Hsp90 is simultaneously enhanced

Hspa5 Deficiency Blocks Intestinal Enterocyte Differentiation in Graft-Versus-Host Disease

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as an effective therapy for hematological malignancies. Graft-versus-host disease (GVHD) is a life-threatening complication, and the intestine is one of the major sites affected. Currently, immunosuppressive agents remain the preferred choice for GVHD treatment. However, treatment resistance still occurs in a large population, indicating that some tissue damage may be irreversible. Methods: The murine transplant and GVHD model were established as previously described. To characterize the intestinal damage mediated by radiation and GVHD, murine small intestine tissues were collected at day 14 after transplantation. Primary small intestinal crypts were isolated from each group and cultured for five consecutive days to evaluate their ability to proliferate and differentiate. Single-Cell analysis was performed to characterize the impaired intestinal repair after injury. Results: At day 14, the villus-to-crypt ratio was partially recovered in the BM group, but remained imbalanced in the GVHD group. We observed a significant decrease in the expression of Reg1, a gene expressed by enterocytes (EC), in the GVHD groups compared to the BM group. we further utilized a 3D organoid model to investigate intestinal growth and differentiation. The organoid size from the GVHD group was comparable to the BM and control groups. However, the number of buds per organoid was significantly reduced in the GVHD group compared to the BM and control groups. Immunofluorescence staining confirmed that cells in GVHD-derived organoids exhibited high proliferation (stained by Mki67) but impaired differentiation of enterocyte (stained by Aldob). To gain insights into the cellular mechanisms influencing enterocyte differentiation, we dissociated murine small intestinal tissues from each group into single cells and sequenced them using the 10X Genomics platform. Through pseudochronological analysis, each cell was ordered along the predicted lineages and assigned a pseudotime. We found that enterocytes in GVHD exhibited a long and discontinuous trajectory, suggesting explicit blockage of EC differentiation. Functional enrichment analysis showed that pathways related to cell growth and differentiation, such as MAPK, ERBB, and inositol phosphate metabolism, were highly enriched in M2 of the control and BM groups. In contrast, MAPK was dramatically downregulated at the early stage of ISC differentiation in the GVHD group. Next, we analyzed the differentially expressed genes in EP between the GVHD and BM groups. Endoplasmic reticulum (ER) chaperone-related genes from the heat shock protein (Hsp) 70 family (Hspa1a/1b/5), Hsp90 family (Hsp90aa1/ab1), and Hsp40 family (Dnajb1) were significantly decreased in GVHD compared to BM. Notably, Hspa5 (also known as GRP78 or Bip), which serves as a sensor of glucose levels and misfolded proteins, exhibited reduced protein levels, along with decreased IRE1ɑ phosphorylation (p-IRE1ɑ) and Xbp1s, as well as decreased PERK phosphorylation (p-PERK) and ATF6 expression. To validate our conjecture, we utilized small intestinal organoids cultured for 3 days for further experimentation. RT-qPCR demonstrated that energy restriction downregulated the mRNA expression of Hspa5 and other chaperones. Furthermore, we treated intestinal organoids with HA15, a BIP inhibitor, which resulted in significantly enlarged area but fewer buds. Consistent with our previous findings, Hspa5 was downregulated in the HA15-treated group, along with decreased expression of Hsp family and enterocyte markers. Since Hsp family plays key roles in restoring cellular homeostasis, we wondered if upregulating Hspa5 could protect intestinal cells from outside stimuli such as pro-inflammatory cytokines. When intestinal organoids were cocultured with IFN-g, we found that Bix, the selective Bip inducer, protected organoids from apoptosis by increasing Hsp70 expression. Conclusion: Given that immunosuppression partly resolves GVHD, strategies aimed at restoring intestinal homeostasis should be given sufficient attention. Our study reveals that intestinal enterocyte differentiation is blocked in GVHD, possibly due to starvation and energy restriction. Deficiency in Hspa5 results in impaired intestinal repair, and increasing Hsp levels may enhance cell resistance to cytokine storms.

Prolonged ecological changes can affect morphometrics and gene expression profile? Focusing on Hsp-70 and NLHS-induced Hsp-70 of Artemia urmiana

BackgroundIn recent years, many aquatic ecosystems, including Urmia Lake, have undergone severe ecological tensions. This lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated and its salinity has dramatically increased over the last three decades. In the face of the long period environmental stresses, understanding the adaptation and ecological plasticity mechanisms is the most interesting challenges in genetic and applied ecology. These mechanisms may probably be driven by inducing expression of some genes involved in adaptation such as Hsp-70 and also adjusting morphological parameters. But they are yet to be understood. Hence, the present work aimed to study the mechanisms, along with testing the hypothesis that non-lethal heat shocked nauplii originating from drought period can evoke Hsp-70 expression more than those from rainy period. MethodsThis study measured and analyzed morphometrical characters of adult male and female Artemia urmiana over three decades. Then, the influence of three-decade ecological crisis on Hsp-70 and non-lethal heat shock (NLHS)-induced Hsp-70 expression levels of nauplii of Artemia urmiana habiting Urmia Lake using Real-time PCR technique, based on cyst collections in 1994 (rainy period) to 2020 (drought period), was evaluated. ResultsThe morphometrics results showed that the morphological characters were significantly shrunk in 2020 compared to 1994 (CI 95%, p < 0.05). Furthermore, our results depicted that, Hsp-70 expression level was significantly upregulated in response to the prolonged ecological crisis, (CI 95%, P < 0.0001), and also interestingly, the nauplii exposed to longe-term ecological crisis (belong to 2020) were able to increase Hsp-70 expression more than other ones in response to environmental stressors including heat. ConclusionsThe present results showed the involvement of Hsp-70 in the adaptation of Artemia urmiana to long term ecological alteration at the cost of shrinking morphometric parameters.

Prevalence and effects of a parasitic trematode on the blue mussel, Mytilus edulis, in the Boston Harbor

The blue mussel, Mytilus edulis, is a keystone species in the North Atlantic that plays critical roles in nutrient cycling, water filtration, and habitat creation. Blue mussel populations have declined significantly throughout the North Atlantic due to various factors, including habitat loss, pollution, increasing water temperature, and parasites. One parasite is Proctoeces maculatus, a digenetic trematode, which uses M. edulis as an intermediate host. This parasite causes reduced growth, castration, and death in mussels. The range of P. maculatus has expanded northward from Cape Cod, MA to Maine which may be associated with rising temperatures in the Gulf of Maine. To evaluate the negative impacts of P. maculatus on mussels, we analyzed its infections in M. edulis throughout the Boston Harbor, MA. P. maculatus was present in every population and time point analyzed, with approximately 50% of mussels in the harbor infected. The parasite reduced gonadal development in infected mussels, which could lead to decreased fecundity. Severe P. maculatus infections induced a stress response, indicated by increased HSP70 expression. We developed a non-destructive hemolymph-based assay to determine if mussels are infected with P. maculatus, thus speeding up the evaluation process and eliminating the need to sacrifice individuals. With P. maculatus’ continued expansion northward, more mussel populations will be under threat from this parasite.

Antioxidative enzymes, alkaline phosphatases and Hsp70 expression in larvae of Lymantria dispar (Lepidoptera: Erebidae) from unpolluted and polluted forests after chronic cadmium treatment

Long-term exposure of populations to pollution may result in enhanced ability to cope with environmental stress. To compare the responses of two Lymantria dispar populations living in unpolluted and polluted forests (UP and PP, respectively), we chronically exposed larvae to cadmium at concentrations of 50 and 100 μg Cd/g dry food (Cd1 and Cd2, respectively). We examined cadmium accumulation in the midgut and hemolymph, activities of superoxide dismutase (SOD), catalase (CAT), and alkaline phosphatases (ALP) in the midgut, as well as Hsp70 protein expression in the midgut, hemolymph, and brain and evaluated these parameters as biomarkers of cadmium contamination. Larvae from PP, fed a control diet, showed higher activity of SOD and increased Hsp70 expression compared with larvae from UP. Excessive amounts of Cd were accumulated in the midgut of all Cd-fed larvae, whereas Cd content in the hemolymph was elevated only in larvae from PP after Cd2 treatment. In larvae from UP, Cd2 treatment decreased the activity of CAT and induced the expression of Hsp70 in the midgut and hemolymph. In larvae from PP, exposure to both Cd concentrations strongly attenuated SOD and CAT activities, while Hsp70 expression was not induced in any organ/tissue. Cd did not affect ALP activity in either population. Midgut Cd content proved to be a suitable indicator of Cd contamination for both polluted and unpolluted habitats.

Design, synthesis, and biological activity of dual monoamine oxidase A and heat shock protein 90 inhibitors, N-Methylpropargylamine-conjugated 4-isopropylresorcinol for glioblastoma

Monoamine oxidase A (MAO A) and heat shock protein 90 (HSP90) inhibitors have been shown to decrease the progression of glioblastoma (GBM) and other cancers. In this study, a series of MAO A/HSP90 dual inhibitors were designed and synthesized in the hope to develop more effective treatment of GBM. Compounds 4-b and 4-c are conjugates of isopropylresorcinol (pharmacophore of HSP90 inhibitor) with the phenyl group of clorgyline (MAO A inhibitor) by a tertiary amide bond substituted with methyl (4-b) or ethyl (4-c) group, respectively. They inhibited MAO A activity, HSP90 binding, and the growth of both TMZ-sensitive and -resistant GBM cells. Western blots showed that they increased HSP70 expression indicating reduced function of HSP90, reduced HER2 and phospho-Akt expression similar to MAO A or HSP90 inhibitor itself. Both compounds decreased IFN-γ induced PD-L1 expression in GL26 cells, suggesting they can act as immune checkpoint inhibitor. Further, they reduced tumor growth in GL26 mouse model. NCI-60 analysis showed they also inhibited the growth of colon cancer, leukemia, non-small cell lung and other cancers. Taken together, this study demonstrates MAO A/HSP90 dual inhibitors 4-b and 4-c reduced the growth of GBM and other cancers, and they have potential to inhibit tumor immune escape.

Alvespimycin Inhibits Heat Shock Protein 90 and Overcomes Imatinib Resistance in Chronic Myeloid Leukemia Cell Lines.

Heat shock protein 90 (HSP90) facilitates folding and stability and prevents the degradation of multiple client proteins. One of these HSP90 clients is BCR-ABL, the oncoprotein characteristic of chronic myeloid leukemia (CML) and the target of tyrosine kinase inhibitors, such as imatinib. Alvespimycin is an HSP90 inhibitor with better pharmacokinetic properties and fewer side effects than other similar drugs, but its role in overcoming imatinib resistance is not yet clarified. This work studied the therapeutic potential of alvespimycin in imatinib-sensitive (K562) and imatinib-resistant (K562-RC and K562-RD) CML cell lines. Metabolic activity was determined by the resazurin assay. Cell death, caspase activity, mitochondrial membrane potential, and cell cycle were evaluated by means of flow cytometry. Cell death was also analyzed by optical microscopy. HSPs expression levels were assessed by western blotting. Alvespimycin reduced metabolic activity in a time-, dose-, and cell line-dependent manner. Resistant cells were more sensitive to alvespimycin with an IC50 of 31 nM for K562-RC and 44 nM for K562-RD, compared to 50 nM for K562. This drug induced apoptosis via the mitochondrial pathway. In K562 cells, alvespimycin induced cell cycle arrest in G0/G1. As a marker of HSP90 inhibition, a significant increase in HSP70 expression was observed. Our results suggest that alvespimycin might be a new therapeutic approach to CML treatment, even in cases of resistance to imatinib.

Changes in the components of salivary exosomes due to initial periodontal therapy.

Exosomes are membrane vesicles that are present in body fluids and contain proteins, lipids, and microRNA (miRNA). Periodontal tissue examinations assess the degree of periodontal tissue destruction according to the probing depth (PD), clinical attachment loss (CAL), bleeding on probing, and X-ray examinations. However, the accurate evaluation of the prognosis of periodontitis is limited. In this study, we collected saliva from patients before and after initial periodontal therapy (IPT) and compared changes in the clinical parameters of periodontitis with changes in the components of salivary exosomes. Saliva was collected from patients with stage III and IV periodontitis at the first visit and post-IPT. Exosomes were purified from the saliva, and total protein and RNA were extracted. Changes in expression levels of C6, CD81, TSG101, HSP70, and 6 kinds of miRNA were analyzed by western blots and real-time polymerase chain reaction. Patients with increased C6 expression after IPT had significantly higher levels of periodontal inflamed surface area (PISA), miR-142, and miR-144 before and after IPT than patients with decreased C6 expression after IPT. Patients with decreased and unchanged CD81 expression after IPT showed significantly higher PD, CAL, and PISA before IPT than after IPT. Patients with decreased and unchanged TSG101 expression after IPT had significantly higher PD before IPT than after IPT. Patients with increased HSP70 expression after IPT had significantly higher PD and PISA before and after IPT than patients with unchanged HSP70 after IPT. The expression levels of miR-142, miR-144, miR-200b, and miR-223 changed with changes in the levels of C6, CD81, TSG101, and HSP70 in the salivary exosomes of periodontitis patients before and after IPT. The expression levels of proteins and miRNAs in salivary exosomes significantly changed after IPT in periodontitis patients, suggesting that the components of exosomes could serve as biomarkers for periodontitis.

Experimental Analysis of Vaginal Laxity in Rats Treated With a Combination of Er:YAG Fractional Lasers and AMSC-MP

Introduction: Vaginal laxity, a symptom of pelvic floor dysfunction observed in women, has many negative biological and psychological impacts. Laser treatments and stem cell-based therapies are emerging therapeutic methods for treating this condition. This study aimed to determine changes in vaginal laxity in model rats using a combination therapy of erbium-doped yttrium aluminium garnet (Er:YAG) fractional lasers and topical treatment with amniotic membrane stem cell metabolite products (AMSC-MP). Methods: The experimental animal population comprised 36 female white rats (Rattus norvegicus; 2-day-post-vaginal-delivery rats) allocated into the following four groups (n=9): K1, untreated two-day-post-vaginal-delivery rats; K2, two-day-post-vaginal-delivery rats treated with topical gel without AMSC-MP; P1, two-day-post-vaginal-delivery rats treated with Er:YAG fractional lasers and topical gel without AMSC-MP; P2, two-day-post-vaginal-delivery rats treated with Er: YAG fractional lasers and topical gel containing AMSC-MP. Immunohistochemical (IHC) examination was carried out for the expression and activity of heat shock protein 70 (HSP-70), collagen-1, tissue inhibitors of metalloproteinase 1 (TIMP-1) and matrix metalloproteinase 1 (MMP-1), as well as vaginal mucosal thickness. Results: There was a significant difference (P<0.05) in the expression of HSP-70 among all groups except K2 and P1 (P>0.05); there was no significant difference in type I collagen and TIMP-1 expression between the groups (P>0.05); there was a significant difference (P<0.05) in MMP-1 activity, with the activity in the K2 group (5.79±0.83) being higher than that in the P1 group (4.44±1.82) and that in the K1 group (5.74±1.03) being higher than that in the P2 group (4.24±1.55). Also, there was a significant difference in the thickness of the vaginal mucosa in all groups except K2 and P1 (P>0.05). Conclusion: Er:YAG fractional laser and AMSC-MP combination therapy improved vaginal laxity in model rats by increasing Hsp70 expression and vaginal mucosal thickness and decreasing MMP-1 activity.

EXTH-67. PRECLINICAL ASSESSMENT OF THE EFFICACY OF MPT0B640, AN ORALLY BIOAVAILABLE SMALL MOLECULE HSP90 INHIBITOR, AGAINST GLIOMAS

Abstract BACKGROUND Heat-shock protein 90 (HSP90), an evolutionary conserved protein, is a key member of the epichaperome complex involved in maintenance of protein homeostasis during non-stress and stress conditions. It is overexpressed in gliomas and supports tumor cell survival and resistance. Hsp90 inhibition can impact several pathways essential for glioma cell survival and overcome tumor heterogeneity. Here, we examined the activity and toxicity profile of MPT0B640, an orally bioavailable Hsp90 inhibitor, against gliomas and its ability to cross the blood-brain barrier (BBB). METHODS Using genetically heterogeneous glioma stem-like cell (GSC) lines, we determined the effects of MPT0B640 on cell proliferation (Cell Titre Glo assay), apoptosis induction and cell cycle changes (flowcytometry; AnnexinV/PI staining), and alterations in protein expressions (western blot and reverse-phase protein array, RPPA assays). Pharmacokinetic and pharmacodynamic (PK/PD) studies were performed in non-tumor bearing mice to assess BBB permeability and antitumor effects of MPT0B640. RESULTS MPT0B640 inhibited proliferation of both adherent (LN229, A172, U251-HF) and patient-derived GSCs (GSC262, GSC811, GSC11, GSC23, GSC20 and GSC272) in a dose- and time-dependent fashion with an IC50 range of 50-1000 nM. MPT0B640-treated cells underwent apoptosis and/or cell cycle arrest with downregulation of pro-survival signal kinases, phospho-Akt, p-ERK1/2 and p-S6. PK/PD studies showed increased HSP70 expression (a marker of Hsp90 inhibition) and detectable levels of drug in brain specimens from non-tumor bearing mice after a single dose of MPT0B640, indicating BBB permeability and target inhibition of the agent. Results of ongoing animal studies assessing drug toxicity and animal survival with MPT0B640 will be presented. CONCLUSIONS MPT0B640 is a potent HSP90 inhibitor that has potent antitumor activity against genetically heterogeneous gliomas in vitro, effectively crosses the intact BBB and demonstrates target inhibition warranting further investigation for treatment of gliomas.

The Change in Circadian Rhythms in P301S Transgenic Mice is Linked to Variability in Hsp70-related Tau Disaggregation.

Circadian disruption often involves a neurodegenerative disorder, such as Alzheimer’s disease or frontotemporal dementia, which are characterized by intraneuronal tau accumulations. The altered sleep pattern and diurnal rhythms in these disorders are the results of tau pathology. The circadian disturbance in reverse is thought to develop and potentially aggravate the condition. However, the underlying mechanism is not fully understood. In this study, perturbed oscillations in BMAL1, the core clock gene, were observed in P301S tau transgenic mice. Tau fractionation analysis of the hippocampus revealed profound fluctuations in soluble and insoluble tau protein levels that were in opposite directions to each other according to zeitgeber time. Interestingly, a diurnal oscillation was detected in the heat shock 70 kDa protein 1A (Hsp70) chaperone that was in-phase with soluble tau but out-of-phase with insoluble tau. Tau protein levels decreased in the soluble and insoluble fractions when Hsp70 was overexpressed in HEK293T cells. Transfection of the BMAL1 carrying vector was continual with the increase in Hsp70 expression and diminished tau protein levels, and it was effectively attenuated by the knockdown of Hsp70, suggesting that Bmal1 could modulate tau protein by Hsp70. Our results suggest that altered circadian oscillations affect tau status and solubility by modulating Hsp70 expression in an experimental model of tau pathology. These findings suggest Hsp70 as a possible pathogenic link between circadian disruption and aggravations of tau pathology.

Cold stress elevates HSP70, TLR2 and TLR4 of indigenous chicken

The study revealed that cold stress elevates the expression of HSP70, TLR2 and TL4 of indigenous chicken. Increase in expression of HSP70 was most likely to have cryoprotective properties by elevating the TLR2 and TLR4 of indigenous chicken during cold stress.

Whole-Body Cryotherapy Enhances the Expression of Heat Shock Protein 70 and Related Hormones

BACKGROUND: Whole-body cryotherapy (WBC) is used as a conditioning method for athletes. However, the scientific evidence for its effects is still insufficient. OBJECTIVE: To elucidate the effects of transient WBC on the expression of heat shock protein (HSP) 70 and the secretion of related hormones in humans. MATERIALS AND METHODS: The participants in this study were six healthy adult men. WBC was performed for 3 min in a booth at a temperature in the range of -150 to -120°C, and measurements were taken immediately before (Pre), immediately after (Post), and 60 min after WBC (Post60). For measurement of core body temperature (gastrointestinal temperature), participants ingested a capsule-type wireless temperature sensor. The body surface temperature was measured using a noncontact thermometer, and measurements were taken at four sites on the body surface (chest, abdomen, front of the thigh, and front of the lower thigh). Leukocyte count, lactate dehydrogenase, creatine kinase, hemoglobin, hematocrit, adrenaline, noradrenaline, cortisol, adrenocorticotropic hormone (ACTH), erythropoietin, and HSP70 in the collected blood were measured. RESULTS: The results showed a decrease in body surface temperature and an increase in noradrenaline and ACTH immediately after WBC. In addition, the core body temperature decreased 60 min after WBC, accompanied by an increase in HSP70 expression. CONCLUSION: WBC may increase HSP70 expression via noradrenaline and ACTH. The results of this study suggest the usefulness of WBC in triggering protein synthesis and the maintenance of immune function after training.

In vivo evaluation of oxidized multiwalled-carbon nanotubes-mediated hyperthermia treatment for breast cancer

Breast cancer is one of the most common types of cancer that contribute to high mortality worldwide. Hyperthermia (HT) was introduced as one of the alternative treatments to treat breast cancer but has major drawback of damaging normal adjacent cells. This study explores the integration effect of multiwalled‑carbon nanotubes (MWCNTs) in combination with hyperthermia treatment for breast cancer therapy regimes. In this study, acid-functionalized MWCNTs (ox-MWCNTs) were prepared by acid washing methods using H2SO4/HNO3 (98%/68%) with the ratio of 3:1 (ν/ν) and characterized by colloidal dispersibility test, FTIR, TGA, XRD, FESEM and EDX analysis. EMT6 tumor-bearing mice were treated with ox-MWCNTs in combination with local HT at 43 °C. The tumor progression was monitored and the influence of immune response was evaluated. Results from this study demonstrated that mice from ox-MWCNTs in combination with local HT treatment group experienced complete tumor eradication, accompanied by a significant increase in median survival of the mice. Histological and immunohistochemical analysis of tumor tissues revealed that tumor treated with combined treatment underwent cell necrosis and there was a significant reduction of proliferating cells when compared to the untreated tumor. This observation is also accompanied with an increase in Hsp70 expression in tumor treated with HT. Flow cytometry analysis of the draining lymph nodes showed an increase in dendritic cells infiltration and maturation in mice treated with combined treatment. In addition, a significant increase of tumor-infiltrated CD8+ and CD4+ T cells along with macrophages and natural killer cells was observed in tumor treated with combined treatment. Altogether, results presented in this study suggested the potential of ox-MWCNTs-mediated HT as an anticancer therapeutic agent, hence might be beneficial in the future of breast cancer treatment.