Expression Pattern of Genes Related to Heat Shock Proteins, Apoptosis, Antioxidants and Interleukins in Growing Female Murrah Buffaloes Implanted with Melatonin during Summer Season

A BSTRACT Background: Heat stress has deleterious effect in the production trait and biological functions in animals. In tropical continents, melatonin has been shown to retain genomic actions and regulating the expression of several genes necessary for cell survival during stress. So, the study was carried out to observe the effect of melatonin implantation on mRNA gene expression for Heat Shock Proteins, Apoptosis, Antioxidants and Interleukins affected by heat stress during summer season. Methods: Twelve healthy growing female Murrah buffaloes were selected and grouped into control and treatment (melatonin @ 18 mg/50 kg body weight subcutaneously). Peripheral Blood Mononuclear Cells were separated at fortnightly intervals and processed for quantitation of mRNA expression following the double delta Ct method. Result: The fold change in expression pattern of Heat Shock Proteins 60, 70, 90, BAX, Caspase-3, IL-2 and IL-6 were found significantly (P  0.05) higher in control group. Whereas Bcl-2, Mn SOD and CuZn SOD were found significantly (P  0.05) higher in treatment group. Melatonin implantation reduces the stress levels by up-regulating the expression of the antioxidant genes, anti-apoptotic genes and lowering the expression of heat shock protein genes during heat stress. Therefore, melatonin can be implanted to growing buffaloes for coping during heat stress.


INTRODUCTION
Exposure to extreme temperature elicits a series of extreme changes in the animal's biological functions that include reduction in feed intake, efficiency and utilization, hormonal secretions, enzymatic reactions and blood metabolites.Heat shock causes mitochondrial damage and senescence is induced by exhilarated ROS levels by alters cell homeostasis in animals (Nir et al., 2022).The effect of heat stress is exaggerated when it is accompanied with high ambient humidity (Marai et al., 2007).Animal's adaptation to cacophonous environmental conditions and confrontation to stress has been associated with expression of HSPs (Dangi et al., 2012).In tropics, melatonin has been shown to retain genomic actions and regulating the expression of several genes necessary for cell survival during stress (Gechev et al., 2002).Melatonin accrues higher concentration in mitochondria of the cells and managed the mitochondrial homeostasis.Sharma et al. (2013) found that the melatonin reduces oxidative stress and enhance the HSPs transcription.Melatonin is widely tested in the context of regenerative medicine and cell therapy and as antioxidants, they are predicted to improve stem cell fitness and resistance to stress (Nir et al. 2022).
Apoptosis is a physiological process which is associated to a complex network of biochemical pathways where undesirable cells are disposing off during development and other biological processes to ensure a homeostatic balance between cellular proliferation and turnover in every tissue (Choudhury et al., 2012).BAX hasten programmed cell death by binding to alienate the apoptosis repressor Bcl-2 or its adenovirus homolog E1B 19k protein.During stress, BAX undergoes to cause translocation to the mitochondrion membrane, leading to the release of cytochrome-C that then triggers apoptosis by activation of Caspase-3 and thereby apoptosis.Bcl-2 gene regulates cell death by inducing or inhibiting apoptosis (Cleary et al., 1986).Amidst of three superoxide dismutase responsible for destroying free Expression Pattern of Genes Related to Heat Shock Proteins, Apoptosis, Antioxidants and Interleukins in Growing Female Murrah.. superoxide radicals in the body, SOD-I bind with copper and zinc ions.

MATERIALS AND METHODS
The experiment was conducted at Livestock Research Station of ICAR-NDRI, Karnal, Haryana (India) during 2015-17.The dry and wet bulb temperature and relative humidity were recorded twice daily at 7.30 AM and 2.30 PM with dry and wet bulb hygrometer (Zeal, UK) every day during experimental period.The temperature humidity index (THI) was calculated as method described by Mc Dowell (1972).
Twelve healthy growing female Murrah buffaloes (8 to 12 months) were selected and equally divided into control and treatment (melatonin implanted @ 18 mg/50 kg body wt.) group on the basis of their body weight and body condition score.The experimental animals were housed in a custom design animal shed throughout the study.Deworming of animals were done prior to experiment.
PBMC was collected at fortnightly interval for analysis and quantitation of mRNA expression for HSP 60,70,90,IL2,IL6,Bcl2,Bax,Caspase3,MnSOD and CuZnSOD.Isolation of RNA was done by Qiagen RNeasy minikit, U.S. Agarose Gel electrophoresis was run to check RNA bands and the gels were subsequently photographed by Gel Doc System (Bio-Rad, Hercules, CA, USA).Strand cDNA Synthesis was done using kit from Thermo Scientific, U.S.A.The reference and targeted genes were procured from Europhins Genomics India Pvt. Ltd.Relative expression of the studied genes with respect to the THI was transformed in terms of fold change for quantification of mRNA expression following the double delta Ct method according to Livak and Schnittgen (2001).β-actin used as reference gene for normalization of differential expression of mRNA for relative quantification.
The expression of different genes in different samples was estimated as: gene in control The experiment was approved by IAEC constituted vide article no. 13 of CPCSEA rules (Reg.No. 1705/GO/ac/ CPCSEA dt.3/7/2013).Statistical analysis was done using Two-way ANOVA followed by post-hoc Tukey's test.Evaluation of the correlation between all the factors was made, using a correlation coefficient at the level of probability (P0.05).The analysis was performed using software version (9.1) SAS Institute Inc., Cary, NC, USA Copyright© (2011).

Temperature and humidity index
Mean±SE of Temperature Humidity Index (THI) during the experimental period have been depicted in Fig 1 .The 6 th , 7 th and 8 th fortnights showed most stressful period.The higher value for THI is obtained in the 7 th fortnight and lower value is obtained in the 1 st fortnight.Lactating dairy cows experience heat stress when THI rises above 72 and severe when it exceeds 88 (Thatcher et al., 2010).Kumar and Singh (2021) reported significant positive association of THI with RR and RT in growing buffaloes.Factors like level of air movement, sun exposure and duration of these conditions may affect THI values, because animals may experience more severe heat stress at lower temperature and higher relative humidity (Thatcher et al., 2010).

Heat shock proteins
The Expression Pattern of Genes Related to Heat Shock Proteins, Apoptosis, Antioxidants and Interleukins in Growing Female Murrah.. 6, Mn SOD and CuZn SOD and negative correlation with BAX and Caspase-3, presented in Table 1.
Melatonin interacts with numerous cellular proteins such as signalling molecules, transporters, channels and enzymes (Hemati et al., 2020).During heat stress, regulation of expression of HSP60 genes protects cell damage from heat stress through homeostatic mechanism (Vargas-Parada and Solis, 2001).HSP60 mitochondrial protein helps in refolding of proteins and preventing aggregation of denatured proteins.Sharma et al. (2013) revealed increased mRNA expression of HSP60 in melatonin treated group due to modulating effect under heat stress.The mRNA expression of HSP70 was significantly one fold higher in control than the treatment during summer.Expression Pattern of Genes Related to Heat Shock Proteins, Apoptosis, Antioxidants and Interleukins in Growing Female Murrah.. all the HSPs is most sensitive and positively correlated with heat tolerance.Patir and Upadhyay (2007) also observed HSP70 arose in Murrah buffaloes after two hours of heat exposure due to change in the adaptive and physiological mechanism to cope up with the thermal stress and to attain the thermo-tolerance.Manjari et al. (2015) reported an increase in HSP70 expression due to summer stress in different livestock species.HSP90 acts as a co-chaperone of HSP70 and restores the protein recognition (Pratt and Toft, 2003).

Apoptotic genes
The mean values of Bcl-2 mRNA expression in treatment group was significantly (P0.05)higher than the control  Pedreanez et al. (2004) found that treatment with melatonin causes a decrease in the expression levels of pro-apoptotic proteins along with an increase in the expression of Bcl-2.Activation of Caspase-3 is a sign of irreversible apoptosis stage (Fesik and Shi, 2001).Kireev et al. (2013) reported that mitochondrial DNA deletion induced oxidative stress and apoptosis and melatonin was found to protect the damage of neuronal cells (Jou et al., 2007).Juknat et al. (2005) found that melatonin down-regulate the expression of BAX and inhibit Caspase-3 activation in the dentate gyrus of old male rats by inhibiting H 2 O 2 induced apoptosis in cultured rat astrocytes.Ortiz et al. (2001) suggested that anti-apoptotic action of melatonin is one of the mechanisms by which it protects neuronal cells from neurotoxic insults.Espino et al. (2010) reported that melatonin is capable of reducing the activity in caspases-3 and induced by increased concentration of cytoplasmic calcium in human leukocytes, due to the activation of BAX with release of cytochrome-C, thus leading to reduced apoptotic activity.

Antioxidant genes
The mRNA expression of Mn SOD and CuZn SOD have been presented in Fig 4a and 4b respectively and revealed that differential expression in treatment group was significantly (P0.05)higher than the control group.Antolin et al. (1996) reported that in the lacrimal glands of hamsters, melatonin prevents from the porphyrins which are being produced due to the decreased mRNA synthesis of aminolevulinate synthetase caused cell damage and also it tends to increase the levels of Mn SOD and CuZn SOD.The synthesis of glutathione peroxidase enzyme eliminates the free radicals in an organism increases in the brains of mice when treated with melatonin (Weishaupt et al., 2006) and thus this hormone evident to act over other enzymes that provide protection against toxic reactive.Melatonin's antioxidant actions derive from its stimulatory effect on Glutathione Peroxide and Glutathione Reductase and its inhibitory action on NOS (Reiter, 2003).

Interleukins
The mean values of IL-2 and IL-6 expression in control group was significantly (P0.05)higher than the treatment group and have been presented in Fig 5a and 5b.Melatonin has been reported to be pro-inflammatory (Sutherland et al., 2002) whereas anti-inflammatory properties have also been attributed with some workers Cuzzocrea et al. (1999).The results are in accordance with Wang et al. (2015) who rep orted that h eat expo sure sig nific an tly (P<0 .0 5) elevated the level of IL-2 and IL-12. Carrillo-Vico et al. (2003) reported that melatonin regulates production of c ytok in e and acts m ostly on th e im mu n e system .Melatonin activates the production of IL-2, IL-6 and IFNã by T helper cells and monocytes Garcia-Maurino et al. (1997).IL-6 is a potent pro-inflammatory cytokine which induces the expression of reactants in the acute phase and the differentiation and migration of the activated macrophages Perez-Fernandez and Kaski, (2002).This suggests that there is a down-regulation of these genes in antioxidant supplemented group and control animals express severe stress.Vargas and Marino, (2016) found that during severe heat stress or exercise there was efflux o f LP S from G.I. tract w h ic h sw itch ac ute ph ase inflammatory response, including the release of IL-6 which acts during endotoxemic surrounding by debilitating the response through signaling the hypothalamic pituitary adrenal (HPA) axis.Hence, during the torrent of events, there could be release of LPS and provoked response to IL-6 during substantial heat stress.
Expression Pattern of Genes Related to Heat Shock Proteins, Apoptosis, Antioxidants and Interleukins in Growing Female Murrah..

CONCLUSION
Melatonin implantation increased the levels of Bcl-2, Mn SOD and CuZn SOD in treatment group, which appears to be helpful in scavenging free radicals.The higher levels of BAX, Caspase-3, IL-2 and IL-6 in control group indicating the level of heat stress experienced by the animals, while lower levels in treatment group is associated with increased THI might be due to antioxidant activity of melatonin, which ameliorate the impact of summer stress and reduced metabolic heat production.Altogether, the relative mRNA expression of HSP 60, 70 and 90 was significantly higher in control than treatment group.The findings of the study reveal that expression of HSPs were up-regulated during exposure of heat and HSP70 was found to be most induced.These HSPs safeguards the protein folding in a systematic manner and regulation of apoptosis during stressful physiological conditions.An exogenous melatonin administration ameliorated the adverse effects of thermal stress and potentiated the immunity and improved the antioxidant status and abated the apoptosis rate which battens cell survival during heat stress.
Fig 1: Fortnightly temperature humidity index during the experimental period.

Fig 2 :
Fig 2: Changes in HSP60, HSP70 and HSP90 in control and treatment groups of growing Murrah buffaloes during summer season.

Fig 3 :
Fig 3: Changes in Bcl-2, BAX and caspase-3 in control and treatment groups of growing Murrah buffaloes during summer season.

Fig 4 :Fig 5 :
Fig 4: Changes in Mn SOD and CuZn SOD in control and treatment groups of growing Murrah buffaloes during summer season.

Table 1 :
Correlation coefficient among different gene expressions of growing Murrah buffaloes.