Keywords: Oxidative stress, inflammation, polycystic ovary syndrome, Commiphoramukul, clomifene citrate, hyperandrogen
PCOS affected by many factors, but IR play important role in the manifestation of pathogenesis of this disease. A risk factor for developing diabetes, cardiovascular diseases, obesity, dyslipidemia are increasing in this syndrome [4].
Reactive oxygen species (ROS) and inflammation are important in the regulation of many physiological functions of female reproduction, such as development of oocyte, folliculogenesis, ovulation, and steroidognesis [5].
There is a vice-versa relationship occurring in between inflammation and ovarian steroid hormone [6]. In PCOS chronic low-grade inflammation markers such as hscrp, interleukin-18 (IL-18), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6) are elevated. Systemic inflammation occurs in obesity, but the level of circulating pro-inflammatory cytokines are also elevated in PCOS independent of obesity [7].
Glucose induced OS is found in mononuclear cell in polycystic ovary that leads to stimulation of the local inflammatory response which could induce more production of androgens by ovarian theca cells [8]. In some clinical study it was shown that ROS formations have a positive correlation with hyperandrogen production [9]. Insulin resistance and free fatty acid are also involved in production of osin body [10,11].
There are many modern drugs available for induction of ovulation and treatment of infertility in PCOS, such as Clomifene Citrate (CC). It is an anti-androgenic, non-steroidal estrogen agonist [12]. However, these modern medicines have some limitation such as long-term treatment and side effects so there is a need of using herbal supplement with no side effects for management of PCOS. Commiphora mukul (CM) belongs to family Burseraceae. Its gumresin contains steroid, sterol, alcohols and esters. The main active compounds found in CM are guggulusterones Z and E [13]. C.mukul useful in the treatment of obesity, liver dysfunction, leucoderma etc. Its gum has anti-inflammatory, anti-arthritic activity and also used in the treatment of menstrual dysfunction and ovulation disorders [14,15]. Here the present study based on to find positive effect of gum resin of CM on the management of PCOS in isolation and in combination with CC.
The estrous cycle of each rat was monitored by daily examination of vaginalsmear. The bodyweight of each rat was recorded at the start of the experiment and subsequently at 3daysintervaltillautopsy.Twenty-fourhours after the last treatment, animals were euthanized and the serum was kept at -20 0c for hscrp and test oster one assays. One ovary of each rat was fixed in bouin’s fluid for histological study, whereas the other was stored at-40 0c until used for biochemical and immune blot analysis.
Preparation of extract of C.mukul: Dried exudates of the bark of CM are known as “guggulu” in India. Gum-guggulu obtained from an ayurvedic pharmacy of BHU was air-dried. As guggul used as, an ayurvedic medicine from ancient time and its gum resin also have many clinical applications so we prepared water decoction of its gum resin. Plant doses were decided on the basis of toxicity experiment.
Parameters |
Control |
Only LTZ |
LTZ+CM |
LTZ+CC |
LTZ+CC+CM |
Body |
107.9±5 |
153.4±12.90 *** |
120.5±4.78*** |
133.41±6.45*** |
124.90±7.68*** |
Ovary |
0.055±0.0034 |
0.082±0.0035*** |
0.072±0.0049*** |
0.075±0.0037*** |
0.068±0.005*** |
Antral |
6.75±1.1 |
0 *** |
1.5±0.10 *** |
3.43±0.98 *** |
4.75±1.07*** |
Cystic |
0 |
8.5±1.67 *** |
5.1±1.28 *** |
4.25±1.12 *** |
4.18±1.89*** |
Follicular |
267.54±36.56 |
397.35±53.28 |
312.17±41.76*** |
298.11±21.66 |
274.11±36.25 |
Granulosa |
63.18±3.98 |
47.35±7.47* |
55.17±8.11* |
59.46±5.97 |
61.66±6.28 |
Thecalayer |
37.14±3.35 |
28.71±4.25* |
35.16±5.17 |
34.73±3.15 |
36.19±2.98 |
Vascularisation of follicular wall |
- |
+++ |
++ |
++ |
++ |
Disintegration and dispersion of granulose cell |
- |
+++ |
++ |
+ |
+ |
Histomorphometryof ovary: the ovaries fixed in bouin’s fix at ive were subsequently processed for histological study. Thin serial paraffin sections(5mm)of the ovary were stained with hematoxylin-eosin. Nikon microscope (Eclipse 50i, Japan) and Nikon imaging software examined follicular diameter, thickness of the granulosa and theca layers and changes in follicles. Different types (healthy antral, cystic, andatretic) off ollicles were counted in every fifth serial sections of each ovary. The numbers of healthy antral, atretic, and cystic follicle sat different duration of pcos development were compared with those in the control. Follicless howing features, such as hyper trophied granulosa cells, pyknotic cell nucleiin the granulosa cells andan abnormal oocyte were categorized as abnormal follicles. Histological changes were categorized and scored according to the severity of changes in the ovary: No changes= 0; mild = +; moderate = ++; and intense = +++.
The membrane was blocked with 3% BSA (Bovine Serum Albumin) in TBST (Tris buffer saline tween-20, for 4-5 hours at room temperature (RT). The blot was then incubated overnight at (40 C) with primary antibody IL-6 (1:1000) in TBST. Next day, this blot was extensively washed with TBST (5 washes, each of 5 min) and incubated for 2 hrs with secondary antibody at dilutions (1:1000) in TBST at RT. Proteins were detected by the enhanced chemiluminescence (ECL) system (Millipore India Pvt. LTD) in LAS500- imagequant (GE healthcare, hongkong) and the immune blot was quantified by Alpha imager. The blot was stripped and re-probed with housekeeping gene b-actin (1:1000) in a similar manner.
(A) Transverse section of ovary of control rat showing mature follicle with oocyte.
(B) Transverse section of ovary from rat treated with LTZ showing cystic follicles.
(C) Transverse section of ovary from rat treated with LTZ+CM showing cystic follicles.
(D)Transverse section of ovary from rat treated with LTZ+CC showing cystic follicles and Antral follicles.
(E) Transverse section of ovary from rat treated with LTZ+CC+CM showing Antral follicles. (Magnification X 10) AF- Antral follicles, CF- Cystic follicles, GC- Granulosa cell, TLTheca cell layer, O-Oocyte
LTZ- letrozole, CM- Commiphora mukul, CC- Clomifene citrate
Parameters |
SOD in ovary |
CAT in ovary |
LPO in ovary |
IL-6 |
Cystic follicle |
hsCRP |
-0.229 |
-.450* |
.774** |
.822** |
.934** |
Testosterone |
-.575** |
-.600** |
.697** |
.839** |
.716** |
SOD in blood |
.551* |
.702** |
-.714** |
-.873** |
-.794** |
Catalase in blood |
.583** |
.735** |
-.729** |
-.878** |
-.777** |
LPO in blood |
-0.339 |
-.489* |
.937** |
.856** |
.857** |
– No changes= 0 ; mild = + ; moderate = ++ ; and intense = +++
Parameters |
Normal |
Drug vector |
C.mukul treatment (mg/100gm body weight) |
||
20 |
40 |
80 |
|||
Glucose (mg %) |
63.8±2.9 |
60±4.0 |
61±7.3 |
68±4.5 |
65±5.2 |
Aspartate aminotransferase (AST) (IU/L) |
47.3± 2.6 |
45±3.3 |
42±3.8 |
35±2.3 |
44±4.1 |
Alanine aminotransferase (ALT) (IU/L) |
50±2.5 |
58±3.2 |
53.7±3.6 |
59.2±4.5 |
61±4.3 |
Triglyceride (mg%) |
74±3.8 |
72±3.6 |
70±4.5 |
65±3.8 |
69±6.1 |
Urea (mg%) |
43.8±2.2 |
40±2.6 |
45.5±1.8 |
39±3.2 |
43.7±2.9 |
Creatinine (mg%) |
0.63±0.03 |
0.44±0.013 |
0.37±0.02 |
0.40±0.03 |
0.49±0.02 |
*Correlation is significant at the 0.05 level (2-tailed).
Effect of CM treatment on ovarian expression of interleukin- 6 (IL-6) protein and high sensitive C-reactive protein (hscrp): The LTZtreated rats had increased serum level of hscrp as compared to control, whereas LTZ+CC+CM and LTZ+CM treatment significantly (p < 0.001) reduced level of hscrp in serum as compared to LTZ+CC treatment (Figure-4). Ovarian expression of IL- 6 was increased in LTZ treated group whereas it decreases both in LTZ+CM and LTZ+CC+CM treated groups (Figure-5). Effect of cm treatment on serum testosterone level: Serum testosterone level was significantly (p< 0.001) high in LTZ treated group as compared to control where as it became normal in LTZ+CC, LTZ+CM and LTZ+CC+CM treated groups (Figure-4).
The values are the mean± SEM of parameters measured. Difference between control and treated groups is significant :(*p< 0.001.)
LTZ- letrozole, CM- Commiphora mukul, CC- Clomifene citrate
In case of PCOS, number of atretic primordial follicles, atretic growing follicles, atretic graafian follicles, cystic follicles increases as compared to normal ovary so proliferation of granulosa cells increases but disintegration and dispersion of granulosa cell, decreases granulosa cell layer thickness around cystic follicles [24,25]. This is a characteristic feature of PCOS. As granulosa cells convert androgen to estradiol with the help of the aromatase enzyme. As already discussed that letrozole is a aromatase inhibitor, thus administration of letrozole, increased the concentration of androgen, which may be the cause of increased number of atretic follicles in ovary [26]. Detachment of granulosa cells is one of the morphological characteristic features of follicular atresia. It also showed complete an ovulation and significant increase in weight of the ovary. Formation of cystic follicles is positively correlated with hyperandrogenism, low-grade inflammatory marker and OS markers. Letrozole treated group showed elevated level of inflammatory marker in both serum (hscrp) and ovary (IL-6).There is a positive relationship between inflammatory markers (IL-6, hscrp) and hyperandrogenism. Our result is consistent with other studies in which level of TNF-a correlated with hyperandrogenism [25].
The values are the mean± SEM of parameters measured.
Difference between control and treated groups is significant :(*p< 0.001.)
LTZ- letrozole, CM- Commiphora mukul, CC- Clomifene citrate
Clomifene citrate (CC) is a modern drug used for ovulation induction. It is a non-steroidal compound and similar to estrogen. By blocking the estrogen receptor of hypothalamus this triggers the release of follicle steroid hormone (FSH) and luteinising hormone (LH) from the pituitary gland. Rats treated with CC shown reverse morphological features induced by LTZ. Its treatment significantly decreases the numbers of cystic follicles in ovary and increase numbers of healthy antral follicles in ovary. These rats also showed increases in thickness of granulosa and theca cell layer. Treatment of CC also reverses the serum testosterone level as compare to the LTZ treated group. But CC was not improved the low-grade inflammation and oxidative stress induced by LTZ administration. The level of serum hscrp and expression of IL-6 in the ovary was not showing significant difference in CC treated and LTZ treated group. Level of LPO product and activity of SOD and catalase were also not significantly differing in between CC treatment and LTZ treatment. Some studies also suggest that CC induces generation of ROS. Its treatment increases hydrogen peroxide (H2O2) formation and reduced catalase activity in the ovary [28]. Thus treatment of CC was not showing any effect on biochemical parameters. Herbal supplement (C.mukul)showed a positive effect on all above discussed parameters. C.mukul treatment reduces the adverse effect induced by LTZ and improved histological parameters, but ovarian histological features had much better improve in modern drug treated group. Its treatment also normalize the level of serum testosterone as obtained in the CC
The values are the mean ± SEM of parameters measured.
Difference between control and treated groups is significant :(*p< 0.001).
LTZ- letrozole, CM- Commiphora mukul, CC- Clomifene citrate
C.mukul has anti-inflammatory effect and also has antioxidant potential [13].The active compound found in the gum resin of CM isz-guggulsterone. It regulates the expression of different transcription factors such as NF-kb, STAT-3 and also regulates androgen receptor and glucocorticoid receptor [29]. The pathway of low-grade inflammation and oxidative stress are also linked by NF-kb. Z-Guggulsterone down-regulated the NF-kb which decreases the expression of Bcl-2, MMP-9, COX-2 and VEGF [30]. It inhibits NF-kb signalling pathway and release of proinflammatory cytokines (TNF-a, IL-6) [31]. Z-Guggulsterone also suppress lipid peroxidation and the formation of free radicals [32]. Some previous studies also showed that Z-guggulsterone decreases lipid peroxidation and increase the superoxide dismutase activity [33].
On giving combined treatment of CM and CC showed better histological features. Serum testosterone level was also found lowest in combined treatment of both herbal supplement and modern medicine. Markers of low grade inflammation and OS were also at the lowest level as compared to individual treatment of CC and CM.
The values are the mean ± SEM of parameters measured.
Difference between control and treated groups is significant: (*p< 0.001.)
LTZ- letrozole, CM- Commiphora mukul, CC- Clomifene citrate
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