Effect of Mucuna Pruriens Seeds Supplementation on Heat Shock Proteins and Mitochondrial Gene Expression in Beetal Goats in Relation to Seasonal Stress

Sameer Niwas Jadhav (  sameerbiochem@gmail.com ) Guru Angad Dev Veterinary and Animal Sciences University https://orcid.org/0000-0002-8595-4449 Shashi Nayyar Guru Angad Dev Veterinary and Animal Sciences University Chanchal Singh Guru Angad Dev Veterinary and Animal Sciences University C.S Mukhopaddhyay Guru Angad Dev Veterinary and Animal Sciences University B.V. Sunil Kumar Guru Angad Dev Veterinary and Animal Sciences University


Introduction
M.pruriens is a leguminous plant having rich nutritional and phytochemical content. The most available literature of M. pruriens in animals cites the use of processed M.pruriens seeds as unconventional feedstuffs to utilize their nutrient content only. Various phyto-constituents in M.pruriens seeds include alkaloids, tri-terpenes, glycosides, tannins, phytosterols, avonoids, and phenolic & polyphenolic compounds exhibiting varied physiological and pharmacological activities. Several Scienti c studies in humans and laboratory animals have demonstrated the herbal supplement potential of raw powdered M. Pruriens seeds in terms of antioxidant, antistress, anti-cholestrolemic, anti-diabetic, fertility-enhancing, anti-in ammatory, and anti-microbial properties (Misra and Wagner, 2007). Microarray and real-time PCR gene expression analysis of M. Pruriens extract treatment versus the control group in rat heart showed that 59 genes are differentially expressed, which includes gene related to in ammatory, immune response, energy metabolism, apoptosis, eicosanoid synthesis, blood and wounding response, cardiac muscle and nervous system having a protective effect on the heart (Fung et al. 2014). Thus M.pruriens seeds exhibit nutraceutical potential in terms of delivering both nutritional and health bene ts to the animals. Still, no extensive molecular studies have been done in animals to evaluate the antistress potential of seeds in terms of their effect on thermotolerance level, oxidative stress-related mitochondrial functional response.
Heat shock proteins (HSPs) are multigene family proteins that range in molecular size from 10-150 kda found in all major cellular compartments. Heat shock proteins (HSPs) are considered as markers of thermotolerance of animals. They are a highly conserved proteins that are ubiquitously expressed in an animal's body during thermal stress. Out of all the members present in this family, HSP-70 is regarded as the most signi cant indicator of thermal stress. HSP-70 is known to be a highly inducible chaperon and plays a key role in stabilizing the native conformation of proteins and maintenance of cell survivability during thermal stress (Beckham et al., 2004). The expression of HSP-70 genes (HSPA8, HSPA6, HSPA1A, HSPA1L, and HSPA2) showed temperature sensitivity and seasonal variation. Relative expression of HSP-70 genes varied markedly among the heat-and cold-adapted goat breeds with a moderate variation between breeds and showed a good response to increased or decreased ambient temperature (Banerjee et al., 2014). The heat-shock protein (HSP) 90 belongs to the molecular chaperones class that participates in the normal folding, intracellular localization and proteolytic turnover of several proteins, including key regulators of cell growth and survival (Prodrumou and Pearl 2003). Two major cytoplasmic forms, α (inducible) and β (constitutive), of the protein, are recognized. Owing to its inducible expression, HSP-90α form exerts a relevant role in cell homeostatic responses to stressful conditions. Singh et al. (2017) reported that the expressions of HSP-70 and HSP-90 were increased signi cantly in goats that were subjected to heat stress.
Mitochondrial functional status is found to be an important marker in determining adaptive cellular response to heat stress. Mitochondrial DNA is highly sensitive to oxidative stress (Yakes and Van Houten 1997, Barja and Herrero 2000). Mitochondria are the main source as well as the target of free radicals.
Heat stress stimulates the excessive production of free radicals (Bernabucci et al.,2002). Mitochondrial DNA lacks histones and other DNA-associated proteins, making it readily accessible to oxidative damage.
Further mitochondrial DNA is near the ETC where ROS production occurs. Therefore they are an easy target for ROS-induced damage (Kakkar and Singh, 2007). Altered mitochondrial gene expression activates cytochrome c mediated intrinsic cellular apoptotic pathway. An increase in caspase 3 activity was found in mice repeatedly exposed to thermal stress (Kim et al., 2013). Relative gene expression of mitochondrial genes was lowered in karan fries bulls subjected to heat stress in tropical climates (Soren et al., 2018).
Considering the importance of HSP and mitochondrial gene in thermal stress response, research work was carried out to study the effect of M.prurien supplementation on the relative expression of heat shock proteins, and mitochondrial gene in beetal does and goat kids during winter and summer season.

Material And Method
Sixteen adult beetal does and goat kids were separately and randomly assigned to one of two groups: control or M.pruriens supplemented (8 goats in each group). Experiment was carried out during two seasons viz the winter (December-January month) and summer seasons (May-June) on separate goats. Adult does, and goat kids in the treatment groups were fed a standard diet plus 100 mg/kg body weight of raw M. Pruriens powdered seeds /animal/ (dose as per Doromala et al 2015) for 30 days. Control goats were fed with only basal diet as per NRC recommendations. Environmental parameters viz temperature, relative humidity was recorded. THI was calculated as per Tucker et al. (2008). Gene expression studies for the following genes were carried out before M. pruriens supplementation (0th day) and after M. pruriens supplementation (on 30th day) on blood samples in does and kids during winter & summer Season.
Mitochondrial functional stress markers-Cytochrome b (CYT-B) and Cytochrome oxidase subunit-I (COX-I) Gene-speci c primers were designed using online Genescript real-time PCR primer design software available online, and the speci city was checked using NCBI BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The details of primer are described in Table 1. during the summer season, respectively. A climatic environment having an air temperature in the range of 13-27°C, relative humidity of 60-70%, THI of less than 65% (Misra and Puneet,2009) is considered as a comfort zone. During summer and winter, animals were found exposed to an ambient temperature or humidity beyond their comfort zone, which indicates that they were experiencing thermal stress in both seasons while pronounced stress exhibited by animals during the summer season.

Gene expression study
Agarose gel electrophoresis of PCR product was used to con rm the size of the speci c size of ampli ed PCR product using 100 bp DNA ladder (shown in Fig. 1). All the ve genes tested using PCR were shown the expected size of PCR product length. Melt curve analysis conducted after real-time PCR ampli cation run revealed a single melt peak in all tested target genes (Shown in Fig. 2).
Effect of M. pruriens Supplementation on HSP and Mitochondrial gene expression on 30 day relative to day 0 Relative gene expression change in the control and treatment group on 30th day (post feeding) compared to prefeeding (0 day) has been represented in table no 2. Log 2 gene expression of all tested target genes has been graphically represented in Fig. 3. HSP-70 and HSP-90 gene expression were found to be signi cantly decreased in M. pruriens supplemented does and kids during both summer and winter seasons compared to the control group.  In our study, HSP-70 and HSP-90 gene expression were found to be signi cantly decreased while COX -I and CYT-B were found signi cantly increased in M. pruriens supplemented does and kids during both summer and winter season compared to the control group. This protective effect was found more pronounced in goat kids, who are normally found more susceptible to summer stress. Previous research work regarding studying the effect of M.pruriens seed supplementation on HSP gene expression during thermal stress in animals are not known. M.pruriens seeds and known to possess antistress and antioxidant properties (Misra and Wagner, 2007). Anti-stress activity is demonstrated by attenuating behavior anomalies exhibited by olfactory bulbectomised rats by chronic M. Pruriens treatment (Pati et al., 2010). The main anti-stress and neuroprotective compound identi ed in M. pruriens is L-DOPA. According to Manyam et al. (2004), M. pruriens has a signi cant neuroprotective effect and restores norepinephrine in the nigrostriatal system and dopamine levels in the substantia nigra of animal models.
M. pruriens seeds have been shown to exhibit neuroprotective effects by an increase in brain mitochondrial complex I activity and signi cantly restoring dopamine and norepinephrine levels in the parkinsonism animal model (Mundkinajeddu et al., 2021). Phenolics and avonoids in M. pruriens are antioxidant substances helpful in reducing the risk of oxidative stress-related disorders, including cancer and parkinson's disease (Rai et al., 2015). Induced cerebral ischemia in the wistar rat model resulted in signi cant neurological damage in the brain of the rats not treated with methanolic extract of M. pruriens seeds as observed in the histopathology of the isolated brain of rats (Nayak et al., 2017). A decrease in HSP gene expression level in our study indicates the amelioration of thermal stress and increase in adaptive response in Beetal goats.
Mitochondria are main site of production of free radicals as well as main targets of free radicals. There is a signi cant increase in mitochondrial free radical production along with altered mitochondrial protein expression observed during heat stress (Luft & Landau, 1995). Regarding the effect of M.pruriens on mitochondrial functionality, a study has shown that M. pruriens cotyledon powder signi cantly increased the brain mitochondrial complex-I activity as having NADH and coenzyme Q-10 in the cotyledon powder, which are shown to have a therapeutic bene t in parkinson's disease (Manyam et al.,2014). The diabetic rats supplemented with M. pruriens showed a remarkable recovery in antioxidant levels and reduced lipid peroxidation with well-preserved sperm DNA. Mitochondrial membrane potential and mitochondrial function test were also preserved in streptazocin plus M pruriens rat sperm (Suresh et al., 2013). These ndings support our results of preserving mitochondrial function by up-regulating respiratory chain component gene expression in M.pruriens supplemented goats. Up-regulation of the mitochondrial genes found in our study indicate improvement in mitochondrial functional status and mitochondrial respiratory e ciency in M.pruriens supplemented beetal goats compared to control goats which are exposed to heat stress. In conclusion, gene expression study results indicated that supplementing M. pruriens seeds @ 100 mg/kg body weight to goats leads to down-regulation of heat shock response and up-regulation of mitochondrial respiratory chain component gene expression thus having positive impact on adaptive response of Beetal goats against seasonal stress.

Declarations
Funding -Institutional funding Con icts of interest/Competing interests -The authors have no relevant nancial or non-nancial interests to disclose.
Ethics approval -All the experimental protocols using animals have been approved by the Institutional animal ethical committee (IAEC) of the Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana (Punjab), India, and by CPCSEA, New Delhi vide reference letter V-11011(13)/2/2020-CPCSEA-DADF dated 10/02/2020. All the experiments were carried out according to the guidelines of the CPCSEA.
Consent to participate -Not applicable.
Consent for publication -Not applicable.
Informed consent-Not applicable Availability of data and material -All data are available via the corresponding author.
Code availability -Not applicable.
Authors' contributions-All authors contributed to the study conception and design. Material preparation, data organization, and analysis were performed by Sameer N. Jadhav. The rst draft of the manuscript was written by the rst author and all authors commented on previous versions of the manuscript. All authors read and approved the nal manuscript.
22. Yakes, F. M., & Van Houten, B. (1997). Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proceedings of the National Academy of Sciences, 94(2), 514-519. Figure 1 Con rmation of speci c PCR product by agarose gel electrophoresis  Log 2 Gene expression fold on 30 th day (post-feeding) relative to 0 th day (prefeeding)