{"id":51287,"date":"2023-09-30T11:26:06","date_gmt":"2023-09-30T11:26:06","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=51287"},"modified":"2023-10-12T05:04:06","modified_gmt":"2023-10-12T05:04:06","slug":"blumea-balsamifera-leaf-extract-maintain-testosterone-levels-in-hypercholesterolemic-rats-through-antioxidant-mechanism-and-upregulation-of-star-gene-expression","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no3\/blumea-balsamifera-leaf-extract-maintain-testosterone-levels-in-hypercholesterolemic-rats-through-antioxidant-mechanism-and-upregulation-of-star-gene-expression\/","title":{"rendered":"Blumea balsamifera Leaf Extract Maintain Testosterone Levels in Hypercholesterolemic Rats Through Antioxidant Mechanism and Upregulation of StAR Gene Expression"},"content":{"rendered":"\n

Introduction\n<\/strong><\/p>\n\n\n\n

Hypercholesterolemic factors have an essential role in enhancing the creation of free radicals and causing the incorrect formation of lipid peroxide at the tissue level, which may contribute to oxidative stress1,2<\/sup>. Hypercholesterolemia causes an increase in the activity of NADPH oxidase, which in turn causes an increase in the production of superoxide anion, which is one of the reactive oxygen species (ROS) that causes oxidative stress. Hypercholesterolemia also raises blood pressure3<\/sup>. On the other hand, ROS are potentially toxic to cells in the reproductive system such as spermatogenic and Leydig cells4,5<\/sup>. A previous study in hypercholesterolemic rats found a significant reduction in plasma testosterone levels. This decline was caused by a disturbance in the hypothalamic-pituitary-testicular axis, Leydig cell deterioration, a decrease in Leydig cell nuclear diameter, or a decrease in LH levels and testicular activity of 17-hydroxysteroid dehydrogenase6,7<\/sup>. The same thing happened to rats fed a diet high in cholesterol for 50 days, and the total number of Leydig cells and spermatogonium A cells significantly decreased8,9<\/sup>.<\/p>\n\n\n\n

Malondialdehyde (MDA) is a metabolite with high\nreactivity with several studies showing its role in carcinogenesis, liver and kidney disease,\ndiabetes mellitus, cardiovascular, neurovascular, and various effects on cellular\nlevels 10<\/sup>. MDA levels can be used as a biomarker of changes in lipid\noxidation in tissues. On the other hand, MDA is not only produced in the\nprocess of lipid peroxidation but it is a common by-product of eicosanoid\nsynthesis in small amounts 11<\/sup>.<\/p>\n\n\n\n

In addition to the influence of MDA levels, the\ntestosterone that is produced by changing cholesterol through the process of\nsteroidogenesis initiated by the protein Steroidogenic Acute Regulatory Protein\n(StAR) has an important role in the functioning of the male reproductive system\n12<\/sup>,13<\/sup>. The steroidogenesis process can be blocked by\ninhibiting StAR protein function due to disturbance of the\nhypothalamic-pituitary-testicular axis, causing decreased LH production due to\naging and free radical damage14,15<\/sup>. Several experiments on mice\ngiven a high-fat diet showed a decrease in LH hormone secretion and decreased\nsteroidogenesis enzyme activity including StAR activity 16\u201318<\/sup>.<\/p>\n\n\n\n

Hypercholesterolemic medicines\nwere created and distributed in the market during the previous several decades;\nnevertheless, the adverse effects made these treatments unsuitable for\nlong-term usage. On the other hand, these side effects are the main cause of\nmore than 24 million deaths expected by 2030 19<\/sup>. A diet with the\ntendency to consume high-fat and high-cholesterol foods is at risk of causing\nhypercholesterolemia.<\/p>\n\n\n\n

The use of natural ingredients as\nantihypercholesterolemic agents such as herbal is highlighted 20<\/sup>. Blumea balsamifera<\/em>, often known as Sembung, is a plant having a\nhigh antioxidant capacity21<\/sup>. Balinese in Indonesia generally use B. balsamifera<\/em> leaves as a traditional\ndrink called “Loloh”. This traditional plant may reduce blood\ncholesterol levels because of its high antioxidant content and several main\nsecondary metabolites such as flavonoids, phenols, tannins, and alkaloids 21,22<\/sup>.\nAs antioxidants, flavonoid compounds found in sembung leaf extract are reported\nto work in cell membranes by capturing unsaturated fatty acid-free radicals and\nconverting them into hydroperoxy polyunsaturated fatty acids (PUFA-OOH) which\nare not free radicals 23<\/sup>. Ring B in flavonoid compounds has a\nhydroxy group that can\nproduce hydrogen so that it stabilizes free radicals 24<\/sup>.\nFlavonoid compounds in sembung leaf extract also act as anti-hypercholesterolemic\nby reducing body  LDL  and increasing the LDL receptor density in the\nliver and binding to apolipoprotein B 25<\/sup>.<\/p>\n\n\n\n

The effectiveness of B. balsamifera<\/em> leaf extract (BBLE) on the reproductive function of\nmale Wistar rats treated with a high-fat diet showed a significant increase in the diameter of\nthe seminiferous tubules, spermatogonium-A cells, pachytene spermatocytes, and\nspermatid-16 after administration of BBLE at a dose of 4 mg\/day for 50 days21<\/sup>.\nThis demonstrated the potential of BBLE to preserve the HPT axis in balance and\nminimize the\ndamaging impacts of free radicals caused by high-cholesterol diets. However,\nfurther investigation is needed regarding the impact of oxidative stress by\nhigh cholesterol diets and its prevention by direct BBLE on the testicular\ncells as a site for testosterone production and spermatogenesis.<\/strong><\/p>\n\n\n\n

Materials\nand methods<\/strong><\/p>\n\n\n\n

Ethical clearance<\/strong><\/p>\n\n\n\n

The Research Ethics Committee of the Faculty of Medicine, Udayana University (UNUD) \/ Central General Hospital (RSUP) Sanglah, Denpasar has permitted for this research with ethical approval with Protocol No. 2021.03.1.0276.<\/p>\n\n\n\n

Research\ndesign <\/strong><\/p>\n\n\n\n

This experimental research used a Randomized\nPosttest-Only Control Group Design. For 50 days, 36 adult male Wistar rats (Rattus norvegicus)<\/em> were randomly\ndivided into two groups: Control (HCD + sterile distilled water) and BBLE (HCD +\nBBLE 4 mg\/mL\/BW rats orally)20<\/sup>.<\/p>\n\n\n\n

Preparation\nof BBLE<\/strong><\/p>\n\n\n\n

Fresh B.\nbalsamifera<\/em> leaves of medium size and green were collected from plantations\nin Luwus Village, Tabanan that were previously determined as species at the National\nResearch and Innovation Agency (BRIN), Bali Botanical Garden, Tabanan, Bali. Using a grinding\nmachine, washed, dried, and crushed B\nbalsamifera<\/em> leaves. B. balsamifera<\/em>\nleaf powder was weighed \u00b1 250 and macerated in 70 % ethanol for 24 hours furthermore filtered. The\ncollected filtrate was then evaporated with a vacuum rotary evaporator with a\ntemperature of 50\u2103, speed of 80 rpm, and\npressure of 80 kPa to obtain crude extract. The crude extract standards were adjusted\nto the Indonesian Herbal Pharmacopeia II Edition 2017, namely yield not less\nthan 10.6%, the compound identity of flavonoids (+), water content not more\nthan 14%, and total ash not more than 6.7% 26<\/sup>.<\/p>\n\n\n\n

Rats models and treatment <\/strong><\/p>\n\n\n\n

The model animals were male Wistar rats that met the inclusion requirements (aged 12-14 weeks, body weight ranged from 150-200 grams). Wistar rats were randomly selected and acclimatized for 1 week. Rats were fed a regular diet that includes protein (20-25%), fat (5%), starch (45-50%), crude fiber (5%), ash (4%), and vitamins and minerals during the acclimation period. Furthermore, the high-cholesterol diet (HCD) contains a combination of 10% (100 gr) lard oil, 5% (50 gr) duck egg yolk, and up to 1,000 gr of standard feed. Ad libitum feeding of up to 20 gr\/day.<\/p>\n\n\n\n

The control group was given an HCD and distilled water. Whereas the BBLE group acquired up to 4 mg\/mL\/body weight per day of B. balsamifera<\/em> leaf extract, each rat was given 1 mL containing 4 mg of extract orally for 50 days16<\/sup>. After 50 days, the testicular organs were surgically taken for assay of tissue MDA levels, StAR mRNA expression, and Leydig cell number. While serum testosterone was examined through blood taken from the orbital sinus. <\/p>\n\n\n\n

Malondialdehyde\n(MDA)<\/strong><\/p>\n\n\n\n

Tissue MDA was measured using the QuantiChrom TBARS Assay Kit (BioAssay Systems, USA) (Cat. No. DTBA010). As much as 20 mg of mashed testicular tissue samples were taken and put into a test tube. Add 1 mL of chilled 20% TCA then vortexed and centrifuged at 3,500 rpm for 10 minutes. The supernatant was taken and put into another test tube that already contained 2 mL of 0.67% TBA. All tubes were put into the tube rack, then put in a water bath at 100\u00b0C for 10 minutes. After that, it was taken out and cooled in a vessel filled with ice water. The result of the reaction was taken as much as 1 ml and put in the cuvette. The absorbance of the sample was then measured with a spectrophotometer at the maximum wavelength (OD max = 532 nm). The number that appears on the screen was converted into a formula to get the MDA level (\u00b5mol\/mL). The TBARS concentration of the sample was  calculated using the following equation:<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

R sample and R blank were  OD 535 nm or fluorescence intensity values \u200b\u200bof sample and H2<\/sub>O blank (standard), n was the sample dilution factor (n = 3 for proteinate sample).<\/p>\n\n\n\n

StAR mRNA\nExpression<\/strong><\/p>\n\n\n\n

Examination of StAR\nmRNA expression was carried out in several stages including mRNA extraction\n(RNeasy Mini Kit Qiagen, USA), Reverse Transcriptase (RNA to cDNA), Primary\noptimization and Running Samples, and StAR gene Amplification.<\/p>\n\n\n\n

Testosterone\nlevels<\/strong><\/p>\n\n\n\n

Testosterone levels were measured using the Enzyme Linked\nImmunosorbent Assay (ELISA) with the Rat Testosterone BT-Lab Kit (BioAssay,\nUSA) (Cat.No. E0259Ra). Briefly, all reagents, samples, and standards were\nprepared. After adding the sample, and standard antigen, and biotinylated it,\nit was incubated for 60 minutes at 37\u00b0C before being aspirated and washed 5\ntimes before being incubated for 60 minutes at 37\u00b0C with avidin-HRP. After\nthat, aspirate and wash 5 times before adding substrate solution A and\nsubstrate solution B and incubating for 10 minutes at 37\u00b0C in dark conditions.\nAfter adding a stop solution, the OD value was measured at 450 nm in 15\nminutes.<\/p>\n\n\n\n

Histopathology of Testis<\/strong><\/p>\n\n\n\n

Rat testes were fixed in 10% formaldehyde solution and passed through a series of ethanol baths and then cleaned using toluene, and then embedded in paraffin. Sample tissue was sectioned at 5 \u00b5m and stained with Hematoxylin and Eosin (H&E). Sections were examined with a light microscope at magnifications of 100\u00d7 and 400\u00d7 26-30<\/sup>.<\/p>\n\n\n\n

Data analysis <\/strong><\/p>\n\n\n\n

MDA levels, StAR gene expression, testosterone hormone, and Leydig cell count were statistically analyzed using SPSS 22 for Windows software (IBM, USA). Independent T-Test and sequential path analysis were used to determine the effect of treatment between the control and BBLE groups as well as the direct and major effect of tissue MDA levels and StAR mRNA expression on Leydig cell number and testosterone secretion. Graph views were processed using GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA) 22<\/sup>.<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

MDA levels <\/strong><\/p>\n\n\n\n

Testicular MDA levels of\nhypercholesterolemic rats differed significantly (p<0.05) in the two groups\n(Table 1). The MDA level of the testicular tissue of the control rats was 8.61 \u00b1\n0.61 \u03bcm and significantly higher than the BBLE group which was 5.27 \u00b1 0.82 \u03bcm (Figure 1). Our\nresults confirm that BBLE apart from having an anti-hypercholesterolemic effect\nhad antioxidant properties.<\/p>\n\n\n\n

Currently,\nmany studies prove a\ndecrease in male fertility due to high cholesterol foods. The development of herbal\nmedicines to treat hypercholesterolemia continues to be carried out, including\nresearch on sembung (B. balsamifera<\/em>)\nplants with leaves that are\ntraditionally used in many areas, especially tropical areas such as Indonesia.\nThe positive effect of BBLE on hypercholesterolemia subjects had not been comprehensively\nstudied, especially as indicated by markers of oxidative stress in the\nreproductive system. The chemical composition and several parameters of BBLE\nreproductive performance in rats induced by high cholesterol feed had previously been studied\nand this raw material with the potential to be developed in biomedical industries22<\/sup>.<\/p>\n\n\n\n

Table 1: MDA tissue levels, StAR mRNA expression, Leydig cell counts, and testosterone levels in the control and treatment groups<\/strong>.<\/p>\n\n\n\n\n\n\n\n\n\n
\n

 <\/strong><\/p>\n

Groups<\/strong><\/p>\n<\/td>\n

\n

Mean\u00b1SD<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Testicular tissue MDA levels (\u03bcmol\/mL)<\/strong><\/p>\n<\/td>\n

\n

StAR mRNA expression (fold change)<\/em><\/strong><\/p>\n<\/td>\n

\n

Leydig cells counts<\/strong><\/p>\n<\/td>\n

\n

Testosterone levels<\/strong><\/p>\n

(ng\/L)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Control<\/p>\n<\/td>\n

\n

8. 61 \u00b1 0. 61<\/p>\n<\/td>\n

\n

1<\/p>\n<\/td>\n

\n

9. 07 \u00b1 0. 95<\/p>\n<\/td>\n

\n

310. 98 \u00b1 4. 94<\/p>\n<\/td>\n<\/tr>\n

\n

BBLE<\/p>\n<\/td>\n

\n

5. 27 \u00b1 0. 82<\/p>\n<\/td>\n

\n

3. 09<\/p>\n<\/td>\n

\n

13. 07 \u00b1 1. 47<\/p>\n<\/td>\n

\n

426. 02 \u00b1 9. 37<\/p>\n<\/td>\n<\/tr>\n

\n

P value<\/em><\/p>\n<\/td>\n

\n

0. 00<\/p>\n<\/td>\n

\n

0. 03<\/p>\n<\/td>\n

\n

0. 00<\/p>\n<\/td>\n

\n

0. 00<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Note: The value of p<0.05 showed  significantly different interpretations\nbetween the control and BBLE groups.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: MDA levels in hypercholesterolemic rats (n=18) (mean standard<\/strong>\u00b1<\/strong>deviation).*revealed a significant difference (p < 0.05).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The low\nlevels of MDA in the BBLE group can be caused by the secondary metabolites\ncontained therein that were dominated\nby the flavonoid group 21,22<\/sup>. Based on the results, the flavonoids in BBLE restored the antioxidant defense system and\nreduced lipid\nperoxidation by binding to free radicals and turning them into more stable\ncompounds 30<\/sup>. In contrast, in the control group,\nhypercholesterolemia can trigger lipid peroxidation31<\/sup>. BBLE has also\nbeen shown in other research to reduce blood sugar levels and enhance the Langerhans\ncell in on\npancreas of hyperglycemia mice32<\/sup>. The presence of flavonoids is also\nthought to affect the increase in glycogenic and glycolytic pathways that work\noutside the pancreas 33<\/sup>.<\/p>\n\n\n\n

The number of  Leydig cells <\/strong><\/p>\n\n\n\n

The number of Leydig cells in the control group was 9.07 \u00b1 0.95, which tended to be lower than the BBLE group, that was 13.07 \u00b1 1.47 (Figure 2). Interestingly, the effect of a high-cholesterol diet affected the proliferation of Leydig cells in the testes. The histological appearance of Leydig cells in both groups can be seen in Figure 3.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: <\/strong>Leydig cell counts in hypercholesterolemic rats (n=18) <\/strong>(mean standard<\/strong>\u00b1<\/strong>deviation). * revealed a significant difference (p < 0.05) between the two groups.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Leydig cells histology (arrows) in both groups in this study.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The repair effect on the Leydig cells given BBLE in this study was through three main mechanisms. I) maintaining the working of the HPT axis as evidenced by the increased expression of the StAR gene in Leydig cells due to optimal stimulation of the Luteinizing hormone (LH). II) protecting the testicular organs from exposure to free radicals as evidenced by the MDA levels of the testicular tissue that was given low BBLE, and III) the mechanism of action of the flavonoids found in BBLE had a high probability of binding to androgen receptors in the testicular Leydig cells34<\/sup>.<\/p>\n\n\n\n

Testosterone Levels<\/strong><\/p>\n\n\n\n

Testosterone levels in the\ncontrol group showed a significant difference (p<0.05) compared to the BBLE\ngroup (Table 1). Testosterone levels in the BBLE group were 426.02 \u00b1 9.37 ng\/L,\nit was higher than the control group which was 310.98 \u00b1 4.94 ng\/L (Figure 4).\nOur findings reveal that there was a positive relationship between a\nhigh-cholesterol diet given for 50 days and a decrease in testosterone hormone\nsecretion shown in the control group. Likewise, the ability of BBLE is\nimportant to highlight because it can maintain the function of Leydig cells for\ntestosterone biosynthesis.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 4: <\/strong>Testosterone levels in hypercholesterolemia rats (n=18) <\/strong>(mean standard<\/strong>\u00b1<\/strong>deviation). * revealed a significant difference (p < 0.05) between the two groups.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Testosterone is a hormone produced by testicular interstitial cells or Leydig cells through steroidogenesis processes. The administration of BBLE can effectively maintain the function of Leydig cells which correlates with the increase in the amount of testosterone in this study. It has been confirmed by our previous studies, which observed that providing Wistar rats with a high-fat diet for 50 days increased the number of spermatogenic cells and the diameter of the seminiferous tubules. The testosterone hormone testosterone, released by the testicular Leydig cells, promoted spermatogenesis in male animals35<\/sup>. The important role of testosterone hormone especially in spermatogenesis was to initiate, maintain and restore spermatogenesis36<\/sup>. Cholesterol is the main ingredient in the biosynthesis of the testosterone hormone37<\/sup>. Interestingly, the accumulation of cholesterol in mice with hypercholesterolemia had inhibition of testosterone synthesis due to decreased activity of steroidogenesis enzymes, as well as increased levels of stress biomarkers in the endoplasmic reticulum and activation of ATF-638<\/sup>.<\/p>\n\n\n\n

StAR mRNA Expression<\/strong><\/p>\n\n\n\n

The results showed that administration of BBLE for 30 days was able to up-regulate StAR gene expression by threefold (fold change) when compared to the control group (Figure 5). We suspected that the phytochemical compounds in BBLE that were dominated by flavonoids from our previous researchcan maintain StAR gene expression that in turn affects the performance of steroidogenesis thereby maintaining testosterone production22<\/sup>.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 5: <\/strong>The StAR mRNA expression in hypercholesterolemic rats in the control and BBLE groups. The * sign showed that
there was an up-regulation of the StAR mRNA in the BBLE  group compared to the control.<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

StAR triggers the movement of cholesterol, the major component of steroidogenesis, from the mitochondrial outer membrane to the inner membrane in male animals. In this study, StAR gene expression was up-regulated threefold (fold change) in the BBLE group compared to the control group. Flavonoid compounds in BBLE were thought to play an important role in increasing StAR activity by suppressing the protein performance of DAX-1, an AHC critical region on the X-chromosome gene, that was a protein repressor for StAR gene transcription 39<\/sup>. The effectiveness of flavonoid compounds in increasing StAR activity was also revealed by Martin and Touaibia through the inhibition of COX-2 (cyclooxygenase-2) expression. Likewise, inhibition of COX-2 expression had been found to inhibit the action of the StAR protein in aging mouse Leydig cells39<\/sup>.<\/p>\n\n\n\n

Path analysis <\/strong><\/p>\n\n\n\n

The results of pathway analysis showed that MDA levels, and StAR mRNA expression had a significant direct effect (p<0.05) on the Leydig cell numbers. The contribution of MDA levels and StAR gene expression to the number of Leydig cells was 89.4%, while the remaining 10.6% was contributed by other not examined variables. The direct effect of testicular tissue MDA levels, StAR mRNA expression, and the number of Leydig cells was significant on serum testosterone hormone secretion (p<0.05). MDA levels, StAR mRNA expression values\u200b\u200b, and the number of Leydig cells contributed as much as  94.2% to testosterone secretion. The indirect effect of tissue MDA levels on testosterone secretion through the number of Leydig cells was significant from the direct effect. However, the indirect effect of StAR mRNA expression values \u200b\u200bon testosterone secretion was not significant compared to the direct effect (Table 2).<\/p>\n\n\n\n

Table 2: Effect of testicular tissue MDA levels, StAR gene expression, and the number of Leydig cells on testosterone levels in hypercholesterolemic Wistar rats<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Variable<\/strong><\/p>\n<\/td>\n

\n

Type of Effect<\/strong><\/p>\n<\/td>\n

\n

R-square <\/em>value<\/strong><\/p>\n<\/td>\n

\n

The amount of Effect (\u03b2)<\/strong><\/p>\n<\/td>\n

\n

p-value<\/em><\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

MDA            Leydig cell        <\/p>\n<\/td>\n

\n

Direct<\/p>\n<\/td>\n

\n

0,894<\/p>\n<\/td>\n

\n

-0. 712<\/p>\n<\/td>\n

\n

0. 00<\/p>\n<\/td>\n<\/tr>\n

\n

Ct-StAR           Leydig cell         <\/p>\n<\/td>\n

\n

Direct <\/p>\n<\/td>\n

\n

-0. 331<\/p>\n<\/td>\n

\n

0. 00<\/p>\n<\/td>\n<\/tr>\n

\n

MDA           testosterone     <\/p>\n<\/td>\n

\n

Direct <\/p>\n<\/td>\n

\n

0,942<\/p>\n<\/td>\n

\n

-0. 540<\/p>\n<\/td>\n

\n

0. 00<\/p>\n<\/td>\n<\/tr>\n

\n

Ct-StAR           testosterone       <\/p>\n<\/td>\n

\n

Direct <\/p>\n<\/td>\n

\n

0. 287<\/p>\n<\/td>\n

\n

0. 01<\/p>\n<\/td>\n<\/tr>\n

\n

sel Leydig         testosterone<\/p>\n<\/td>\n

\n

Direct <\/p>\n<\/td>\n

\n

0. 634<\/p>\n<\/td>\n

\n

0. 00<\/p>\n<\/td>\n<\/tr>\n

\n

MDA           testosterone<\/p>\n<\/td>\n

\n

Indirect<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

-0. 451<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Ct-StAR           testosterone<\/p>\n<\/td>\n

\n

Indirect<\/p>\n<\/td>\n

\n

 <\/p>\n<\/td>\n

\n

-0. 209<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The results showed that the administration of BBLE had a protective effect against oxidative stress directly in the testes as evidenced by lower MDA levels. Reducing oxidative stress had the effect of regenerating Leydig cells that produce testosterone22<\/sup>. On the other hand, administration of BBLE also resulted in up-regulation of StAR gene expression with a direct influence on testosterone biosynthesis by Leydig cells.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The results show a protective effect against oxidative stress by decreasing MDA levels in testicular tissue in hypercholesterolemia Wistar rats. The results of this study also prove the effectiveness of the BBLE antioxidant that acts directly on the testes and improves Leydig cell regeneration and stimulates the regulation of StAR gene expression thereby increasing testosterone secretion. Further, research is still needed to examine several steroidogenesis markers that can complete information about BBLE\u2019s ability to increase testosterone secretion.<\/p>\n\n\n\n

Acknowledgment <\/strong><\/p>\n\n\n\n

The author would like to\nthank Mr. I Gede Wiranata, M.Si from the Laboratory of Pharmacology, Faculty of Medicine, Udayana\nUniversity (UNUD) who has assisted in this research through the maintenance and\nintervention of BBLE in experimental animals. The author also thanks the Laboratory\nof Biochemistry and Molecular Biology of the Faculty of Medicine, Udayana\nUniversity (UNUD), Histopathology Laboratory, Denpasar Veterinary Center\n(BBVET), and Laboratory of Molecular Biology of Institute of Tropical Disease\nCenter (ITD) of Universitas Airlangga (UNAIR) who has assisted in the provision\nof reagents and analysis of research samples.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors reported no declarations\nof interest. <\/p>\n\n\n\n

Funding\nSource<\/strong><\/p>\n\n\n\n

This research was funded by the\nMinistry of Research, Technology and Higher Education of the Republic of\nIndonesia (KEMENRISTEKDIKTI) through the Domestic Postgraduate Education Scholarship\nProgram (BPPDN) with Contract Number: B\/276\/D3.2\/KD. 02.00\/2019.<\/p>\n\n\n\n

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