eugenia jambolana extract versus n-acetyl cysteine against...
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CHAPTER 6: EXPERIMENTAL
CHAPTER 3
Protective effects of Eugenia jambolana extract versus N-acetyl cysteine against cisplatin
induced damage in rat testis
Chapter 6: Experimental Chapter 3
116
3.1 Introduction
“The protection against CIS induced testicular damage as a result of various abovementioned
interventions is primarily based on strengthening mechanisms of cell survival by augmenting
cell’s own internal resources to meet such a challenge (Anand et al., 2014). However, though
antioxidants are reported and recognized as beneficial, all of the previous studies with herbal
extracts had never used any known antioxidant for comparison (Anand et al., 2014). In addition,
despite reports of mortality in animals, various doses of cisplatin (2.5 to 10 mg/kg b.w) were
utilized, both for short term (10 days) and other studies extending even beyond 30 days
(Atessahin et al., 2006b, (Anand et al., 2014). In all such studies, deleterious effects on
spermatogenesis due to cisplatin treatment ranged from partial disruption to complete arrest. The
extent of revival of spermatogenesis following herbal or other interventions similarly varied
widely (Anand et al., 2014). Separate assessment of Leydig cell function in cisplatin treated
animals has never been reported (Anand et al., 2014). Accordingly, the present study was
carefully planned to evaluate the protective effects of fruit pulp of Eugenia jambolana extract, in
comparison with a known antioxidant, N-acetyl cysteine (NAC) in short-term intervention
studies with CIS-treated adult rats (Anand et al., 2014). The molecular mechanisms associated
with cell survival and steroidogenesis were also extensively studied to assess their impact on the
overall testicular function (Anand et al., 2014).”
3.2. Experimental Plan
“Adult male rats (Holtzman strain) weighing 200-220 g were used (Anand et al., 2014). Sixty
animals were divided into six groups of ten animals each (Anand et al., 2014). The animals were
housed under control temperature (25±20C) and photoperiodic conditions (12 hr light:dark cycle)
with food and water ad libitum (Anand et al., 2014). With prior approval from Institutional
Animal Ethical Committee (IAEC), experiments were conducted under strict compliance of the
guidelines issued by the Committee for the purpose of Control and Supervision of Experiments
on Animals (CPCSEA), India. EJE, Cisplatin (Dabur, India) and NAC (Amresco, Ohio, USA)
were procured and administered to animals either alone or in combination as described below
(Anand et al., 2014). The animals were housed with proper husbandry conditions and monitored
every day (Anand et al., 2014).
Chapter 6: Experimental Chapter 3
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Gr 1: Saline (i.p)/alternate day
Gr 2: CIS (5 mg/kg b.w, i.p) single injection on day 1
Gr 3: EJE (25 mg/kg b.w, i.m)/alternate day
Gr 4: CIS (5 mg/kg b.w, i.p) single injection on day 1 + EJE (25 mg/kg
b.w i.m.)/alternate day
Gr 5: NAC (150 mg/kg b.w, i.p) twice a week (day 1 and 4)
Gr 6: CIS (5 mg/kg b.w, i.p) single injection on day 1 + NAC (150 mg/kg
b.w, i.p) twice a week (day 1 and 4)
While one testis from each animal was immediately fixed in buffered formalin, the other testis
was snap frozen in liquid nitrogen and stored at -80°C till used for biochemical and molecular
analysis (Anand et al., 2014). Under mild anaesthesia, blood was collected from the tail vein of
rats on the day of sacrifice; serum was separated and stored at -20°C till assayed for hormones
(Anand et al., 2014).”
3.3 Material and Methods
“The details of the material and protocols used for the experiments have been listed in the
preceding chapter, Chapter 2.”
3.4 Results
3.4.1 Effect on Seminiferous epithelium and hormone levels
“One time cisplatin treatment showed very little effect on the seminiferous epithelium and
spermatogenesis in the histology preparation by the end of 7 days (Fig.1, Anand et al., 2014).
However, significant alterations in the serum reproductive hormone levels were observed. Rise
in follicle stimulating hormone (FSH) was associated with a significant decline in luteinizing
hormone (LH) and testosterone levels (Anand et al., 2014). EJE rather than NAC
supplementation to the CIS treated rats was more effective in restoring the hormones to near
normal levels (Anand et al., 2014). In contrast, NAC supplementation to CIS treated rats
stimulated a rise in FSH but attenuated both LH and testosterone levels (Fig.2A, B, C) (Anand et
al., 2014). While EJE or NAC, given alone, had no significant effect on serum FSH levels, the
inhibitive effect of NAC on serum LH and testosterone was evident in the backdrop of
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significant reduction of both hormones in CIS+NAC as compared with control levels (Fig.2A, B,
C) (Anand et al., 2014).”
3.4.2 Evaluation of the presence of functional Leydig cells
“Evaluation on the status of functional Leydig cells present in the testis was carried out through
immunolocalization using 3β-hydroxysteroid dehydrogenase (3β-HSD) antibody (Fig.3A) and
also through analysis of gene expression specifically for 3β-HSD and steroidogenic acute
regulatory protein (StAR) (Anand et al., 2014). One time cisplatin treatment significantly
depleted the numbers of functional Leydig cells (Fig.3B) which were restored only after EJE
(Fig.3D) or NAC (Fig.3F) supplementation (Anand et al., 2014). EJE (Fig.3C) or NAC (Fig.3E)
alone demonstrated no significant beneficial effect as the number of functional Leydig cells was
comparable with that of the controls (Fig.3A) (Anand et al., 2014). 3β-HSD, protein (Fig.3G)
and transcript (Fig.3H) levels followed an identical trend with attenuation of expression due to
cisplatin treatment (Anand et al., 2014). Restoration back to normal levels was observed either
with EJE or NAC intervention. While StAR transcripts are favorably upregulated (Fig.3H), its
protein expression (Fig.3G), on the other hand, was comparatively unaffected following EJE or
NAC intervention to CIS treated rats (Tables 3.1, 3.2) (Anand et al., 2014).”
3.4.3 Redox status of testis
“Redox status in the testis was evaluated simultaneously by measuring malondialdehyde, total
glutathione and antioxidant capacity, hemeoxygenase-1 (HO-1) concentration and activities of
antioxidant enzymes (Anand et al., 2014). Expression analysis of few of the antioxidant enzyme
was also studied. A rise in lipid peroxidation following cisplatin treatment coincided with the
increased formation of thiobarbituric acid reactive substances (TBARS, Fig.4A) or HO-1
(Fig.4B) in the testis (Anand et al., 2014). Intervention with either EJE or NAC was able to
contain the rise and to bring it down to control levels (Anand et al., 2014). Cisplatin, on the other
hand, significantly depleted the total glutathione (Fig.5A) and antioxidant capacity (Fig.5B) in
the testis and both parameters were restored after supplementation with either EJE or NAC
(Anand et al., 2014).
The activities of all the antioxidant enzymes, superoxide dismutase (SOD, Fig.6A), catalase
(Fig.6B), glutathione-s-transferase (GST, Fig.7A), glutathione reductase (GR, Fig.7B) and
glutathione peroxidase (GPx, Fig.7C) demonstrated a significant decline in the testis of cisplatin
Chapter 6: Experimental Chapter 3
119
treated rats compared to vehicle treated controls (Anand et al., 2014). Complete restoration in
enzyme activities was seen only when EJE or NAC was supplemented to CIS treated rats
(Anand et al., 2014). The efficiency of restoration was not identical when expression analysis of
specific antioxidant enzyme genes was compared with respect to EJE versus NAC. Catalase
protein and transcript levels were attenuated after CIS treatment but found restored after NAC
supplementation. EJE, in contrast, was less effective in the restoration of catalase gene
expression as seen both in western blot and PCR analysis (Fig.6C, D). Besides catalase,
expression analysis of all other antioxidant enzyme genes such as MnSOD, Cu/ZnSOD (Fig. 6C,
D) and GR (Fig. 7D, E), demonstrated a comparable pattern of restoration either with EJE or
NAC supplementation. GST protein expression in western blots too revealed a very much similar
trend (Fig.7 D) (Anand et al., 2014).”
3.4.4 Apoptotic induction of germ and Leydig cells
“The seminiferous epithelium depicted significantly more TdT-mediated
deoxyuridinetriphosphate nick end-labeling (TUNEL) positive germ cells from the CIS treated
group (Fig.8B) compared to vehicle treated controls (Fig.8A) (Anand et al., 2014). This was
further supported from the data on apoptotic index (Fig.8G) representing each category of
treatment (Anand et al., 2014). No apparent increase in germ cell apoptosis was seen with either
EJE (Fig.8C) or NAC (Fig.8E) given alone (Anand et al., 2014). The rise in germ cell apoptosis
in CIS treated rats was significantly brought down following either EJE (Fig.8D, G) or NAC
(Fig.8F,G) supplementation (Anand et al., 2014).
Considering the fact that there was a significant reduction both in serum testosterone and the
number of functional Leydig cells after CIS treatment, a fluorescent based kit was further
utilized to confirm TUNEL positivity, if any, among interstitial cells. Besides germ cells,
TUNEL positive cells were also resolved in the interstitium much more in number in CIS treated
(Fig.9B) compared to other, vehicle treated control (Fig.9A), EJE (Fig.9C) or NAC (Fig.9E)
alone, CIS+EJE (Fig.9D) and CIS+NAC (Fig.9F) groups (Anand et al., 2014). In order to make
a quantitative analysis of the extent of apoptotic induction in Leydig cells, the cells were isolated
and evaluated for TUNEL positivity in vitro. Accordingly, a large percentage (~18%) of Leydig
cells were found TUNEL positive in the CIS (Fig.10B, G) treated as compared to control group
(Fig.10A, G). EJE (Fig.10D, G) or NAC (Fig.10F, G) intervention to CIS treated rats
significantly (p<0.001) attenuated the number of TUNEL positive Leydig cells. Given alone,
Chapter 6: Experimental Chapter 3
120
EJE (Fig.10C, G) or NAC (Fig.10E, G) had no significant effect on Leydig cell apoptosis
(Anand et al., 2014).”
3.4.5 Leydig cell function in vitro
“Leydig cell function was assessed through hCG-induced testosterone production in vitro
(Fig.11) (Anand et al., 2014). Steroidogenesis leading to testosterone production in Leydig cells
was seen severely curtailed (~50%) in CIS as compared to the vehicle treated control group.
Individual treatments either with EJE or NAC showed little effect on the hCG-induced
testosterone production (Anand et al., 2014). On the other hand, testosterone production
improved in CIS treated rats receiving EJE (69.5% compared to CIS group) or NAC (60.5%
compared to CIS group) supplementation (Anand et al., 2014).”
3.4.6 Expression analysis of apoptotic markers
“Both upstream and downstream markers of extrinsic, intrinsic and other pathways of metazoan
apoptosis were analyzed using testicular tissue. Caspase 3 activity (Fig.12A) and protein
expression in western blots were seen identically raised following CIS treatment but brought
down to near control levels after EJE or NAC intervention (Anand et al., 2014). The expression
of the cleaved form of DNA repair enzyme, poly ADP ribose polymerase (PARP), demonstrated
a very much identical trend (Fig.12B) (Anand et al., 2014). Extrinsic markers, caspase 8 activity
(Fig.13A) and expression analysis of protein (Fig.13B) and transcript (Fig.13C) levels for
caspase 8, Fas and FasL, all demonstrated a beneficial modulation countering the rise in the
activity and expression of these markers when EJE or NAC was intervened after CIS treatement.
Intrinsic markers, caspase 9 activity (Fig.14A) and expression of Bad (Fig.14B), caspase 9
(Fig.14B, C) and Bax (Fig.14B, C) too displayed a similar trend. Bcl-2, the anti-apoptotic
marker, down regulated (Fig.14B, C) after CIS treatment, restored back to control levels after
EJE or NAC intervention (Fig.14B, C) (Anand et al., 2014).
Positive regulation of cisplatin effect by EJE or NAC was also seen with respect to analysis of
markers in other pathways of apoptosis. Anti-apoptotic Akt and pAkt (Fig.14D), down regulated
as a result of CIS treatment recovered after EJE ( Akt, 31.6%, pAkt, 38%) or NAC (Akt/pAkt,
26.5%) supplementation (Table 3.1) (Anand et al., 2014). NFkB which acted as a pro-apoptotic
factor and up-regulated with cisplatin treatment, was countered and brought down by EJE but not
significantly by NAC supplementation (Fig.14D, Table 3.1) (Anand et al., 2014). No significant
Chapter 6: Experimental Chapter 3
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change was observed for JNK or p-JNK either with cisplatin or any other treatments (Fig.14E)
(Anand et al., 2014). The upstream marker c-Jun, in the JNK pathway, however, was over
expressed in CIS treated group but depleted to near control levels with EJE or NAC intervention
(Fig.14E) (Anand et al., 2014).”
3.5 Discussion
“The above findings substantiate that intervention with either antioxidant, NAC or plant extract,
EJE protects the damage induced by cisplatin in the adult testis. The adverse effect of cisplatin is
not only limited to germ cell apoptosis in the seminiferous epithelium but also extends to impact
Leydig cell survival and function in the interstitium too. The molecular mechanism of action
against cell death due to apoptosis is found mainly channeled through favorable modulation of
marker proteins in the pathways of apoptosis under the backdrop of augmentation of
antioxidative defense in the target tissue (Anand et al., 2014).
Testicular dysfunction is a common long-term sequel of cytotoxic chemotherapy used in
treatment of various types of malignancies. However, the degree of dysfunction is proportional
to duration, dose and the nature of the drug used. Cisplatin chemotherapy has been reported to
produce long-lasting adverse effects on spermatogenesis (Petersen et al., 1994, Howel and
Shalet, 2001). As a heavy metal coordination compound, cisplatin produces cross-links,
including the DNA cross-links that are presumably responsible for its antineoplastic effect
(Boekelheide, 2005). Large scale, short and long term studies in animals confirm identical effects
of cisplatin on spermatogenesis. Cisplatin at a dose of 7 mg/kg bw administered to rats for 5 and
50 days has been reported to affect spermatogenesis; severety being directly proportional to the
duration of treatment. The hypospermatogenesis induced after 5 days of CIS treatment is
supported equally by significant decrease in the testis weight (Atessahin et al., 2006). In the
present study, however, no significant change in the testis weight and spermatogenesis (data not
shown) is observed even after 7 days of one time treatment of the drug (5 mg/kg b.w).
Considering the relatively long spermatogenic cycle in the rat (~50days), histological evaluations
of testis against short-term interventions are unlikely to yield any distinguishable signal in this
regard. Still, the observed differential response of the drug on spermatogenesis may be attributed
to a comparatively lower dose of cisplatin, as utilized in the present study (Anand et al., 2014).
Alterations in the hormonal profile are also common in the individuals receiving cispaltin
treatment. Cisplatin-administered to non-seminomatous testicular cancer subjects depicts a rise
Chapter 6: Experimental Chapter 3
122
in gonadotropin but fall in serum testosterone levels (Strumberg et al., 2002). In animals too,
adult rats exposed to cisplatin (7-9 mg/kg b.w intravenously) show a significant attenuation in
serum and intratesticular testosterone 7 days after the exposure. The effect is reportedly
reversible following hCG intervention. Gonadotropin levels, both LH and FSH, however, remain
unaffected (Maines and Mayer, 1985, Maines et al., 1990). In the present study, while serum
testosterone and LH have declined, FSH is estimated significantly higher in the CIS treated
group compared to vehicle treated controls. This is for the first time that such a differential
response for gonadotropins was observed after CIS treatment and may represent a part of the
immediate homeostatic mechanism within the gonadal-pituitary loop for maintaining testicular
function close to normal levels, as presently observed (Anand et al., 2014).
Intervention studies to protect the testicular function from cisplatin-induced damage have earlier
been attempted. From herbal extracts (Amin et al., 2008, Azu etal., 2010, Yildrim et al., 2011) to
chemotherapy protecting compounds such as amifostine (Lirdi et al., 2008), to those with
antioxidant properties, like lycopene (Atessahin et al., 2006a) and melatonin (Atessahin et al.,
2006b) are all reportedly tried mostly in animal studies with variable efficacies but without
utilizing a known recognized antioxidant for comparison. In the present work, NAC, a
recognized antioxidant is taken as a case control and the plant extract EJE was simultaneously
intervened to examine the beneficial effects in comparison with the known antioxidant. EJE has
been shown to possess strong antioxidant properties (Anand et al., 2013; Vasi and Austin, 2009).
It is interesting to note that in the restoration of normalcy for reproductive hormone levels, EJE
is found to be more effective than NAC. NAC supplementation to CIS treated rats fails not only
to bring down the FSH but also to restore the declining LH and testosterone anywhere near to
control levels (Fig.2) (Anand et al., 2014).
The cisplatin-induced decline in serum testosterone is reported to be associated with a decreased
number of LH receptors on Leydig cells in which p450 side-chain cleavage activity has also been
found inhibited (Maines et al., 1990). But, falling testosterone levels may not be only due to
inhibition of Leydig cell function but may also be due to a loss in the number of functional
Leydig cells in situ. This aspect is investigated for the first time in the present study utilizing 3β-
HSD antibody as the marker for identifying functional Leydig cells. CIS administration is seen to
severely deplete the number of functional Leydig cells which are restored to near control levels
only after either EJE or NAC supplementation (Fig.3A-F). That cisplatin primarily effect Leydig
cell function is further supported by the fact that the 3β-HSD gene expression (protein and
Chapter 6: Experimental Chapter 3
123
transcript levels) is found significantly downregulated but restored only after antioxidant NAC or
EJE intervention (Fig.3G,H). The CIS-induced attenuation in Leydig cell function is further
confirmed from in vitro studies in which isolated Leydig cells are challenged with hCG and later
assayed for efficiency in the form of testosterone released into the medium. EJE administration
to CIS rats is seen to provide a better protection from cisplatin stimulating a higher (~10% more
than NAC) testosterone production (Fig.11). Despite the restoration in the number of functional
Leydig cells, the failure of NAC restoring serum testosterone in the CIS+NAC group (Fig.2C)
may be due to the relative insensitivity of NAC to serum LH which continues to show no
improvement all through (Anand et al., 2014).
Alteration in the redox status in the testicular cells has been consistent with CIS treatment
(Kandemir et al., 2011, Salem et al., 2012). In the present study, it is extensively evaluated by
biochemical measurement of HO-1 protein, TBARS levels, total antioxidant capacity (TAC) and
total glutathione levels in conjunction with evaluation of activity and expression of antioxidant
enzymes. The effect of EJE in restoring the levels of HO-1 protein, TBARS levels (Fig. 4A) and
TAC (Fig. 5B) in the CIS treated rats is comparable with that of NAC supplementation.
Improvement in the activities of all the antioxidant enzymes analysed in the present study
followed an identical trend (Fig.6 and 7). However, total glutathione, fully recovered to control
levels by EJE, is seen almost doubled with NAC intervention (Fig.5A). Such drastic elevation in
glutathione levels is never beneficial and may be considered as one such adverse effects
associated with NAC treatment (van Klaveren, et al., 1997). In the system presently utilized,
NAC-induced high glutathione manifestation resulting either through toxic effects of cysteine or
through other mechanism/s needs further confirmation in future studies. Analysis of catalase
gene expression, however, reveals an opposite trend as NAC supplementation to CIS treated
animals shows superior restoration in protein and transcript levels than EJE (Fig.6C, D) (Anand
et al., 2014).
Strengthening the antioxidant defense is instrumental for augmentation of cell survival both
within the seminiferous tubule and intertubular regions. While cisplatin-induced germ cell
apoptosis has been reported before (Zhang et al., 2001, Seamen et al., 2003), simultaneous
apoptotic induction in the germ (Figs.8,9) along with Leydig cells (Fig.9) is revealed for the first
time in the present study. The findings are further confirmed with the identification of TUNEL
positive cells in vitro (Fig.10) from CIS-treated rats using the streptavidin-horseradish
peroxidase (HRP)-TUNEL assay which, somehow, has not been sensitive enough to detect the
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same signal in histological sections (Fig.8). Intervention with either EJE or NAC to CIS-treated
rats has successfully countered the rise in the number of germ (Fig.8G) or Leydig cell (Fig.10G)
apoptosis (Anand et al., 2014).
The dynamics of apoptotic induction are primarily regulated by activities and expressions of
marker proteins associated with the pathway of metazoan apoptosis. It is observed that down-
stream apoptotic markers, caspase 3 (activity and expression) and PARP are significantly
upregulated after CIS treatment. But, intervention with EJE or NAC has helped to counter the
rise and bringing it down to control levels (Fig.12A, B). Identical modulation is seen with respect
upstream markers of extrinsic (casapase 8, Fas and FasL, Fig.13) and intrinsic (Caspase 9, Bcl-2,
Bax and Bad, Fig.14A-C) pathway of apoptosis (Anand et al., 2014).
Analyzing the markers for other pathways of apoptosis, again for the first time, it is seen that Akt
and pAkt, being antiapoptotic, are down regulated after CIS treatment but reversed after EJE or
NAC supplementation (Fig.14D). In the present system, cisplatin is seen inducing
overexpression of NFkB which probably acts as an apoptotic inducer but later downregulated by
EJE or NAC supplementation (Fig.14D). The observation is consistent with findings earlier
reported (Kim et al., 2006). NFkB gets activated by different stimuli and appears to be mediated
through the production of reactive oxygen species (ROS). NFkB exists in the cytosol as a pre-
formed trimeric complex. The P50/P65 protein dimer is associated with an inhibitory protein
known as I-kB. Oxidants trigger a change in the cell that results in phosphorylation of the I-kB
subunit. After I-kB is phosphorylated, a process of the proteolytic digestion of this subunit is
activated. When the inhibitor subunit is dislodged from the P60/P65 heterodimer the activator
NFkB can migrate to the nucleus and bind to DNA, thereby initiating transcription (Blackwell
and Christman, 1997, Finkel, 1998) (Anand et al., 2014).
The tyrosine kinase c-Abl plays an important role in stress response to DNA damaging agents. It
belongs to the nonreceptor tyrosine kinases and contains nuclear localization motifs and nuclear
export signals shuttling between the nucleus and cytoplasm. Nuclear import of c-Abl was shown
to be necessary for DNA damage-induced apoptosis (Preyer et al., 2007). It is activated in
response to cisplatin causing activation of c-Jun-N-terminal kinase (JNK)/stress-activated protein
kinase (SAPK) (Kharbanda et al., 1995). Though in the present system, no significant change in
JNK expression is observed following CIS treatment, it does activate upstream marker protein c-
Jun which is, however, favorably modulated by antioxidant NAC or EJE supplementation
(Fig.14E) (Anand et al., 2014).
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125
In conclusion, this study clearly indicates that CIS-mediated adverse effects in the testicular
function and cell survival are countered both by the antioxidant NAC or EJE possessing similar
properties by augmenting antioxidant defense and favorably modulating molecular pathways
associated with steroidogenesis and cell death by apoptosis. On the basis of the information
obtained, a model representing the mechanism of NAC versus EJE action ameliorating the
adverse effects of cisplatin on rat testis is proposed (Fig.15). The therapeutic application of the
present findings however need further elucidation in future clinical studies (Anand et al., 2014).”
Chapter 6: Experimental Chapter 3
126
“Fig. 1. Representative testis sections stained with hematoxylin and eosin after (A) saline,
(B) CIS alone one time (single inj. 5mg/kg b.wt.), (C) EJE alone (25 mg/kg b.w), (D) CIS+EJE,
(E) NAC alone (150 mg/kg b.w), and (F) CIS+NAC. No significant change is observed in any of
the treated sections. X400
B C
D E F
A
50 μm 50 μm
50 μm 50 μm
50 μm
50 μm
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127
Fig. 2. Serum levels of FSH (A), LH (B) and testosterone (C) on Day 8th of one time (single inj.
5mg/kg b.wt.) cisplatin (CIS) treatment with or without EJE (25 mg/kg b.w)/NAC (150 mg/kg b.w)
supplementation.
* **
##
A
*
*** ***
***
B
** ** *
#
C
Control CIS EJE CIS+EJE NAC CIS+NAC
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128
Fig. 3. Detection of the presenc
immunolocalization of 3β-HSD in representative testicular sections from (A) vehicle control, (B)
CIS, (C) EJE alone, (D) CIS+EJE, (E) NAC alone and (F) CIS+NAC. x 400.
B C
D E F
A
50 μm 50 μm 50 μm
50 μm 50 μm 50 μm
3β-HSD
StAR
β-Actin
Cisplatin - + - + - + - + - + - +
EJE - - + + - - - - + + - -
NAC - - - - + + - - - - + +
547 bp
500 bp
539 bp
42 kDa
30 kDa
42 kDa
G H
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Fig. 4. Evaluation of redox status in testes of CIS treated rats with or without EJE/NAC
supplementation.
***
### ###
A
***
## #
B
** ###
###
** ###
Chapter 6: Experimental Chapter 3
130
Fig. 5. Evaluation of redox status in testes of CIS treated rats with or without EJE/NAC
supplementation.
A
**
# ##
B
***
### ###
### ###
** ###
** ###
Chapter 6: Experimental Chapter 3
131
Fig. 6. Assessment of activities and expression of antioxidant enzymes in CIS treated rats with or
without EJE/NAC supplementation.
A
***
### ### ###
###
B
***
*
###
** ###
###
Cu/ZnSOD
MnSOD
Catalase
β-Actin
CIS - + - + - + - + - + - +
EJE - - + + - - - - + + - -
NAC - - - - + + - - - - + +
C D
277bp
326 bp
970 bp
539 bp
23 kDa
25 kDa
64 kDa
42 kDa
*** ###
Chapter 6: Experimental Chapter 3
132
Fig. 7. Assessment of activities and expression of antioxidant enzymes in CIS treated rats with or
without EJE/NAC supplementation.
A
***
### ### $$
### $$
*** ### $$$
B
***
### ###
## ##
C
**
## ## # #
GST
GR
β-Actin CIS - + - + - + - + - + - +
EJE - - + + - - - - + + - -
NAC - - - - + + - - - - + +
D E
296 bp
539 bp
28 kDa
65 kDa
42 kDa
Chapter 6: Experimental Chapter 3
133
Fig. 8. Analysis of TUNEL positive cells () in testicular sections of rats treated with (A)
saline, (B) CIS, (C) EJE alone, (D) CIS+EJE, (E) NAC alone and (F) CIS+NAC. x 400
B C
D E F
A
50 μm
50 μm
50 μm
50 μm
50 μm
50 μm
***
### ### ###
###
G
Chapter 6: Experimental Chapter 3
134
Fig. 9. Fluorescein based detection of TUNEL positive cells in the seminiferous tubule () and
interstitium ().
A B C
E F D
50
50 μm 50 μm 50 μm
50 μm 50 μm
Chapter 6: Experimental Chapter 3
135
Fig. 10. Assay of Leydig cell TUNEL positivity in vitro. Isolated Leydig cells after 7 days of (A)
vehicle control, (B) CIS alone, (C) EJE alone (D) CIS+EJE, (E) NAC alone and (F) CIS+NAC
treatment.
B A C
D E F
50 μm 50 μm 50 μm
50 μm 50 μm 50 μm
G
***
### ### ###
###
Chapter 6: Experimental Chapter 3
136
Fig. 11. The hCG induced testosterone production from Leydig cells in vitro is stimulated in
CIS+EJE (~60%) versus CIS+NAC (~43%) group.
**
*
*
##
##
Chapter 6: Experimental Chapter 3
137
Fig. 12. (A) Rise in the activity and (B) expression of caspase-3 and cleaved PARP in CIS
treated group is brought down by EJE or NAC supplementation.
***
###
###
### ###
A
Caspase 3
PARP
β-Actin
Cisplatin - + - + - +
EJE - - + + - -
NAC - - - - + +
B 35 kDa
17 kDa
116 kDa
85 kDa
42 kDa
Chapter 6: Experimental Chapter 3
138
Fig. 13. EJE or NAC modulation of extrinsic pathway of apoptosis.
***
### ###
### ###
A
Cisplatin - + - + - + - + - + - +
EJE - - + + - - - - + + - -
NAC - - - - + + - - - - + +
Caspase 8
Fas
Fas L
β-Actin
B C
48 kDa
48 kDa
40 kDa
42 kDa
123 bp
351 bp
238 bp
539 bp
Chapter 6: Experimental Chapter 3
139
Fig. 14. EJE or NAC modulation of intrinsic and other pathways of apoptosis.
***
### ### ### ###
A
Cisplatin - + - + - + - + - + - +
EJE - - + + - - - - + + - -
NAC - - - - + + - - - - + +
Bad
Caspase 9
Bcl-2
Bax
β-Actin
B C
132 bp
293 bp
483 bp
539 bp
25 kDa
46 kDa
35 kDa
27 kDa
23 kDa
42 kDa
Akt
pAkt
NFkB
β-Actin
Cisplatin - + - + - +
EJE - - + + - -
NAC - - - - + +
D
62 kDa
60 kDa
65 kDa42
kDa
E JNK
pJNK
c-Jun
β-Actin
Cisplatin - + - + - +
EJE - - + + - -
NAC - - - - + +
54 kDa
46 kDa
54 kDa
46 kDa
39 kDa
42 kDa
Chapter 6: Experimental Chapter 3
140
Fig. 15. A proposed model depicting the mechanism of NAC versus EJE action ameliorating the
adverse effects of cisplatin on rat testis. EJE modulation of NFκB has been shown (----).
Upregulation (), downregulation () and inhibition ( | ) are indicated.”
Chapter 6: Experimental Chapter 3
141
Table 3.1: Densitometric analysis showing ratio of different protein expression to β-Actin ±
SEM.
Proteins
Protein/β-Actin Ratio
Control
Treatment
Cisplatin (5mg/kg
b.wt)
EJE (25mg/kg
b.wt)
Cisplatin (5mg/kg
b.wt) + EJE (25mg/kg
b.wt)
NAC (150 mg/kg b.wt)
Cisplatin (5mg/kg
b.wt)+ NAC (150mg/kg
b.wt) 3β-HSD 0.85+0.0088
0.7134 + 0.009*
0.9453 + 0.042 ###
0.951 + 0.031 ###
0.854 + 0.024 #
0.921 + 0.003 ###
StAR 0.9421+0.009
0.8826+ 0.006
1.0334+ 0.047
0.9696+ 0.065 0.9907+ 0.006 0.9256+ 0.003
Cu/Zn SOD
0.8209+0.008
0.5678+ 0.004 ***
0.7392+ 0.033 #
0.9061+ 0.061 ###
0.7755+ 0.004 ##
0.7774+ 0.002 ##
Mn SOD 0.9224+0.008
0.7887+ 0.005
1.0704+ 0.048 ##
1.0725+ 0.072 ##
0.9767+ 0.006 #
0.9454+ 0.003
Catalase 0.9002+0.008
0.6532+ 0.024 ***
0.8214+ 0.037 #
0.8214+ 0.055 #
0.7393+ 0.004 *
0.836+ 0.003 ##
GST 0.4582+0.004
0.3706+ 0.003 ***
0.4352+ 0.020 ##
0.4727+ 0.001 ###
0.5676+ 0.003 ***, ###
0.374+ 0.001 ***
GR 0.8102+0.006
0.7457+ 0.014
0.9422+ 0.008 **, ###
0.9783+ 0.033 **, ###
0.8549+ 0.031 0.8362+ 0.017 #
Caspase 3 0.7670+ 0.007
0.9148+ 0.006 **
0.7336+ 0.006 ###
0.7090+ 0.047 ###
0.656+ 0.004 *, ###
0.584+ 0.002 ***, ###
PARP 0.8421+ 0.021
0.9422+ 0.007 ***
0.8704+ 0.013 ##
0.8612+ 0.001 ##
0.7431+ 0.004 ***, ###
0.8008+ 0.002 ###
Caspase 8 0.6676+ 0.006
0.7778+ 0.005 *
0.7074+ 0.032
0.6542+ 0.044 #
0.6091+ 0.004 ##
0.6583+ 0.002 #
Fas 0.6568+ 0.007
0.8662+ 0.006 ***
0.6183+ 0.028 ###
0.6422+ 0.017 ###
0.6592+ 0.004 ###
0.8188+ 0.003 ***
FasL 0.9699+ 0.009
1.1857+ 0.008 **
0.8067+ 0.036 *, ###
0.8841+ 0.059 ###
0.9158+ 0.005 ###
0.8912+ 0.003 ###
Caspase 9 0.7883+ 0.008
0.9474+ 0.007 **
0.7909+ 0.009 ##
0.7996+ 0.054 ##
0.9863+ 0.006 ***
0.9145+ 0.003 *
Bcl-2 0.8292+ 0.008
0.6894+ 0.004 *
0.8819+ 0.009 ##
0.9419+ 0.063 ###
0.8623+ 0.005 ##
0.8322+ 0.003 #
Bax 0.73+ 0.007
0.918+ 0.006 ***
0.750+ 0.004 ###
0.7788+ 0.001 ***, ###
0.9870+ 0.006 ***, ###
0.8292+ 0.003 ***, ###
Bad 0.39+ 0.004
0.4560+ 0.003
0.4103+ 0.019
0.4571+ 0.031 0.4919+ 0.003 **
0.4248+ 0.002
Chapter 6: Experimental Chapter 3
142
Bak 0.6935+ 0.006
0.8138+ 0.006
0.7614+ 0.035 **
0.8688+ 0.058 ***
0.9335+ 0.005 0.7976+ 0.003
Akt 0.9644+ 0.009
0.7557+ 0.022 ***
1.0205+ 0.046 ###
0.9965+ 0.035 ###
0.9622+ 0.005 ##
0.96+ 0.003 ##
pAkt 1.1001+ 0.011
0.8689+ 0.006 *
1.1232+ 0.051 ##
1.1667+ 0.078 ##
1.0677+ 0.006 #
1.0681+ 0.003 #
NFκB 0.6848+ 0.006
0.8396+ 0.006 **
0.7450+ 0.034
0.7004+ 0.047 #
0.6997+ 0.004 #
0.8777+ 0.003 **
JNK 0.8647+ 0.008
0.8839+ 0.006
0.9654+ 0.044
0.9391+ 0.063 0.9095+ 0.005 0.9272+ 0.003
pJNK 0.6566+ 0.006
0.6168+ 0.004
0.4807+ 0.022
0.4728+ 0.032 0.5922+ 0.003 0.8181+ 0.002
c-Jun 0.3523+ 0.003
0.4868+ 0.003 ***
0.3677+ 0.017 ###
0.4164+ 0.028 *, #
0.4124+ 0.002 #
0.4414+ 0.001 **,#
*p<0.05, **p<0.01, ***p<0.001 compared to untreated control
#p<0.05, ##p<0.01, ###p<0.001 compared to Cisplatin treated sample
Chapter 6: Experimental Chapter 3
143
Table 3.2: Densitometric analysis of RT-PCR showing ratio of gene expression to β-Actin ±
SEM.
Genes
Target Gene/β-Actin Ratio
Control
Treatment
Cisplatin (5mg/kg
b.wt)
EJE (25mg/kg
b.wt)
Cisplatin (5mg/kg b.wt) +
EJE (25mg/kg
b.wt)
NAC (150 mg/kg b.wt)
Cisplatin (5mg/kg
b.wt)+ NAC
(150mg/kg b.wt)
3β-HSD 0.8985+ 0.012
0.6077+ 0.012***
0.9181+ 0.037###
0.8863+ 0.023###
0.9447+ 0.05###
0.8368+ 0.027###
StAR 0.5191+ 0.003
0.4156+ 0.004***
0.4671+ 0.017*, #
0.6038+ 0.015***,
###
0.5497+ 0.007###
0.5305+ 0.001###
Cu/Zn SOD 1.0822+ 0.018
0.6125+ 0.045***
1.2032+ 0.041###
1.0632+ 0.035###
1.1347+ 0.015###
1.1464+ 0.006###
Mn SOD 0.8082+
0.004 0.6347+
0.007*** 0.8112+
0.029### 0.7256+
0.018*, # 0.8381+
0.012###
0.8854+ 0.002*,
###
Catalase 0.7071+
0.003 0.385+ 0.004***
0.5994+ 0.021***,
###
0.4672+ 0.012***,
##
0.6781+ 0.009###
0.7215+ 0.001###
GPx 0.8826+ 0.004
0.6004+ 0.006***
0.6182+ 0.022***
0.6122+ 0.015***
0.5171+ 0.007***,
##
0.5744+ 0.001
GR 0.6056+ 0.004
0.4520+ 0.025***
0.6457+ 0.022###
0.6046+ 0.019###
0.5582+ 0.011###
0.5534+ 0.009###
Caspase 8 0.6214+ 0.003
1.0564+ 0.012***
0.8951+ 0.032***,
###
0.9707+ 0.003***,
#
0.9942+ 0.014***
0.6723+ 0.002###
Fas 0.6145+ 0.005
0.7982+ 0.024**
0.6606+ 0.024##
0.6711+ 0.031##
0.6213+ 0.012###
0.6394+ 0.002##
FasL 0.6315+ 0.015
0.8110+ 0.005**
0.6958+ 0.027#
0.6369+ 0.026##
0.5867+ 0.031###
0.6112+ 0.028###
Caspase 9 0.5778+ 0.018
0.9183+ 0.011***
0.5497+ 0.032###
0.5742+ 0.027###
0.4905+ 0.027*,
###
0.5522+ 0.025###
Bcl-2 0.7367+ 0.032
0.5482+ 0.009***
0.7700+ 0.026###
0.7839+ 0.019###
0.7845+ 0.024###
0.6945+ 0.002*, ##
Bax 0.6058+ 0.003
0.7354+ 0.008***
0.6344+ 0.023###
0.5983+ 0.015###
0.7211+ 0.010***
0.5551+ 0.001###
*p<0.05, **p<0.01, ***p<0.001 compared to untreated control
#p<0.05, ##p<0.01, ###p<0.001 compared to Cisplatin treated sample