Nanocurcumin Innovation as an Anti-Apoptosis of Ovarian Granulosa Cells in White Rats Exposed to Lead Acetate (PbAc)

Introduction: Exposure to Pb causes increased apoptosis of ovarian granulosa cells through oxidative stress mechanism. Curcumin has protective effects on reproductive organs, anti-apoptotic, antioxidant in normal cells. Curcumin in innovated nano form can function as an effective anti-apoptosis in ovarian granulosa cells of rats due to PbAc exposure. Methods: Thirty female rats were divided into 3 groups, the negative control group (the rats receiving distilled water, in each 90 minutes receiving corn oil), positive control group (the rats receiving PbAc of 30 mg/kg BW, in each 90 minutes receiving corn oil), the experimental group, in which the rats receiving PbAc of 30 mg/kg BW, and in each 90 minutes receiving nanocurcumin of 200 mg/kg BW. All groups received treatment orally once a day for 30 days. On day 31 the rats to granulosa cell apoptosis examination using Tunnel method. Results: Rate of apoptosis was in the positive control group (5.4 ± 0.8%/micro) and the lowest was in the experimental group (1.1 ± 0.5%/micro) and the negative control group (1.2 ± 0.6). The experimental group showed the same p value as the negative control group (p = .095) and different p value (p = .010) from the positive control group. These findings indicated that the innovation of curcumin in nano form at a dose of 200 mg/KgBW reduced apoptosis of rat ovarian granulosa cells due to PbAc exposure. Conclusion: The innovation of curcumin in nano form has the potential as an effective natural anti-apoptosis in rats ovarian granulosa cells exposed to PbAc.


INTRODUCTION
Exposure to lead acetate (Pb) causes increased apoptosis of ovarian granulosa cells through oxidative stress mechanism. The hydroxyl radicals (OH*), which are formed as a result of lead exposure, can translocate to ovarian granulosa cells and stimulate P53 production. P53 reacts with the mitochondrial membrane and activates pro-apoptosis (Bax) and causes a decrease in anti-apoptotic (Bcl-2 and Bcl-x) which causes the release of cytochrome c into the cytosol of granulosa cells. In the cytosol, cytochrome c binds to Apaf-1 (apoptosis-activating factor 1) to form a caspase recruitment domain (CARD) that stimulates caspase 9 in granulosa cells, and caspase 9 stimulates caspase-3, an effector that carries out granulosa cell apoptosis [1].
Curcumin, which is derived from the turmeric plant, is a compound that has potential as an antioxidant and antiinflammatory, able to inhibit the reduction of the risk of cancer and other malignancies. In doxorubicin to induce apoptosis of HL60 cells [3].  [6]. Therefore, innovation is needed to increase bioavailability, longer circulation and better permeability, so that in this study curcumin was formulated in the form of nanoparticles.
The purpose of nanocurcumin formation is to produce smaller, evenly distributed particles that have high bioavailability and stability [7] which are able to penetrate the target cell wall so as to prevent oxidative stress and apoptosis [8].

Chemical materials
The lead acetate used in this study had linear   Figure 2.

Preparation of nanocurcumin solution
Corn oil is the best solvent for nanocurcumin compared to butter, milk and water, so this study used corn oil as a solvent [9]. The solution was made by dissolving 2 g of nanocurcumin with corn oil to become 200 ml, so that 1 ml of the solution contained 10 mg of nanocurcumin.

Experimental animals
This study had passed the ethical test of the

Statistical analysis
Data were presented with mean ± standard deviation. The comparative test was carried out using Kruskall-Wallis Test to determine the differences between groups, followed by the Mann-Whitney test to determine the differences between the groups.

Characteristics of nanocurcumin size
The discrepancy between the results of the curcumin diameter analysis was possible because the software processing of the SEM images (500 x magnification) had limited sample area selection, so the particle size could not be accommodated as a whole.
Examination with SEM is basically an examination by analyzing the surface. The profile obtained was surface data with a thickness of about 20 μm from the surface. In particle analysis using PSA, the measured particle is a single particle.

Table 1
Innovation of nanocurcumin as anti-apoptotic granulosa cells in rat ovaries due to lead acetate exposure (mean ± standard deviation)

DISCUSSION
This study proved that exposure to lead acetate of 30 mg/kg BW in white rats increased ovarian granulosa cell apoptosis.
The mean expression of apoptosis in control group (C+) was higher than that in control group (C-) and in the experimental group.
This finding was in line with the study [14] that administration of intraperitoneal CARDs combine to form apoptosome complexes, then bind to pro-caspase 9 and activate it to become caspase 9 (initiator caspase). Caspase 9 will activate procaspase 3 into caspase 3 which is an effector caspase that carries out apoptosis [16].  [20]. The antioxidant activity of curcumin compounds takes place because the formation of free radicals is inhibited by the curcumin compounds by suppressing the activity of cytochrome p450 [21].
Ovarian granulosa cell apoptosis of white rats exposed to lead can be protected by and Apaf-1, called apoptosome, resulting in the prevention of caspase-9 stimulation. As a result, caspase-3 decreases and ends with decreased apoptosis [23].

CONCLUSION
The innovation of curcumin in nano form (nanocurcumin) can be used as an effective anti-apoptotic in white rat ovarian granulosa cells due to lead acetate exposure.

CONFLICT OF INTEREST
There is no conflict of interest in this research.

ACKNOWLEDGEMENT
The authors would like to thank the team of the Physics Laboratory Unit, Universitas Airlangga, Surabaya Indonesia, which has assisted in the manufacture of nanocurcumin.
Thanks are also conveyed to the team of the