Prevention of Testicular Damage by Indole Derivative MMINA via Upregulated StAR and CatSper Channels with Coincident Suppression of Oxidative Stress and Inflammation: In Silico and In Vivo Validation

Abstract

Cis-diamminedichloroplatinum (II) (CDDP) is a widely used antineoplastic agent with numerous associated side effects. We investigated the mechanisms of action of the indole derivative N'-(4-dimethylaminobenzylidene)-2-1-(4-(methylsulfinyl) benzylidene)-5-fluoro-2-methyl-1H-inden-3-yl) acetohydrazide (MMINA) to protect against CDDP-induced testicular damage. Five groups of rats (n = 7) were treated with saline, DMSO, CDDP, CDDP + MMINA, or MMINA. Reproductive hormones, antioxidant enzyme activity, histopathology, daily sperm production, and oxidative stress markers were examined. Western blot analysis was performed to access the expression of steroidogenic acute regulatory protein (StAR) and inflammatory biomarker expression in testis, while expression of calcium-dependent cation channel of sperm (CatSper) in epididymis was examined. The structural and dynamic molecular docking behavior of MMINA was analyzed using bioinformatics tools. The construction of molecular interactions was performed through KEGG, DAVID, and STRING databases. MMINA treatment reversed CDDP-induced nitric oxide (NO) and malondialdehyde (MDA) augmentation, while boosting the activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD) in the epididymis and testicular tissues. CDDP treatment significantly lowered sperm count, sperm motility, and epididymis sperm count. Furthermore, CDDP reduced epithelial height and tubular diameter and increased luminal diameter with impaired spermatogenesis. MMINA rescued testicular damage caused by CDDP. MMINA rescued CDDP-induced reproductive dysfunctions by upregulating the expression of the CatSper protein, which plays an essential role in sperm motility, MMINA increased testosterone secretion and StAR protein expression. MMINA downregulated the expression of NF-κB, STAT-3, COX-2, and TNF-α. Hydrogen bonding and hydrophobic interactions were predicted between MMINA and 3β-HSD, CatSper, NF-κβ, and TNFα. Molecular interactome outcomes depicted the formation of one hydrogen bond and one hydrophobic interaction between 3β-HSD that contributed to its strong binding with MMINA. CatSper also made one hydrophobic interaction and one hydrogen bond with MMINA but with a lower binding affinity of -7.7 relative to 3β-HSD, whereas MMINA made one hydrogen bond with NF-κβ residue Lys37 and TNF-α reside His91 and two hydrogen bonds with Lys244 and Thr456 of STAT3. Our experimental and in silico results revealed that MMINA boosted the antioxidant defense mechanism, restored the levels of fertility hormones, and suppressed histomorphological alterations.

Keywords: antineoplastic agent; cisplatin; indole; molecular docking; molecular interactome; reproductive hormones; testicular tissues.

Figure 1

Effect of various treatments on organ weights. *, **** p < 0.05 and p < 0.0001 versus Control respectively, ^{#,##, ####} p < 0.05, p < 0.01 add p < 0.0001 versus CPDD. ns = non-significant.

Figure 2

MMINA upregulated protein expression levels of the key testosterone synthesis factors and related hormones in rat testes. (A) Effect on reproductive hormones; (i): testosterone, (ii): LH. (B) Effect of MMINA on transcriptional regulation of the steroidogenic hormone biosynthesis gene. rtPCR analysis of StAR (i), CYP11A1 (ii), and 3β-HSD (iii) mRNA expression. (C)-StAR, CYP11A1, and 3β-HSD protein content were determined by Western blot analysis. Values were expressed as mean ± standard deviation (n = 7). **, **** p < 0.05 and p < 0.0001 versus control, respectively, and ⁺⁺, ⁺⁺⁺⁺ p < 0.05 and p < 0.0001 versus CDDP. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests using graph pad prism version 9. STAR: steroidogenic acute regulatory protein, CYP11A1: P450 side-chain cleavage enzyme, HSD17B: 17β-hydroxysteroid dehydrogenase. Uncropped blots are presented in Supplementary File S3.

Figure 3

MMINA treatment enhanced sperm motility and activate the CatSper channel. (a): The percentage of motile spermatozoa in different treatment groups. (b): Western blot analysis indicating the protein expression of CatSper 1 and CatSper 2 channels in various treatment groups. MMINA administration prominently upregulates the expression of both channels in comparison to the CDDP alone exposed group. (c,d) Indicated the mRNA expression of CatSper 1 and 2 genes. Data are mean ± SEM, (n = 7). **, **** indicated significant variation at p < 0.01 and p < 0.0001, respectively, versus Control group and ^##, ^#### indicated significant variation at p < 0.05 and p < 0.0001, respectively, versus CDDP group. ns= non-significant. Uncropped blots are presented in Supplementary File S3.

Figure 4

Comparison of fold change expression of target proteins and genes in various treatment groups. (a) RT-PCR analysis to determine the mRNA expression of STAT 3, NFκb, COX 2, and TNF-α. The error bars indicated the standard error of the mean expression level. Data are mean ± SEM (n = 7). *,**, **** indicated significant variation at p < 0.05, p < 0.01 and p < 0.0001 versus Control group, respectively, and ++++ indicated significant variation at p < 0.0001 versus CDDP group. (b) Protein expression analysis by Western blotting: β-actin was used as an endogenous control for the assessment of protein loading. Uncropped blots are presented in Supplementary File S3.

Figure 5

Light micrographs of testicular tissues of rats treated with MMINA and CDDP. Sections were stained with hematoxylin and eosin (40x). (a) Photomicrograph of the testicular tissues from various treatment groups. Control group showing typical seminiferous tubules structure at all stages of spermatogenic and the interstitial cells with Leydig cells filling the space between the seminiferous tubules. The CDDP (12 mg/.kg b.w) treatment group showed degenerative alterations in spermatogenic cells and the detachment of the spermatogenic epithelium. With empty lumen, the MMINA + CDDP group indicated a significant protective effect of MMINA against CDDP-induced morphological alterations and increase spermatogenesis. MMINA alone group showed a healthy histological structure similar to the control group. (b) Morphometric analysis of testicular tissue alterations in experimental groups was performed using Image J software. STD: seminiferous tubule diameter, STEH: Seminiferous tubule epithelial height, TL: Tubular lumen, STA: Seminiferous tubular area, IS: i2terstitial space, LS: Lumen with sperms. Data are mean ± SEM, (n = 7). *** indicated significant variation at p < 0.0001, respectively, versus Control group, and #, ###, #### indicated significant variation at p < 0.05, p < 0.001, and p < 0.0001 versus the CDDP group, ns= non-significant, respectively. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison tests.

Figure 6

Molecular interactions between MMINA and pro-inflammatory proteins TNF-alpha, STAT3, COX-2, and NF-κβ. (a) The MMINA-COX2 complex has predominant hydrophobic interactions, whereas (b) the MMINA-NF-κβ complex and (c) the MMINA-STAT3 complex has hydrogen bonding, and (d) the MMINA-TNFalpha complex has both hydrogen bonding and hydrophobic interactions. Semi-circles in red color depict hydrophobic interactions, green dotted lines show hydrogen bonding, and green numbers indicate the distance between hydrogen-bonded ligand and amino acid. Purple-lined structure denotes ligand, while orange line structures represent amino acids. The surface view of the protein and ligand complex is shown below the 2D figure. Ligand is indicated with purple color.

Figure 7

Proposed pathways of MMINA effect on the signal transduction of inflammatory biomarkers. Cytokine/JAK-STAT signaling and TNFalpha/NF-κβ signaling mainly contribute to the manifestation of inflammation. MMINA downregulates the expression of NF-κβ, TNFalpha, and STAT3 at mRNA and protein levels and reduces inflammation. COX2 pathway crosstalk also activates the NF-κβ signaling and contributes to inflammation. MMINA also regulates COX2 expression and inhibits this pathway.

Figure 8

Molecular interactions between MMINA and 3β-HSD, CatSper, CYP20A1, and StAR. (a) MMINA-3β-HSD and (b) MMINA-CatSper complex has one hydrophobic interaction and one hydrogen bond, whereas hydrophobic interactions predominated in (c) MMINA-CYP20A1 and (d) MMINA-StAR complex. Semi-circles in red color depict hydrophobic interactions, green dotted lines show hydrogen bonding, and green numbers indicate the distance between hydrogen-bonded ligand and amino acid. Purple lined structure denotes ligand, while orange line structures represent amino acids. The surface view of the protein and ligand complex is shown below the 2D figure. Ligand is indicated with purple color.

Figure 9

Proposed pathways of MMINA mediated expression of StAR, CYP17A1, 3βHSD, and CatSper in increasing testicular efficiency. StAR, CYP17A1, and 3βHSD are modulators of progesterone and testosterone production. Their expression is mediated by calcium ions and cAMP/PKA signaling mediated transcription factors (SF1, Sp1, Plox1, CREB, and NGF1β) activation and their nuclear translocation. This group of transcription factors promotes StAR, CYP17A1, and 3βHSD gene expression. These genes then mediate the production of progesterone from cholesterol and testosterone from progesterone. Progesterone further mediates sperm motility by activating the sperm-specific calcium ion channel CatSper via ABHD14/2. MMINA contributes by activating CatSper and StAR, CYP17A1, and 3βHSD gene expression.