{"id":57039,"date":"2024-03-20T11:18:59","date_gmt":"2024-03-20T11:18:59","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=57039"},"modified":"2024-04-01T19:21:15","modified_gmt":"2024-04-01T19:21:15","slug":"comparative-assessment-of-the-effectiveness-of-hsp70-hif-1%ce%b1-system-modulators-after-prenatal-hypoxia","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no1\/comparative-assessment-of-the-effectiveness-of-hsp70-hif-1%ce%b1-system-modulators-after-prenatal-hypoxia\/","title":{"rendered":"Comparative Assessment of the Effectiveness of HSP70 \/ HIF-1\u03b1 System Modulators after Prenatal Hypoxia"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Prenatal hypoxia (PH),\ncharacterized by insufficient oxygen supply to the developing fetus, poses a\nsignificant threat to fetal development and, depending on the timing and level\nof exposure, may be the cause of neonatal mortality or the development of\npathological neuropsychiatric changes manifested after birth at early and later\nages 1, 2, 3, 4<\/sup>. Under conditions of chronic PH, the main negative\nchanges occur in the brain, which is the most aerobic organ of the body. Oxygen\ndeficiency induces oxidative and nitrosative stresses, resulting in an\nelevation of ROS levels to excitotoxicity, mitochondrial and endothelial\ndysfunction, and damage to developing neurons 5, 6, 7, 8<\/sup>. It is\nknown that endogenous neuroprotective mechanisms – thiol-disulfide system, heat\nshock proteins and HIF-1 – are activated in neurons in response to hypoxic\ndamage. There are works devoted to the role of the complementary HSP70\/HIF-1\nsystem in the implementation of mechanisms in response to acute hypoxia and\nischemia 9, 10, 11<\/sup>. The cellular response to hypoxic stress results\nin the activation of stress-reactive proteins, among which a special role is\nattributed to heat shock proteins (HSPs), namely HSP 70, which are molecular\nchaperones 9, 12, <\/sup>. They participate in protein folding, prevent\naggregation of denatured proteins and promote cell survival under stress\nconditions; therefore, they can be considered as potential therapeutic targets\nfor pharmaceutical correction of the effects of PH. It is also known that HSP\n70 prolongs the lifespan of HIF-1 and, through it, influences the activation\nand regulation of compensatory energy shunts under hypoxia 9, 11, 12, 13<\/sup>.\nCurrently, the neuroprotective effects of HSP 70 modulators – tamoxifen, HSF-1,\nglutaredoxin, L-lysine 3-methyl-1,2,4-triazolyl-5-thioacetate (angiolin) – are\nknown 11, 14, 15, 16<\/sup>. The above provides theoretical support for\nconsidering HSP 70 modulators as neuroprotective agents and requires conducting\npreclinical studies.<\/p>\n\n\n\n

Aim: To identify a possible correlation between offspring survival and stages of expression of endogenous neuroprotective factors (HSP70 and HIF-1) after chronic prenatal hypoxia with course administration of potential HSP70 modulators (angiolin, piracetam, thiotriazoline, nicomex, cerebrocurin, tamoxifen, L-arginine, glutoredoxin, HSF-1, and mildronate).<\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

The experiment was conducted on white females rats which were\nobtained from the nursery of the Institute of Pharmacology and Toxicology of\nthe Academy of Medical Sciences of Ukraine (n=50, weight 220-240 g, age 4.5\nmonths). In our research, we were guided by the the national “General\nEthical Principles of Animal Experiments” (Ukraine, 2001) and Directive\n2010\/63\/EU of the European Parliament and of the Council on the protection of\nanimals used for scientific purposes (EU, 2010). The protocols of experimental\nstudies and their results were approved by the decision of the Commission on\nBioethics of ZSMU (Minutes No. 33 of June 26, 2019). The animals were housed in standard\nvivarium conditions. A model of chronic PH of medium severity induced by sodium\nnitrite was selected for the experiment. PH was induced by administering a\ndaily intraperitoneal injection of sodium nitrite solution (50 mg\/kg) to\npregnant female rats from the 16th to the 21st day of pregnancy 17<\/sup>.\nThis model causes histological, neurochemical, metabolic disorders of the CNS\nand cognitive-mnestic deficits in offspring 18, 19<\/sup>. In the intact\ngroup, pregnant females received saline solution in the same manner. To study\nthe effect of drugs, the offspring were divided into groups (Table 1).<\/p>\n\n\n\n

Table 1: Experimental groups<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Group No<\/strong><\/p>\n<\/td>\n

\n

Prenatal conditions<\/strong><\/p>\n<\/td>\n

\n

Administered drugs<\/strong><\/p>\n<\/td>\n

\n

Method of administra-tion<\/strong><\/p>\n<\/td>\n

\n

Manufacturer<\/strong><\/p>\n<\/td>\n

\n

Dosage<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/strong><\/p>\n<\/td>\n

\n

Normal<\/p>\n

(Intact)<\/p>\n<\/td>\n

\n

–<\/p>\n

Physiological solution<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

5 mL\/g<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n

(control)<\/p>\n<\/td>\n

\n

–<\/p>\n

Physiological solution<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n

\n

5 mL\/g<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Thiotriazolin (tiazotic acid) (2.5% solution for injection)<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Arterium, Ukraine<\/p>\n<\/td>\n

\n

50 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Tamoxifen*<\/p>\n<\/td>\n

\n

intranasally<\/p>\n<\/td>\n

\n

Zdorovye, Ukraine<\/p>\n<\/td>\n

\n

0.1 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Angiolin ((S)-2,6-diaminohexanoic acid 3-methyl-1,2,4-triazolyl-5-thioacetate)<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Farmatron, Ukraine<\/p>\n<\/td>\n

\n

50 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

6<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Glutaredoxin<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Sigma-Aldrich, USA<\/p>\n<\/td>\n

\n

200 \u00b5g\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

7<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Cerebrocurin<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

NIR, Ukraine<\/p>\n<\/td>\n

\n

150 \u00b5l\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

8<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

L-arginine (42% solution for injection)<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Yuria-Pharm, Ukraine<\/p>\n<\/td>\n

\n

200 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

9<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Nikomex (2-ethyl-6-methyl-3-hydroxypyridine succinate, solution for injection 50 mg\/mL)<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Nikopharm, Ukraine<\/p>\n<\/td>\n

\n

100 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

10<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

HSF-1 (recombinant)<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Sigma-Aldrich, USA<\/p>\n<\/td>\n

\n

50 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

11<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Mildronate ((2-(2-carboxyethyl)-1,1,1-trimethylhydrazinium, 10% solution for injection)<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Grindex, Latvia<\/p>\n<\/td>\n

\n

50 mg\/kg<\/p>\n<\/td>\n<\/tr>\n

\n

12<\/strong><\/p>\n<\/td>\n

\n

PH<\/p>\n<\/td>\n

\n

Piracetam (20% solution for injection)<\/p>\n<\/td>\n

\n

intra-peritoneally<\/p>\n<\/td>\n

\n

Farmak, Ukraine<\/p>\n<\/td>\n

\n

500 mg\/kg<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

* – intranasal gel with active substance content of 1 mg\/1 mL, manufactured on the basis of tablets 20 mg ex temporae at the Department of Drug Technology of ZSMFU.<\/em><\/p>\n\n\n

Rat pups were administered drugs from day 1 to day 30 of life. For dosage calculation, the instructions for the drugs and data from previous studies were used 9, 14, 15<\/sup>.<\/p>\n\n\n\n

Animals (10 from each group) were removed from the\nexperiment on the 30th and 60th days of life using thiopental anesthesia (40\nmg\/kg).<\/p>\n\n\n\n

Preparation of\nBiological Material<\/strong><\/p>\n\n\n\n

Blood was obtained from the abdominal artery by syringe,\nplasma was separated by centrifugation with Ependorff 5804R centrifuge at +4\u00b0C\nat 1500 rpm for 20 min. Blood was obtained from the abdominal artery by\nsyringe, plasma was separated by centrifugation with Ependorff 5804R centrifuge\nat +4\u00b0C at 1500 rpm for 20 min. The brains were fixed in Bouin’s fixative for\n24 h and processed according to standard histological techniques, then embedded\nin Paraplast (MkCormick, Hunt Valley, MD, USA). For real-time PCR, serial\nhistologic brain sections 5 \u03bcm thin were sectioned on a Microm-325 rotary microtome\n(Microm Corp., Munich, Germany) and processed with o-xylene and ethanol.<\/p>\n\n\n\n

Enzyme-Linked\nImmunoassay<\/strong><\/p>\n\n\n\n

HSP70 and HIF-1 protein concentrations in plasma were\nmeasured by solid-phase sandwich immunosorbent assay using ELISA Kit test\nsistems: AMP’DR HSP70 high-sensitivity ELISA kit # ENZ-KIT-101-0001, Enzo\n(Solna, Sweden) for determination of HSP70 level; AMP&#39;D HSP70\nhigh-sensitivity (Enzo, Sweden) for determination of HIF-1 concentration. HSP70\nand HIF-1 concentrations were expressed in ng\/mL.<\/p>\n\n\n\n

Polymerase Chain\nReaction in Real-Time<\/strong><\/p>\n\n\n\n

Quantitative real-time PCR was employed to assess HSP70\nmRNA expression using Maxima SYBR Green\/ROX qPCR Master Mix (2x)\n(ThermoScientific, Waltham, MA, USA) with gene-specific primers. The analysis\nwas conducted on a CFX96\u2122 Real-Time PCR Detection System by Bio-Rad\nLaboratories, Inc., USA. Fluorescence intensity was automatically registered\nthrough the automatic SybrGreen channel at the end of elongation stage of each\ncycle. Beta-actin (Actb) served as the reference gene for determining the\nrelative change in the expression level of the target gene. Quantification was\nperformed by the comparative delta Ct method.<\/p>\n\n\n\n

Statistical\nAnalysis<\/strong><\/p>\n\n\n\n

Statistical analysis of the experimental data was\nperformed with “STATISTICA 13.0 TIBCO Software Inc.” and\n“Microsoft Office Excel 2016”. The normality of the results was\nchecked by the Shapiro-Wilk and Kolmogorov-Smirnov tests. For comparative\nanalysis, we used the parametric Student’s t-test for normal distributions and\nthe Mann-Whitney U-test for non-normal distributions. ANOVA dispersion analysis\nwas used for normal distributions and Kruskal-Wallis test for non-normal\ndistributions to compare independent variables in more than two selections.\nCorrelation analysis based on Pearson’s or Spearman’s correlation coefficient\nwas used to analyze correlations between parameters. For all analyses,\ndifferences were considered significant at p<0.05 (95%).<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

According to the results of the conducted experiment it\nwas established that in the blood plasma of healthy animals proteins HSP70 and\nHIF-1 are present in relatively constant concentrations without significant age\ndifferences. Chronic PH resulted in a 5.6-fold decrease in HSP70 level and a\n10.6-fold decrease in HIF-1 plasma concentration on the day 30 of life, and on\nthe day 60 these parameters remained significantly lower than the values of the\nintact group (Figure 1a), demonstrating \nthe long-term after effects of prenatal hypoxia in postnatal\ndevelopment.<\/p>\n\n\n\n

Course administration of the studied drugs, except\nthiotriazoline and piracetam, increased the blood level of HSP70 with the most\nsignificant values in the groups of animals receiving angiolin (2-fold),\nglutaredoxin (by 95%), cerebrocurin (2.6-fold), L-arginine (2.25-fold), nikomex\n(2.37-fold), HSF-1 (2.36-fold).<\/p>\n\n\n\n

Long-term results of pharmacological correction of PH\nconsequences showed positive effects in the groups of animals administered thiotriazolin,\nmildronate, glutaredoxin with maximum indicators compared to the control in the\ngroups after administration of angiolin, glutaredoxin and cerebrocurin.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Concentration of HSP70 (a) and HIF-1\u03b1 (b) in the blood plasma of rats after PH and course of drug administration on the day 30 and day 60 of life (M \u00b1 m, n = 10);<\/strong> *,<\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

In all\ngroups, an increase in plasma HIF-1 concentration was observed during the first\nmonth, with maximum values in the groups of animals receiving cerebrocurin\n(10.8-fold), angiolin (8.1-fold), and HSF-1 (6.6-fold). At the end of the\nsecond month, the level of HIF-1 increased in all experimental groups except\nthose receiving HSF-1and mildronate. In animals treated with angiolin and\ncerebrocurin, this parameter remained maximum, corresponding to the intact\nparameters. (Figure 1b). A correlation relationship between HIF-1 and HSP70\nconcentrations levels was established (Pearson correlation coefficient R = 0.70\nfor day 30 of life and R = 0.68 for day 60 of life).<\/p>\n\n\n\n

PH reduces the level of HIF-1\u03b1 mRNA\nexpression by 37%, and when corrected by the studied drugs, this parameter\nincreases manifold, exceeding intact values, with maximum results in the groups\nafter treatment with cerebrocurin, thiotriazoline, angiolin and HSF-1 (Table 2).\nThe results of HSP70 mRNA expression generally correlate with HIF-1\u03b1 mRNA\nexpression levels (R = 0.71) with maximum results in the groups after treatment\nwith angiolin, cerebrocurin, HSF-1, glutaredoxin and mildronate.<\/p>\n\n\n\n

Table 2: Expression levels of HIF-1\u03b1 mRNA and HSP70 mRNA<\/strong> in the brain of rats after PH on day 60 <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Groups<\/strong><\/p>\n<\/td>\n

\n

mRNA HIF1\u03b1 levels<\/strong><\/p>\n<\/td>\n

\n

mRNA HSP70 levels<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

Intact<\/p>\n<\/td>\n

\n

1.00\u00b10.002<\/p>\n<\/td>\n

\n

1.00\u00b10.003<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

Control<\/p>\n<\/td>\n

\n

0.331\u00b10.002<\/p>\n<\/td>\n

\n

0.409\u00b10.008<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

Thiotriazolin<\/p>\n<\/td>\n

\n

2.06\u00b10.001*1<\/sup><\/p>\n<\/td>\n

\n

2.13\u00b10.006*1<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

Tamoxifen<\/p>\n<\/td>\n

\n

1.63\u00b10.004*1<\/sup><\/p>\n<\/td>\n

\n

1.98\u00b10.001*1<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

Angiolin<\/p>\n<\/td>\n

\n

4.61\u00b10.004*1<\/sup><\/p>\n<\/td>\n

\n

5.21\u00b10.004*1<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

6<\/p>\n<\/td>\n

\n

Glutaredoxin<\/p>\n<\/td>\n

\n

2.80\u00b10.002*1<\/sup><\/p>\n<\/td>\n

\n

3.88\u00b10.001*1<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

7<\/p>\n<\/td>\n

\n

Cerebrocurin<\/p>\n<\/td>\n

\n

5.24\u00b10.002*1<\/sup><\/p>\n<\/td>\n

\n

7.11\u00b10.008*1<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

8<\/p>\n<\/td>\n

\n

L-arginine<\/p>\n<\/td>\n

\n

0.37\u00b10.0011<\/sup><\/p>\n<\/td>\n

\n

0.568\u00b10.003*<\/p>\n<\/td>\n<\/tr>\n

\n

9<\/p>\n<\/td>\n

\n

Nikomex<\/p>\n<\/td>\n

\n

0.77\u00b10.006*<\/p>\n<\/td>\n

\n

0.68\u00b10.004*<\/p>\n<\/td>\n<\/tr>\n

\n

10<\/p>\n<\/td>\n

\n

HSF-1<\/p>\n<\/td>\n

\n

4.70\u00b10.730*1<\/sup><\/p>\n<\/td>\n

\n

4.37\u00b10.051*1<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

11<\/p>\n<\/td>\n

\n

Mildronate<\/p>\n<\/td>\n

\n

1.30\u00b10.022*1<\/sup><\/p>\n<\/td>\n

\n

3.73\u00b10.140*1<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

12<\/p>\n<\/td>\n

\n

Piracetam<\/p>\n<\/td>\n

\n

0.603\u00b10.003*<\/p>\n<\/td>\n

\n

0.563\u00b10.005*<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

*, significantly different from control group, p<0.05; 1<\/sup>, significantly different from intact group, p<0.05.<\/em><\/p>\n\n\n

We have established a correlation between the levels of HIF-1 and HSP70 protein concentration in plasma and survival of animals after PH. Modeling of chronic PH increased offspring mortality 10.5-fold compared to offspring obtained after normal physiological pregnancy (69.56% and 6.67%, respectively) (Figure 2). The majority of lethal cases were registered in newborns and at the age of 9-12 days. PH negatively affects the processes of neural tissue formation, disrupting proliferation and migration of neuroblasts, which leads to pathological changes in the morphofunctional organization of the brain, the consequences of which are manifested after birth.<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 2: Survival rates (%) of offspring after PH and its pharmacological correction (a) and correlation analyses of offspring survival rates and HSP70 concentration in blood plasma on the day 30 (b) and day 60 (c) of life,<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Course\nadministration from the first day of life of pharmacological agents with\npotential and proven neuroprotective effect reduced the mortality of offspring\nafter PH action. The highest offspring survival rates were recorded in the\ngroups of rats that were given mildronate (76.97%, n=39), angiolin (64.29%,\nn=42), thiotriazoline (63.41%, n=41), HSF-1 (62.96%, n=40) and cerebrocurin\n(62.86%, n=35). The results of the correlation analysis of offspring survival\nrates and HIF-1 and HSP70 concentrations for the first month of life showed a\nhigh positive correlation (R=0.73) for HIF-1 and a lower one with HSP70 plasma\nconcentration (R=0.63). On the 60th day, on the contrary, the correlation\ncoefficient between rat survival and HSP70 level was R=0.80 and for HIF-1 it\ndecreased to R=0.51 (Figure 2). Offspring survival after PH depends on the\nefficiency of the complex action of endogenous mechanisms of neuroprotection,\nand HIF-1\/HSP70 dependent mechanisms play a key role in these processes.<\/p>\n\n\n\n

Discussion<\/strong><\/p>\n\n\n\n

The use of sodium nitrite in the PH\nmodel causes maternal hemic hypoxia due to methemoglobin formation and tissue\nhypoxia through dissociation of products of oxidation and phosphorylation\nprocesses. This leads to disturbances in the uteroplacental circulation system,\nresulting in the formation of persistent oxygen deficiency in the fetus, which\nleads to hypoxic organ damage 18, 20<\/sup>. It is known that oxygen\ndeficiency leads to abnormal development of the fetal brain 2, 3<\/sup>. PH\nleads to postnatal individual physical and intellectual disorders, early\nmortality and disability. PH causes persistent disorders of transcriptional\nprocesses, protein synthesis, mitochondrial function, and transmitter\nautocoidosis 5, 7<\/sup>. In response to PH, endogenous defense mechanisms\nare activated in the cells. Important links of these mechanisms are the HIF-1\nfactor and HSP70 proteins 9, 21, 22, 23, 24<\/sup>. It is known that\nhypoxia-induced factors (HIFs) play the role of transcription factors and\nregulate the expression of genes encoding the synthesis of proteins involved in\nthe physiological response to hypoxia\/ischemia. Thus, HIF-1 stimulates\nerythropoiesis, activates additional ATP synthesis shunts, and increases the\nconcentration of reduced glutathione 25, 26<\/sup>. HIF-1 is able to\nenhance HSP70 expression indirectly through HSF-1 activation to maintain\ncellular redox homeostasis. In turn, by possessing chaperone activity, HSP70 is\nable to stabilize HIF-1\u03b1 and prolong its activity under hypoxic and\npost-hypoxic conditions 9, 27, 28<\/sup>. HSP70 proteins are molecular\nchaperones actively involved in the mechanisms of cellular response to\nischemia, stress, hypoxia, temperature increase 9, 12, 29<\/sup>. The\nregulation of HSP70 expression occurs with the participation of heat shock\nfactor (HSF-1) 24, 30<\/sup>. Under hypoxic conditions, HSP70 proteins\nperform cytoprotective functions by preventing the aggregation of misfolded\nproteins, promoting protein refolding and protein translocation across cell\nmembranes. The chaperone activity of HSP70 also extends to stabilization of\ncell membranes, preserving cellular integrity 9, 12, 29, 31<\/sup>. In\naddition, HSP70 inhibits apoptosis by interaction with key regulators of the\napototic pathway, caspases 21, 32<\/sup>. HSP70 in acute ischemia\nconditions are able to influence the activity of compensatory energy shunts by\npreserving HIF-1 38<\/sup>. We found that chronic PH causes a decrease in\nHIF-1 mRNA expression in rat target organs and inhibition of HIF-1-dependent\nprotective and compensatory-adaptive mechanisms 15, 19<\/sup>. There is\ninformation about multidirectional changes in HIF-1 expression in different\nforms of hypoxia\/ischemia. In intensive hypoxia against the background of\noxidative stress activation and energy deficit, HIF-1 decreases due to its\nubiquitylation and lower synthesis 35, 38<\/sup>. It is also known that\nsubtotal cerebral ischemia causes suppression of HSP70 expression. The\ncorrelation between HSP70 deficiency in the brain and the severity of\nneurological disorders in cerebral ischemia was found. In this study, we also\nfound a correlation between HSP70 deficiency and early postnatal lethality of\nnewborn rats after PH. All this leads us to consider HSP70 as an important\ncomponent of endogenous neuroprotection, the level of which determines the fate\nof the fetus and the prospects of health and social development of the neonate.\nTherefore, we have proposed the modulation of HSP70 expression by\npharmacological agents as a promising approach to neuroprotection after PH.<\/p>\n\n\n\n

As a result of this study, it was\nfound that the most significant effects on the expression of HSP70 and HIF-1\nand the reduction of offspring lethality after PH were demonstrated by\nangiolin, cerebrocurin and HSF-1. The results obtained can be explained by the\navailable data on the mechanisms of action of these pharmacological agents.<\/p>\n\n\n\n

The effect of Angiolin on cell\nprotective mechanisms under PH conditions that we have identified is not\ncontradicted by other studies 33, 34<\/sup>. It was demonstrated that\nAngiolin exhibits antioxidative, cardioprotective, neuroprotective and endothelioprotective\nproperties in conditions of myocardial and cerebral ischemia. The mechanism of\nits neuroprotective action is based on regulation of HSP70 expression,\nespecially in mitochondria, mitoprotective action and improvement of cell\nenergy metabolism due to activation of mitochondrial-cytosolic compensatory\nshunts. Angiolin increases NO bioavailability, regulates eNOS and iNOS\nexpression, and inhibits the formation of cytotoxic forms of NO9, 15, 35<\/sup>.\nThese effects are based on the peculiarities of the molecular structure of\nAngiolin, which provides opportunities to form complexes with NO, as well as to\nregulate SH\/SS equilibrium and influence GSH-dependent neuroprotective\nmechanisms. Angiolin increases the level of glutathione reduced in the cytosol\nand mitochondria of neurons during cerebral ischemia. Through the regulation of\nglutathione, Angiolin regulates the synthesis and stability of HSP70 9, 35<\/sup>.\nOur in vitro experiments show that GSH depletion of neurons leads to a decrease\nin HSP70 levels 35<\/sup>.<\/p>\n\n\n\n

Cerebrocurin contains regulatory\nneuropeptides and can activate subtotal transcription factors and influence the\nexpression of genes encoding growth factors, chaperone proteins and antioxidant\nenzymes. Cerebrocurin is known to have antioxidant, neuroprotective, nootropic\nand mitoprotective activities. 14, 36, 37<\/sup> The neuroprotective effect\nof Cerebrocurin is attributed to its ability to significantly increase the\nexpression of HSP70 and intramitochondrial Mn-SOD 35, 38, 39<\/sup>.<\/p>\n\n\n\n

HSF-1 is the main transcriptional regulator\nthat initiates the cellular response to stresses of different nature. Stress\nfactor leads to increase of ROS in the cell, which triggers HSF-1 activation,\nand it binds to promoters of HSE genes, activating first of all HSP protein\ngenes. HSF-1 targets are also genes responsible for metabolic processes; it\ntakes part in the regulation of apoptosis processes, intracellular transport\nand signaling 25, 40<\/sup>.<\/p>\n\n\n\n

Understanding the protective role of\nHIF-1 and HSP70 in hypoxia opens pathways for potential therapeutic\ninterventions (Figure 3). Strategies aimed at enhancing the expression or\nactivity of HIF-1 and HSP70 may represent novel approaches to pharmacological\ncorrection of the effects of PH.<\/p>\n\n\n\n

We have experimentally substantiated\nthe expediency of using HSP70 modulators as neuroprotective agents. We also\nshowed the most promising ways of pharmacological modulation of HSP70 – through\nthe increase of SH-dependent mechanisms (Angiolin) and through activation of\ntranscription factors (Cerebrocurin, HSF-1).<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Pharmacological modulation of the endogenous neuroprotective mechanisms associated with HSP70\/HIF-1\u03b1 after PH.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

PH modeling leads to increased lethality of offspring\nduring 2 months of life against the background of decreased concentrations of\nHSP70 and HIF-1\u03b1 in blood plasma and decreased brain HIF-1\u03b1 mRNA expression\nlevels in experimental animals. Offspring survival after PH was positively\ncorrelated with HIF-1 and HSP70 expression levels. The course administration of\nHSP70\/HIF-1\u03b1 modulators to rats that underwent PH reduces postnatal lethality,\nincreases blood plasma concentrations of HSP70 and HIF-1\u03b1, and positively\naffects the expression level of HIF-1\u03b1 mRNA in the rat brain.<\/p>\n\n\n\n

The most significant effect of modulation of the HSP70\/HIF-1\u03b1 related endogenous neuroprotective mechanisms after PH was obtained after treatment with angiolin (50 mg\/kg), cerebrocurin (150 mg\/kg) and HSF-1 (50 mg\/kg). Therefore, they can be considered as the most promising drugs for new therapeutic strategies of pharmacological correction of the consequences of chronic PH.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

The authors would like to acknowledge the Zaporizhzhia State Medical and Pharmaceutical University for providing some facilities in carrying out the research.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare no conflict of interest.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

This research received no external funding.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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