Open Veterinary Journal, (2024), Vol. 14(11): 2860-2865

Research Article

10.5455/OVJ.2024.v14.i11.14

Effect of nano curcumin administration on IL-6 expression in placental organs and fetal weight exposed to stress during pregnancy in mice

Viski Fitri Hendrawan¹*, Yudit Oktanella¹, Habib Syaiful Arif Tuska¹ and Intan Firdha Olien Noor Al Ichsan²

¹Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya University, Malang, Indonesia

²Students of Bachelor’s Degree, Faculty of Veterinary Medicine, Universitas Brawijaya, Malang, Indonesia

*Corresponding Author: Viski Fitri Hendrawan. Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Brawijaya University, Malang, Indonesia. Email: viski [at] ub.ac.id

Submitted: 04/08/2024 Accepted: 05/10/2024 Published: 30/11/2024

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Abstract

Background: Stress can cause an increase in proinflammatory cytokines, IL-6, which plays a role in the inflammatory response and causes changes in the placenta, causing a low risk of the fetus being born. Giving nanocurcumin, which functions as an anti-inflammatory and antioxidant, is expected to reduce cortisol levels which increase during pregnancy.

Aim: This study aims to determine the effect of stress during pregnancy on pregnant mice, namely IL-6 expression and fetal body weight.

Methods: This study used 25 mice that were given 5 treatment groups, K(-) with no treatment, K(+) with an induced disturbance of 135 dB for 40 minutes with a continuous pattern, P1, P2, and P3 were given a disturbance of 135 dB for 40 minutes. With a continuous pattern and nanocurcumin doses of 14 mg/kg BW, 21 mg/kgBW, and 24.5 mg/kgBW. IL-6 expression in placental organs was measured using immunohistochemistry and fetal body weight was weighed using a digital balance. Data were analyzed using the Kruskal Wallis test and continued with the Parwise Comparison test.

Results: The results of analysis of IL-6 expression in the placenta showed no significant difference in the P3 group compared to the negative control group. The results of fetal weight analysis showed no significant difference in groups P2 and P3 with the negative control group.

Conclusion: The conclusion of this study is the administration of nanocurcumin in group P3 has been proven to increase IL-6 to a level comparable to the negative control group (p < 0.05). Similarly, the administration of nanocurcumin in groups P2 and P3 can increase fetal weight to a level comparable to the negative control group (p < 0.05)

Keywords: Stress, Cortisol, Nanocurcumin, Pregnant mice, IL-6.

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Introduction

As unwanted sounds, noise can cause health problems and discomfort for hearing (O’Donnell et al., 2023). High noise intensity of 90–105 dBA and occur continuously can cause elevated levels of cortisol in the blood of Mice. This is because the condition of mice experiencing stress due to noise that will affect the working system of the hypothalamus and endocrine glands will be disrupted. When noise-induced stress occurs, the hypothalamus will secrete Corticotropin Releasing Hormone to stimulate an increase in Adeno Corticotropin Hormone, cortisol, and cause the secretion of epinephrine (Chen et al., 2022).

Cytokines are responsible for regulating immune responses and interacting with the central nervous system (Meek et al., 2001). Cytokines can function to modulate the immune and inflammatory systems, whether they are pro-inflammatory or anti-inflammatory.

Proinflammatory cytokines include Interleukin-Interleukin-6 (IL-6) (Xu et al., 2023). Curcumin can work to induce Mitogen Activated Protein cinase. The function of MAP cinase is to block Nf-kB, which is a transcriptional factor to trigger cytokines, so that by blocking the Nf-kB pathway by MAP kinase, it will suppress cytokine production (Ghasemi et al., 2019).

Stress conditions during pregnancy can cause inhibition of fetal brain development, increased risk of prematurity, low birth weight, and neonatal abnormalities that can endanger the health of the fetus (Kusumarini et al., 2017). Curcumin supplementation can restore the condition when there is tissue damage and fetal weight loss induced by diet. In addition, curcumin administered during pregnancy was able to increase birth weight, and reduce inflammation and damage in newborn mice (Filardi et al., 2020).

Curcumin has low oral bioavailability and has low solubility. Currently, several dosage forms of curcumin have been developed enough to enhance the action of curcumin, among others, curcumin in the form of nanoparticles or curcumin in nano form. Nanoparticles have the advantage of being able to penetrate the spaces between cells and can increase the bioavailability of drugs that have low solubility (Khasanah and Husni 2016).

The mechanism of curcumin inhibits the production of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. These cytokines are associated with stress responses and inflammation. Curcumin can block the activation of NF-κB, a protein complex that controls the transcription of deoxyribonucleic acid (DNA) and plays a key role in inflammatory responses. By inhibiting NF-κB, curcumin reduces the expression of genes involved in inflammation. Curcumin suppresses the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase, enzymes that are involved in inflammatory processes (Di et al., 2020). Curcumin acts as a scavenger of free radicals, which are reactive molecules that can cause oxidative stress and damage to cells. By neutralizing these free radicals, curcumin reduces oxidative stress. Curcumin increases the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, which protect cells from oxidative damage (Lin et al., 2019).

By reducing inflammation and oxidative stress, curcumin can improve blood flow to the placenta, ensuring better delivery of nutrients and oxygen to the fetus. Curcumin’s anti-inflammatory effects can reduce placental inflammation, which is often linked to poor fetal growth. High-stress levels can increase cortisol, a hormone that can adversely affect fetal growth. Curcumin’s ability to reduce stress and inflammation may help normalize cortisol levels, promoting better fetal development. Curcumin may help maintain healthy progesterone levels, a hormone crucial for pregnancy maintenance and fetal growth. Curcumin supports gut health by reducing inflammation and promoting a healthy microbiome, which can enhance the mother’s nutrient absorption and availability to the fetus (Deb et al., 2020).

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Materials and Methods

This study was conducted in the Laboratory of the University of Airlangga and using a complete randomized design performed 5 repetitions each treatment. This study consisted of 5 treatment groups consisting of, control (−consisted of pregnant mice without any treatment. The control (+) consisted of pregnant mice given a noise of 135 dB for 40 minutes with a continue pattern. Treatment 1 (P1) consisted of pregnant mice given a noise of 135 dB for 40 minutes with a continue pattern and given nanocurcumin dose of 14 mg/kgBW. Treatment 2 (P2) pregnant mice were given a noise of 135 dB for 40 minutes with a continue pattern and given nanocurcumin dose of 21 mg/kgBW. Treatment 3 (P3) consisted of pregnant mice given a noise of 135 dB for 40 minutes with a continue pattern and given nanocurcumin dose of 24.5 mg/kgBW. The treatment was carried out on the 6th–18th day of pregnancy.

Preparation of pregnant mice

The research procedure begins by inducing female mice aged 3 months using the hormone Pregnant Mare Serum Gonodotropin (PMSG or Foligon) at a dose of 5 IU. Forty-eight hours later injected with the hormone Human Chorionic Gonadotropin and directly mated with male mice by polymatting. Seventeen hours after the female mice were mated, a vaginal plug examination was performed. Female mice with a positive vaginal plug were determined to be 0 days pregnant.

Immunohistochemical staining of the placenta

Immunohistochemical staining procedure can be done by histopathological preparation of placenta soaked in a solution of xylol 2 times, then the dehydration process of the preparation using alcohol stratified sequentially (96%, 90%, 80%, and 70%). Next, using a PBS solution with a pH of 7.4, the preparation is washed 3 times for 5 minutes each. Soaked preparate into 3% hydrogen peroxide (in distillate water) for 20 minutes, washed again using a solution of PBS with a pH of 7.4 as much as 3 times each for 5 minutes, then the preparation in soak into a solution of BSA for approximately 10–30 minutes at room temperature, and washed again using a solution of PBS with a pH of 7.4 as much as 3 times each for 5 minutes. Added antibody IL-6 for 1 hour at room temperature and incubated for 1 Night. After one night, the drainage is washed using a pH 7.4 PBS solution 3 times for 5 minutes each. Coloring is done using hematoxilin eosin for 5 minutes, and washed using aqueous solution 3 times each for 5 minutes. The mounting process was carried out using entellan, then observed using a microscope with magnification of 100 and 400x (Pan et al., 2023).

The weight of the fetus

Fetal data collection was done by weighing the fetus after the mice were euthanized, the fetus was weighed using a digital balance sheet that has a level of accuracy of 0.01 g. Fetuses that have external defects in the form of dwarfism can usually be seen if the fetus has an average weight that does not reach 2/3 part of the average normal fetal weight (Wuyts et al., 2021).

Statistical analysis

The parameters used in the study were the expression of Interleukin 6 (IL-6) and fetal weight. The data were analyzed quantitatively using the Statistical Package for the Social Science application with a 99% confidence level. IL-6 expression and fetal weight were analyzed using Kruskall-Wallis non-parametric test and continued using Parwise comparison test.

Ethical approval

This research license is obtained from the research ethics commission of Universitas Brawijaya (NO : 028-KEP-UB-2023).

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Results

Results of the effect of nanocurcumin administration on IL-6 expression in the placenta of pregnant mice under stress conditions

The results of Kruskall-Wallis test analysis of IL-6 expression showed significant differences in five treatment groups with a significance value of less than 0.01 (p < 0.01) which means that H0 is rejected and HI is accepted so that there is a significant difference between the expression of IL-6 (Rozi and Maulidiya 2022). Furthermore, a follow-up Parwise comparison test was observed to determine the significant differences between the treatment groups. The results of the Parwise comparison test on the expression of IL-6 showed that the administration of nanocurcumin had no significantly different effect on the expression of IL-6 in the P3 group compared to the K (−) group. Based on the results of the study, the expression of IL-6 in Group P3 administration of nanocurcumin dose of 24.5 mg/kgBW has the lowest average (25.04%) with a decrease of 3.3% from Group K(−) which has an average expression of IL-6 (25.92%).

Data on immunohistochemical results of IL-6 examination of the placenta after analysis are presented in Table 1.

Figure 1 shows the distribution of IL-6 expression in immunohistochemical staining techniques. The presence of IL-6 expression is visualized in a brownish color. The brownish color that is formed is the result of the bond between the antigen and the primary antibody in the form of secondary antibodies, namely Strep Avidin Horseradish Peroxidase and diamino benzidine substrate (Pan et al., 2023). The result of IL-6 antigen expression in mouse placental cells can be seen in Figure 1.

Results of the effect of nanocurcumin administration on fetal weight in pregnant mice under stress conditions

In this study, a fetal weight examination was carried out by weighing the fetus using a digital balance sheet. Each fetus was weighed to determine the average birth weight (Dillasamola et al., 2018). The data on the results of the examination of body weight after analysis are presented in Table 2.

The results of the Kruskall-Wallis test analysis of fetal body weight in Table 2 showed significant differences in five treatment groups with a significance value of less than 0.01 (p < 0.01). Furthermore, a follow-up test Parwise comparison Test was observed to determine the significant differences between the treatment groups.

The results of the Parwise Comaprasion Test in Table 2 showed significant differences between treatment groups. The lowest mean fetal weight in the sample P1 (nanocurcumin 14 mg/kgBw) with mean fetal weight 0.68% 0.31 g, where group K (−) (without treatment) had significant differences with group P1 and no significant difference with group P2 (21 mg/kgBW) with mean body weight 0.95% 0.23% and P3 (24.5 mg/kgBW) with mean body weight 1.03% 0.33%, might be attributed to insufficient dosage or duration of nanocurcumin administration. At lower doses or shorter treatment periods, nanocurcumin may not reach the therapeutic threshold required to produce noticeable anti-inflammatory and fetal growth-promoting effects. The results showed that the P3 Group had a fairly good result in influencing fetal weight given noise, because it had results close to negative control.

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Discussion

Expression of IL-6 in the placenta is expressed by cytotrophoblast cells and endothelial cells in response to inflammation (Konwar et al., 2019). When inflammation occurs, the body will secrete cytokines, resulting in an increase in cytokines. Interleukin 6 acts as a marker to determine the presence of infection or inflammation that occurs during pregnancy (Omere et al., 2020). Nanocurcumin has anti-inflammatory function by inhibiting NF-kB. NF-kB will attach to the wall of DNA and will form a binding complex between NF-kB DNA which will then regulate the release of cytokine Interleukin 6. Nanocurcumin can work by inhibiting the binding between NF-kB DNA, so that the inflammatory response can be inhibited (Aurellia et al., 2022).

Table 1. Results of examination of IL-6 expression in Placenta.

Fig. 1. Expression of IL-6 (orange arrow ↑) in placental organ preparations of pregnant mice (magnification 400x) with IHC staining, (K-), Control negative group; (K+), Control positive given 135 db noise administration for 40 minutes with continue pattern, (P1) 135 db given noise administration for 40 minutes with continue pattern and nanocurcumin dose of 14 mg/kgBW, (P2) given 135 db noise administration group for 40 minutes with continue pattern and nanocurcumin, and (P3) given 135 db noise administration for 40 minutes with continue pattern and nanocurcumin dosage of 24.5 mg/kgBW.

The nutritional and metabolic conditions of the mother are linearly related to the weight of the fetus. The placenta plays an important role in the nutrition of the fetus and the growth of the fetus because nutrients from circulation need to be transported across the placenta to cross the fetal circulation (Roland et al., 2014). Placental vascularization plays an important role in the organ’s capacity to provide optimal oxygen and nutrient supply to the fetus. Some pregnancy complications can affect the placental vessels (Kretschmer et al., 2020). Pregnant mothers who are exposed to excessive stress can experience changes in the secretion of excessive stress hormones that can trigger inflammation.

Table 2. Results of the parwise comparison test for fetal weight.

Decreased levels of IL-6 expression in the placenta and fetal weight response are thought to be due to the active compounds contained in nanocurcumin. It can be proven in previous studies that nanocurcumin has many biological activities such as controlling the inflammatory response by decreasing the activity of the enzyme COX-2 through inhibition of NF-kB (Karthikeyan et al., 2020). Nanourkumin contains a wide range of active substances such as saponins efficacious as antineoplastic (anticancer) while beta-carotene, polyphenols, and flavonoids function as antioxidants and anti-inflammatory (Anggraeni et al., 2020). In addition, nanokurkumin contains essential oils that help the performance of bile and pancreas so as to increase the absorption of food in the intestine and increase appetite (Prihandini et al., 2018). One way to increase growth is to optimize the functioning of the digestive tract. The availability of digestive enzymes affects the effectiveness of enzymes in digesting feed and will affect growth. One way to optimize digestive enzymes is by administering nanocurcumin (Damayanti et al., 2022).

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Conclusion

This study concluded that both administration of nanocurcumin to pregnant mice treated with 135 dB noise at a dose of 24.5 mg/kgBW showed a decrease in IL-6 expression compared to negative controls and administration of nanocurcumin to pregnant mice treated with 135 dB noise at a dose of 24.5 mg/kgBW showed a response to body weight compared to negative controls.

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Acknowledgments

The researcher would like to thank all the authors for their contribution in this journal.

Conflict of interest

The authors declare no conflict of interest.

Funding

This study was funded by internal funding of DPP SPP year 2024 Faculty of Veterinary Medicine, Brawijaya University.

Author’s contributions

VHF, YO, HSAT, and IFONAI drafted the manuscript. VFH and YO revised and edited the manuscripts. HSAT and IFONAI took part in preparing and critically checking the manuscript. VFH and IFONAI edited the references. All authors read and approved the final manuscript.

Data availability

All references are open access, so data can be obtained from the online web.

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