Heat shock factor 1 inhibition enhances the effects of modulated electro hyperthermia in a triple negative breast cancer mouse model.

Female breast cancer is the most diagnosed cancer worldwide. Triple negative breast cancer (TNBC) is the most aggressive type and there is no existing endocrine or targeted therapy. Modulated electro-hyperthermia (mEHT) is a non-invasive complementary cancer therapy using an electromagnetic field generated by amplitude modulated 13.56 MHz frequency that induces tumor cell destruction. However, we have demonstrated a strong induction of the heat shock response (HSR) by mEHT, which can result in thermotolerance. We hypothesized that inhibition of the heat shock factor 1 (HSF1) can synergize with mEHT and enhance tumor cell-killing. Thus, we either knocked down the HSF1 gene with a CRISPR/Cas9 lentiviral construct or inhibited HSF1 with a specific small molecule inhibitor: KRIBB11 in vivo. Wild type or HSF1-knockdown 4T1 TNBC cells were inoculated into the mammary gland's fat pad of BALB/c mice. Four mEHT treatments were performed every second day and the tumor growth was followed by ultrasound and caliper. KRIBB11 was administrated intraperitoneally at 50 mg/kg daily for 8 days. HSF1 and Hsp70 expression were assessed. HSF1 knockdown sensitized transduced cancer cells to mEHT and reduced tumor growth. HSF1 mRNA expression was significantly reduced in the KO group when compared to the empty vector group, and consequently mEHT-induced Hsp70 mRNA upregulation diminished in the KO group. Immunohistochemistry (IHC) confirmed the inhibition of Hsp70 upregulation in mEHT HSF1-KO group. Demonstrating the translational potential of HSF1 inhibition, combined therapy of mEHT with KRIBB11 significantly reduced tumor mass compared to either monotherapy. Inhibition of Hsp70 upregulation by mEHT was also supported by qPCR and IHC. In conclusion, we suggest that mEHT-therapy combined with HSF1 inhibition can be a possible new strategy of TNBC treatment with great translational potential.

The beneficial effect of nanomethionine supplementation on growth performance, gene expression profile, and histopathology of heat-stressed broiler chicken.

This study investigated the effects of nanomethionine (nano-meth) on performance, antioxidants, and gene expression of HSP70, HSP90 and Heat Shock factor-1 (HSF-1) from the liver, and TLR4 from the jejunum, of broiler chickens reared under normal temperatures or under heat stress. Three hundred 1-day-old chicks were randomly assigned to 5 treatment groups. Group 1 served as control. Under normal temperature, birds in group 2 received nano-meth (10 mL/L of drinking water) from d1 until the experiment ended. Group 3 birds were heat-stressed (HS) and did not receive any supplementation. Group 4 received nano-meth in the same dose from d1 old until experiment ended, and the birds were exposed to HS. Group 5 birds were HS and received supplementation of nano-meth during the HS period only. Nano-meth improved (P < 0.0001) final body weight, weight gain, feed conversion ratio, and also decreased (P < 0.0001) the effect of HS on growth performance. Reduction (P < 0.0001) in malondialdehyde and changes in antioxidant enzymes GPX and CAT activity indicated the antioxidant effect of nano-meth. Nano-meth supplementation caused an increase in the expression of HSP70 , HSP90 and HSF1, and a downregulation of TLR4 gene expression. Additionally, nano-meth-supplemented groups showed marked improvement in the histological liver structure, intestinal morphology and villus height compared to control or HS groups.

Effects of melittin on production performance, antioxidant function, immune function, heat shock protein, intestinal morphology, and cecal microbiota in heat-stressed quails.

The purpose of this study was to investigate the effects of melittin on production performance, antioxidant function, immune function, heat shock protein, intestinal morphology, and cecal microbiota of heat-stressed quails. A total of 120 (30-day-old) male quails were randomly divided into 3 groups. Each group consisted of 4 replicates with 10 birds per replicate. The ambient temperature of the control group (group W) was 24°C ± 2°C. The heat stress group (group WH) and the heat stress + melittin group (group WHA2) were subjected to heat stress for 4 h from 12:00 to 16:00 every day, and the temperature was 36°C ± 2°C for 10 d. The results showed that compared with the group W, heat stress significantly decreased growth performance, serum and liver antioxidative function, immune function, intestinal villus height (VH) and villus height-to-crypt depth ratio (VH/CD), and cecal microbiota Chao and ACE index (P < 0.05). The crypt depth (CD) in the small intestine, and HSP70 and HSP90 mRNA levels in the heart, liver, spleen, and kidney were significantly increased (P < 0.05). Dietary melittin significantly increased growth performance, serum and liver antioxidative function, immune function, intestinal VH and VH/CD, and cecal microbiota Shannon index in heat-stressed quails (P < 0.05). Melittin significantly decreased small intestinal CD, and HSP70 and HSP90 mRNA levels in the viscera (P < 0.05). Furthermore, dietary melittin could have balanced the disorder of cecal microbiota caused by heat stress and increased the abundance and diversity of beneficial microbiota (e.g., Firmicutes were significantly increased). PICRUSt2 functional prediction revealed that most of the KEGG pathways with differential abundance caused by high temperature were related to metabolism, and melittin could have restored them close to normal levels. Spearman correlation analysis showed that the beneficial intestinal bacteria Anaerotruncus, Bacteroidales_S24-7_group_norank, Lachnospiraceae_unclassified, Shuttleworthia, and Ruminococcaceae_UCG-014 increased by melittin were positively correlated with average daily feed intake, the average daily gain, serum and liver superoxide dismutase, IgG, IgA, bursa of Fabricius index, and ileum VH and VH/CD. In sum, our results demonstrate for the first time that dietary melittin could improve the adverse effects of heat stress on antioxidant function, immune function, heat shock protein, intestinal morphology, and cecal microbiota in quails, consequently improving their production performance under heat stress.

Research Note: Probiotic, Bacillus subtilis, alleviates neuroinflammation in the hippocampus via the gut microbiota-brain axis in heat-stressed chickens.

High ambient temperature (heat stress, HS) is one of the critical environmental factors causing gut microbiota dysbiosis and increasing gut permeability, consequently inciting neuroinflammation in humans and various animals including chickens. The aim of this study was to examine if a probiotic, Bacillus subtilis, can reduce neuroinflammation in heat-stressed broiler chickens. Two hundred and forty 1-d-old broiler chicks were randomly assigned to 48 pens among 4 treatments in 2 identical, thermal-controlled rooms (n = 12): Thermoneutral (TN)-regular diet (RD), TN-PD (the regular diet mixed with a probiotic at 250 ppm), HS-RD, and HS-PD. The probiotic diet was fed from d 1, and HS at 32°C for 10-h daily was applied from d 15 for a 43-day trial. Results showed that compared to the TN broilers, the HS broilers had higher hippocampal interleukin (IL)-6, toll-like receptor (TLR)4, and heat shock protein (HSP)70 at both mRNA and protein levels regardless of dietary treatment (P < 0.05). In addition, the HS-PD broilers had higher levels of hippocampal IL-8 (P < 0.05) than the TN-PD broilers. Within the HS groups, compared to the HS-RD broilers, the HS-PD broilers had lower levels of IL-6, IL-8, HSP70, and TLR4 (P < 0.05) in the hippocampus. Within the TN groups, the TN-PD broilers had lower IL-8 at both mRNA expressions and protein levels (P < 0.05) but higher TLR4 protein levels (P < 0.05) in the hippocampus as compared to the TN-RD broilers. These results indicate that dietary supplementation of the Bacillus subtilis-based probiotic may reduce HS-induced brain inflammatory reactions in broilers via the gut-brain-immune axis. These results indicate the potential use of probiotics as a management strategy for reducing the impact of HS on poultry production.

Manganese alleviates heat stress of primary cultured chick embryonic myocardial cells via enhancing manganese superoxide dismutase expression and attenuating heat shock response.

Manganese (Mn) is an essential trace element that has been shown to attenuate the adverse effects of heat stress in the heart of broiler breeders and embryos. However, the underlying molecular mechanisms involving this process remain unclear. Therefore, two experiments were conducted to investigate the possible protective mechanisms of Mn on primary cultured chick embryonic myocardial cells exposed to heat challenge. In experiment 1, the myocardial cells were exposed to 40 °C (normal temperature, NT) and 44 °C (high temperature, HT) for 1, 2, 4, 6 or 8 h. In experiment 2, the myocardial cells were preincubated with no Mn supplementation (CON), 1 mmol/L of Mn as the inorganic MnCl2 (iMn) or organic Mn proteinate (oMn) under NT for 48 h, and then continuously incubated under NT or HT for another 2 or 4 h. The results from experiment 1 showed that the myocardial cells incubated for 2 or 4 h had the highest (P < 0.0001) heat-shock protein 70 (HSP70) or HSP90 mRNA levels than those incubated for other incubation times under HT. In experiment 2, HT increased (P < 0.05) the heat-shock factor 1 (HSF1) and HSF2 mRNA levels as well as Mn superoxide dismutase (MnSOD) activity of myocardial cells compared with NT. Furthermore, supplemental iMn and oMn increased (P < 0.02) HSF2 mRNA level and MnSOD activity of myocardial cells compared with the CON. Under HT, the HSP70 and HSP90 mRNA levels were lower (P < 0.03) in iMn group than in the CON group, in oMn group than in iMn group; and the MnSOD mRNA and protein levels were higher (P < 0.05) in oMn group than in the CON and iMn groups. These results from the present study indicate that supplemental Mn, especially oMn, could enhance the MnSOD expression and attenuate heat shock response to protect against heat challenge in primary cultured chick embryonic myocardial cells.

Synergistic effect of standardized extract of Asparagus officinalis stem and heat shock on progesterone synthesis with lipid droplets and mitochondrial function in bovine granulosa cells.

Progesterone (P4) is a well-known steroid hormone that plays a key role in oocyte growth and the maintenance of pregnancy in mammals, including cattle. Heat stress (HS) has an adverse effect on P4 synthesis through an imbalance in the cellular redox status. We have recently revealed that a standardized extract of Asparagus officinalis stem (EAS) increases P4 through non-HS induction of heat shock protein 70 (HSP70) and a synergistic increase of HSP70 by enhancing the intracellular redox balance, which was adversely affected by HS in bovine granulosa cells (GCs). Bovine GCs collected from bovine ovarian follicles were cultured at 38.5 °C and 41 °C for 12 h with or without 5 mg/mL EAS. After treatment, cells and culture suppernatant were collected for the analysis. Enzyme-linked immunosorbent assay (ELISA) was performed to detect in P4 levels. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to detect expression of steroidogenesis related genes. Fluorescence staining was used to detect mitochondrial activity and lipid droplet. P4 level was increased by EAS treatment in association with increase in steroidogenic acute regulatory protein (STAR), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), mitochondrial membrane activity and lipid droplet both under non-HS and HS conditions. Notably, synergistic effect of EAS with HS co-treatment was observed to show a greater increase in P4 synthesis when comparison with EAS treatment under non-HS condition. Furthermore, inhibition of HSP70 significantly reduced EAS-induced P4 synthesis, mitochondrial activity and synthesis of lipid droplets. These results suggest that P4 synthesis by EAS is mediated by the steroidogenesis pathway via HSP70-regulated activation of STAR and 3ß-HSD, together with improved mitochondrial activity and lipid metabolism in bovine GCs. Moreover, effect of EAS has a synergistic effect of with HSP70-regulated steroidogenesis pathway.

Dietary intake of Spirulina platensis alters HSP70 gene expression profiles in the brain of rats in an experimental model of mixed stress.

Spirulina platensis has gradually gained more attention for its therapeutic, antioxidant, and anti-inflammatory potential worldwide. However, the current molecular knowledge about the effects of spirulina on stress-related genes is rather limited. The effects of dietary intake of spirulina on the HSP70 gene expression were assessed in a controlled in vivo experimental design. Moreover, alterations in serum corticosterone levels, IL-2, IL-4, IFN-Υ, triglyceride, ALT, AST, relative gene expression values, and the correlations between them were evaluated. A total of 36 rats were divided into four groups: control group, stress-only group, spirulina group, and spirulina+stress group. To control the dose administration, S. platensis was applied by a gastric gavage in stress groups. Crowded environment stress and hosting alone stress were applied to the stress-only group and spirulina + stress group. RNA was extracted from brain samples using TRIpure and the relative gene expression assessment was performed using Roche-LightCycler-480-II real-time PCR-System. Gene expression values were remarkably different among the four experimental groups. The differences between stress-only and the spirulina groups were statistically significant (P<0.05). The correlation between the HSP70 gene expression and the IFN-Υ was found to be statistically significant (P<0.05; r=0.50). Results indicate a novel effect of spirulina on the HSP70 expression related to the stress-response. Data presented in this study may be useful for further studies to define not only the molecular genetic aspects through dietary S. platensis but also the effects of spirulina on stress-response and animal welfare.

Zinc supplementation prior to heat shock enhances HSP70 synthesis through HSF1 phosphorylation at serine 326 in human peripheral mononuclear cells.

Zinc supplementation prior to heat shock increases HSP70 (heat shock protein 70) expression, which has cytoprotective effects in tissue cells during inflammation. Effects of zinc deficiency in this regard have been discussed controversially. Whether zinc modulates the expression of HSP70 in the human immune system as well and thus affects cell survival during heat stress is so far largely unknown. Therefore, we investigated the effect of alterations in the cellular zinc status on HSP70 expression and on cellular survival in human monocytes and lymphocytes. Three cell lines (Jurkat, THP-1, and Ramos) and enriched primary human monocytes and lymphocytes from young subjects were subjected to zinc deficiency or supplementation and subsequently heat shock at 42 °C. HSP70 mRNA expression was analyzed by real-time PCR, whereas HSP70 protein expression was analyzed by western blotting. In all cells other than Ramos cells, zinc supplementation and deficiency augmented heat shock-induced HSP70 expression. Further experiments in primary monocytes and lymphocytes indicated that this may be explained by the enhanced phosphorylation of HSF1 (Heat shock factor 1) at Ser326, which plays a significant role in HSP70 induction, as observed in zinc deficient and supplemented cells. While zinc supplementation had negligible effects on cell viability, acute zinc deficiency further increased cell death, induced by heat shock. Our results emphasize the importance of an optimal cellular zinc status. Moreover, we present a possible mechanism behind zinc's influence on HSP70 expression in human leukocytes. Our data form the basis for further in vivo and ex vivo studies to investigate how the zinc status may affect cellular damage in transient high temperature situations.

ß-Elemene Promotes Apoptosis Induced by Hyperthermia via Inhibiting HSP70.

Thermotherapy has been presented as a promising strategy to be used as an effective nonsurgical technique for colorectal carcinoma. Although this strategy presents several advantages, including low toxicity and high repeatability, thermotherapy often needs to be combined with other therapies because residual tumor cells that survive hyperthermal treatment often lead to relapse. In this study, we evaluated the effects of ß-elemene, which has been proven to have the potential to reverse chemotherapy drug resistance, on promoting the antitumor effects of hyperthermia. ß-elemene treatment significantly promoted apoptosis after 2 hours of hyperthermia treatment and blocked cell cycle phases at G1/G0. ß-elemene also significantly decreased colony formation and tumor formation abilities after hyperthermia treatment. ß-elemene treatment significantly decreased HSP70, but not HSP90 or HSP27, induced by hyperthermia treatment without disturbing HSP70 mRNA. It was also found that ß-elemene decreased phosphorylated ERK1/2 induced by hyperthermia. Regain of HSP70 reversed ß-elemene-mediated apoptosis, indicating that ß-elemene may induce apoptosis by decreasing HSP70. Moreover, ß-elemene treatment significantly decreased invasion capacity by decreasing the EMT, which was induced by hyperthermia treatment. Taken together, our results offer a potential strategy for CRC therapy via the combination of hyperthermia and ß-elemene.

Liposome-templated gold nanoparticles for precisely temperature-controlled photothermal therapy based on heat shock protein expression.

Mild temperature photothermal therapy is gaining more and more attention due to high safety, high specificity and moderate efficacy. However, the therapeutical outcome of mild photothermal therapy is limited due to the overexpression of heat shock proteins (HSPs). Therefore, the precise management of HSP expression is the key to improvement of mild temperature photothermal therapy. However, the correlation between HSP expression and photothermal temperature in vivo is still unclear. To precisely control the photothermal temperature by managing the HSP expression, we quantified the HSP expression at different photothermal temperatures after irradiation on liposome-templated gold nanoparticles, which have high photostability, high photothermal conversion efficiency and low temperature fluctuation (smaller than 1 â„ƒ). We found that the expression of HSP70 was least at 47 â„ƒ, which was the optimal temperature for HSP management. We chose to co-administrate HSP70 inhibitor during 47 â„ƒ photothermal therapy, leading to greatly enhanced tumor inhibition. Our precise temperature-controlled photothermal therapy based on HSP expression offers a new strategy for clinical tumor photothermal therapy.

Effects of heat stress on cyto(chemo)kine and inflammasome gene expression and mechanical properties in isolated red and white blood cells from 4 commercial broiler lines and their ancestor jungle fowl.

Commercial broilers have been selected for high growth rate and productivity; however, this has negatively impacted their susceptibility to heat stress (HS). Insight into the molecular mechanisms underlying this vulnerability can help design targeted strategies for improvement of HS tolerance. Red blood cells (RBC) and white blood cells (WBC) were isolated from red jungle fowl and 4 lines of commercial modern broilers. Lines A and B are considered standard-yielding lines, whereas Lines C and D are high-yielding. Cells were cultured at either 37°C or 45°C for 2 h to induce heat stress (HS). Gene expression of cytokines, chemokines, and inflammasome components were measured. Heat shock proteins 27 and 70 (HSPs) in RBC were significantly affected by line (P < 0.05), whereas HSP27 and 60 were affected by temperature (P < 0.05). In WBC, there was a significant line effect on HSP gene expression (P < 0.05), and a significant increase (P < 0.05) in HSP90 in Line D in HS compared to TN conditions. In RBC, there was a main effect of HS on TNFα, CCL4, and CCLL4 (P < 0.05). HS significantly increased IL-8L1 (>30-fold, P < 0.0001) in Line C. Inflammasome genes (NLRP3, NLRC5 and NLRC3) were significantly affected by the line studied (P < 0.05). In WBC, the effect of line was significant for all cytokines, chemokines, and inflammasome components studied (P < 0.05). To examine the mechanical properties of isolated RBC from the 4 commercial lines and jungle fowl, RBC were placed into nematic liquid crystals, where Lines B and D were the most strained, and Line A and the jungle fowl were the least strained. Together, these findings indicate not only the dynamic nature of circulating cells, but the differences in the stress and inflammatory response among commercially available lines and their common ancestor. These profiles have the potential to serve as a future marker for stress responses in broilers, though further study is warranted.

Hepatotoxic effect of lead and hepatoprotective effect of Hydrilla verticillata on hepatic transcriptional and physiological response in edible fish Labeo rohita.

Lead tops the list of developmental toxicants released by industry into the environment according to Commission for Environmental Corporation. Hydrilla verticillata is an aquatic weed and rich source of variable nutrients and chemical constituents like saponins, vitamins, minerals, antioxidants, amino acids, detoxifying agents, etc. This weed is used in a beneficial way to detoxify lead in fish model. The fish were treated with sublethal concentration of lead to induce liver damage and fed with supplementary feed containing 20% of hydrilla dry powder. The present work revealed that lead accumulation is highly toxic to liver. Lead toxicity increased the expressions of cytochrome P450 1 A (CYP1A) and cytochrome P450 3 A (CYP3A) when compared to control. The metal binding stress proteins, heat shock proteins (Hsp60 & Hsp70) and metallothionein were also upregulated due to lead toxicity. Lead intoxicated fish exhibited reduced δ-aminolevulinic acid dehydratase activity in the blood, leading to reduced red blood cell count and changes in cell morphology. The lead toxicity also decreases the level of liver marker enzymes alanine transaminase, aspartate transaminase in serum. In hydrilla supplemented protective group, there was amelioration of these changes. Administration of supplementary feed to the lead intoxicated protective group significantly decreased the expressions of CYP1A, CYP3A, Hsp60, Hsp70 and metallothionein genes. The red blood cell count in lead intoxicated fish was maintained almost normal due to the protective action of Hydrilla verticillata in the diet. The observed increase in the enzymes, which further confirmed the protective effect of Hydrilla verticillata against lead induced toxicity.

Selenium exerts protective effects against heat stress-induced barrier disruption and inflammation response in jejunum of growing pigs.

BACKGROUND: Heat stress (HS) has a negative impact on the intestinal barrier and immune function of pigs. Selenium (Se) may improve intestinal health through affecting selenoproteins. Thus we investigate the protective effect of new organic Se (2-hydroxy-4-methylselenobutanoic acid, HMSeBA) on jejunal damage in growing pigs upon HS and integrate potential roles of corresponding selenoproteins. RESULTS: HS decreased the villus height and increased (P < 0.05) the protein abundance of HSP70, and downregulated (P < 0.05) protein levels of tight junction-related proteins (CLDN-1 and OCLD). HS-induced jejunal damage was associated with the upregulation of four inflammation-related genes and ten selenoprotein-encoding genes, downregulation (P < 0.05) of four selenoprotein-encoding genes and decreased (P < 0.05) the protein abundance of GPX4 and SELENOS. Compared with the HS group, HMSeBA supplementation not only elevated the villus height and the ratio of V/C (P < 0:05), but also reduced (P < 0.05) the protein abundance of HSP70 and MDA content, and increased (P < 0.05) the protein abundance of OCLD. HMSeBA supplementation downregulated the expression of seven inflammation-related genes, changed the expression of 12 selenoprotein-encoding genes in jejunum mucosa affected by HS, and increased the protein abundance of GPX4, TXNRD1 and SELENOS. CONCLUSION: Organic Se supplementation beyond nutritional requirement alleviates the negative effect of HS on the jejunum of growing pigs, and its protective effect is related to the response of corresponding selenoproteins. © 2021 Society of Chemical Industry.

Effects of heat stress on fattening performance, carcass traits, oxidant/antioxidant status, and hepatic heat shock protein 70 levels in different plumage colors of Japanese quail (Coturnix coturnix japonica).

The aim of this study was to investigate the impact of heat stress on production performance and oxidative stress in different plumage colors of Japanese quail. For this purpose, a total of 100 birds were used in this study. The 25 birds belonged to Wild-type (n=25, grey), Tuxedo (n=25, black), Golden (n=25, yellow) and Recessive white (n=25). The birds were reared for 42 days in an environmentally controlled room at 39°C and relative humidity of 60-65%. The body weight, body weight gain (g/bird/day), and feed conversion ratio were not different between the groups (p>0.05). However, the feed intake (g/bird/day) of the Wild-type had a higher value than the Tuxedo (black) group counterparts between 15 and 21 days different (p⟨0.05). There was no significant effect of heat stress on the carcass traits (p>0.05). Spleen weights were different between the groups (p⟨0.05). The yellow group had the highest spleen weight. The highest MDA level was found in the Recessive White variety, followed by Wild-type (grey), Golden (yellow) and Tuxedo (black), respectively. However, there were no statistical differences amongst the groups (p>0.05). There was also no statistical significance in glutathione (GSH) and superoxide dismutase (SOD) levels (p>0.05). The heat shock protein 70 kDa (HSP70) level was significantly different between the groups (p⟨0.001). The highest percentage was observed in the Golden (5.06%) and the lowest in the White (1.43%) variety. There was no superior color variety of Japanese quail regarding fattening performance and carcass traits. It is conceivable that when considering the stress response of the different colors, the Golden group is more sensitive to stress due to the hepatic and cellular level of HSP70.

Long non-coding RNA KCNQ1OT1 facilitates the progression of cervical cancer and tumor growth through modulating miR-296-5p/HYOU1 axis.

Literature reports that lncRNA KCNQ1OT1 is markedly up-regulated in cervical cancer (CC) tissues and cell lines, and KCNQ1OT1 can promote the proliferation and metastasis of CC cells. This current work was designed to investigate the molecular mechanism underlying the participation of KCNQ1OT1 in CC progression. Herein, RT-qPCR was utilized for determining the levels of KCNQ1OT1, miR-296-5p and HYOU1 in clinical tumor tissue specimens and CC cell lines. Then, starBase predicted the complementary binding sites of KCNQ1OT1 and miR-296-5p or miR-296-5p and HYOU1. Dual-luciferase reporter assay/RIP assay validated the interplays among KCNQ1OT1/miR-296-5p/HYOU1. In addition, CCK-8, wound healing and transwell assays were employed to assess the proliferative, migrative and invasive properties of CC cells. Moreover, nude mice xenograft model was established by subcutaneously injection with SiHa cells in order to validate the precise functions of KCNQ1OT1/miR-296-5p/HYOU1 axis in CC in vivo. Besides, Immunohistochemical staining examined Ki-67 expression in xenograft tumors and western blotting analysis detected expressions of MMP2/9 and Wnt/ß-catenin signaling pathway in CC cells and xenograft tumors. Elevated KCNQ1OT1 and HYOU1 as well as reduced miR-296-5p were observed in clinical tumor tissue specimens and CC cell lines. Results revealed that upregulation of miR-296-5p counteracted the enhancing effects of overexpressed KCNQ1OT1 on the proliferative, migrative and invasive abilities of CC cells. Additionally, HYOU1 overexpression abolished the suppressing effects of silenced KCNQ1OT1 on the malignant behaviors of CC cells and tumor growth. To conclude, KCNQ1OT1 could aggravate the malignant behaviors of CC and facilitate tumor growth through modulating miR-296-5p/HYOU1 axis.

Immunohistochemical Expression of AQP2 and HSP70 in Broiler Kidney Tissue Treated with Salix tetrasperma Roxb. Extract under Heat Exposure.

The administration of plant extracts to broilers may be a way to mitigate the effects of heat stress. The importance of AQP2 and HSP70 compounds in maintaining the homeostasis of the chicken body when it is subjected to heat stress is well established. This study aims to determine the effect of giving the ethanolic extract of the leaves of Salix tetrasperma Roxb. on the immunohistochemical expression of AQP2 and HSP70 in exposed and unexposed broiler kidney tissue. This study used 36 samples of 28-day-old chicken kidneys. Chickens were kept in individual cages, provided with feed and drinking water ad libitum. The design used was a completely randomized design with 6 treatments and 6 replications: (a) chickens were reared in conditions exposed to heat (HS + 0); (b) chickens were reared in conditions exposed to heat and given Salix extract at a dose of 50 mg/L drinking water (HS + 50); (c) chickens were reared under heat-exposed conditions and given Salix extract at a dose of 100 mg/L drinking water (HS + 100); (d) chickens were reared in conditions without exposure to heat (n-HS + 0); (e) chickens were reared in conditions without exposure to heat and given Salix extract at a dose of 50 mg/L drinking water (nHS + 50); and (f) chickens were reared in conditions exposed without exposure to heat and given 100 mg/L drinking water (nHS + 100) of Salix extract. Salix extract was given for 24 hours and was renewed every 6 hours. The results showed that giving Salix extract 100 mg/L in drinking water to chickens exposed to heat (HS + 100) reduced the value of the H/L ratio. Giving Salix extract 50-100 mg/L in drinking water caused an upregulated AQP2 expression; on the other hand, it downregulated HSP-70 expression, in chicken kidney tubules both exposed to heat stress and nonexposed to heat stress. In conclusion, exposure to heat stress in broiler chickens and giving Salix extract can increase the formation of aquaporin 2 compounds and suppress the formation of HSP70.

Effect of Selenium and Vitamin E on the Level of Sperm HSPA2+, Intracellular Superoxide Anion and Chromatin Integrity in Idiopathic Asthenoteratozoospermia: A Double-Blind, Randomized, Placebo- Controlled Trial.

PURPOSE: Male infertility accounts for about half of all infertility cases. Asthenoteratozoospermia is a severe form of male infertility. Free radicals play an important role in infertility. In a study we found that asthenoteratozoospermic men had a lower mean percentage of sperm HSPA2+ and higher intracellular anion superoxide than normozoospermia. Antioxidants are thought to be able to counteract the negative effects of free radicals. We explored the efficacy of vitamin E in combination with Se on the level of sperm HSPA2+, intracellular anion superoxide and chromatin integrity in these patients. MATERIALS AND METHODS: 60 patients entered the study. They were randomized to treatment group of oral Se (200 µg) in combination with vitamin E (400 units) for 3 months (n= 30) or placebo (n= 30). Semen samples were obtained and assessed for sperm parameters, intracellular O2-, protamine deficiency, sperm HSPA2+ and apoptotic spermatozoa at baseline and after treatment phase. RESULTS: There were no significant differences in baseline semen parameters, intracellular O2- protamine deficiency, sperm HSPA2+ and apoptotic spermatozoa between the treatment and placebo groups. There was a statistically significant decrease in sperm apoptosis and the level of anion superoxide (P=.001) and an increase in sperm motility and viability (P=.001) in the treated group, but no significant difference was found in the percentage of sperm HSPA2+ and sperm protamine deficiency compared with baseline. Moreover, no significant change was found in these parameters in placebo group after 3 months. CONCLUSION: Our results showed that administration of vitamin E and selenium for three months may improve sperm motility and viability by decreasing intracellular anion superoxide and sperm apoptosis in asthenoteratozoospermic infertile men. We suggest that consuming these supplements before assisted reproductive technology (ART) may improve outcomes in these patients.

Astragalus membranaceus polysaccharides modulate growth, hemato-biochemical indices, hepatic antioxidants, and expression of HSP70 and apoptosis-related genes in Oreochromis niloticus exposed to sub-lethal thallium toxicity.

A 60-day experiment was performed to assess the efficacy of dietary Astragalus membranaceus polysaccharides (ASP) in attenuation of sub-lethal thallium (Tl) toxicity in Nile tilapia. Six experimental groups (in triplicates) were designed where a fish group was raised in clean water and fed basal diet and served as control (CONT), two groups were fed the basal diet supplemented with 0.15% and 0.30% ASP (ASPL and ASPH), Tl-intoxicated group exposed to 1/10 of 96-h LC50 (= 41.9 µg/L), and two other groups were fed 0.15% and 0.30% ASP and concomitantly exposed to 41.9 µg Tl/L (ASPL-Tl and ASPH-Tl). At the end of the experiment, fish behavioral responses, clinical signs, survivability, growth, whole-body composition, intestinal digestive enzymes, serum biochemical parameters, hepatic antioxidative biomarkers, and transcription of stress and apoptosis genes were assessed. Results showed that the whole-body composition, intestinal α-amylase and protease enzymes, serum AST and blood urea levels, and hepatic GSH were not significantly different among groups (P > 0.05). The Tl-intoxicated fish group was off food, had darkened skin, showed restlessness and hyperexcitability, and high mortalities. FBW, WG, SGR and FI were significantly decreased alongside increase FCR in the Tl-exposed group. Tl exposure caused significant increases (P < 0.05) in intestinal lipase enzyme and serum indices such as ALT, creatinine, total cholesterol, triglycerides, glucose, and cortisol levels. Moreover, a significant decreases in hepatic CAT and SOD enzyme activities and significant increases in hepatic MDA contents were also noticed (P < 0.05). Furthermore, Tl exposure induced significant upregulation of hepatic HSP70 and apoptosis-related genes (p53 and caspase 3). Interestingly, dietary supplementation with ASP in ASPL-Tl and ASPH-Tl groups modulated the parameters mentioned above but still not reached the CONT values. Altogether, this study suggests that ASP could be beneficial in the modulation of sub-lethal Tl toxicity effects in Nile tilapia. Additionally, we can conclude that using natural feed supplements such as ASP in aquafeed might be necessary for maintaining the overall health performances of Nile tilapia.

Terpinen4-ol inhibits heat stress induced inflammation in colonic tissue by Activating Occludin, Claudin-2 and TLR4/NF-κB signaling pathway.

Heat stress has severe implications on the health of mice involving intestinal mucosal barrier damage and dysregulated mucosal immune response. This study was designed with long-term heat stress to detect the protective effect of terpinen4-ol on body weight, colon length, organ index, morphological structure, inflammatory cytokines expression, Claudin-2, Occludin, and TLR4 signaling pathway of colonic tissue in mice under heat stress. A study found that oral administration of terpinen4-ol helped against mortality and intestinal inflammation in a mouse model of acute colitis induced by heat stress (40 °C per day for 4 h) exposed for 14 consecutive days. The mice were divided into five groups including control, heat stress, terpinen4-ol low dose (TER LD: 5 mg/kg), medium dose (TER MD: 10 mg/kg), and high dose (TER HD: 20 mg/kg) group. Our study showed that the heat-stress terpinen4-ol group had improved body weight, colon length, and organ index, the number of white blood cells, lymphocytes, and neutrophils in the blood as compared to the heat stress group. In addition, results showed that heat stress upregulated the expression of TLR4, p65, TNF-α, and IL-10. While, in mice receiving the oral administration of terpinen4-ol, the production of TNF-α, IL-10, TLR4, and p65 was suppressed on day 1, 7, and 14 of heat stress. In addition Claudin-2, Occludin mRNA levels were upregulated in mice receiving terpinen4-ol on day 1, 7, and 14 of heat stress. Furthermore, the IL-6, IL-10, TNF-α serum levels were also upregulated in mice under heat stress, but in mice receiving the oral administration of terpinen4-ol, the IL-6, IL-10, TNF-α level was down-regulated on day 1, 7, and 14 of heat stress. Histomorphological examination found that as compared to the control group, the muscle layer thickness and villi height of mice in the heat stress group were significantly reduced, while the changes of the above indicators in the terpinene4-ol groups were improved than those in the heat stress group. In conclusion, the terpinen4-ol has a protective effect on colonic tissue damage induced by heat stress.

Curcumin nano-micelle induced testicular toxicity in healthy rats; evidence for oxidative stress and failed homeostatic response by heat shock proteins 70-2a and 90.

This study explores the effect of curcumin nano-micelle (NCMN) on the testicular anti-oxidant status and heat shock proteins (Hsp) 70-2a and Hsp 90 expression. Therefore, 24 male Wistar rats were divided into control, 7.50 mg/kg, 15 mg/kg, and 30 mg/kg of NCMN-received groups. Following 48 days, the testicular total anti-oxidant capacity (TAC), total oxidant status (TOS), malondialdehyde (MDA) and glutathione (GSH), catalase (CAT) and glutathione peroxidase (GPX) activities, immunoreactivity of 8-oxodG, Hsp70-2a and Hsp90 expressions, germ cell's DNA and mRNA damages, the spermatozoa count, motility and DNA integrity were assessed. With no change in the testicular TAC level, the TOS, MDA and GSH contents were increased in the NMC-received groups. However, CAT and GPX activities were decreased. The NCMN suppressed spermatogenesis, increased immunoreactivity of 8-oxodG, stimulated the Hsp70-2a and Hsp90 expressions, and resulted in severe DNA and mRNA damages. Moreover, the NCMN-received animals exhibited remarkable reductions in the spermatozoa count, motility and DNA integrity. In conclusion, chronic and high dose consumption of NCMN initiates OS, and in response to OS, the Hsp70-2a and Hsp90 expression increases. However, considering enhanced DNA and mRNA damages and suppressed spermatogenesis, HSPs over-expression can neither boost the anti-oxidant system nor overcome the NCMN-induced OS-related damages.