Novel discovery of schisandrin A regulating the interplay of autophagy and apoptosis in oligoasthenospermia by targeting SCF/c-kit and TRPV1 via biosensors

Abstract

Oligoasthenospermia is the primary cause of infertility. However, there are still enormous challenges in the screening of critical candidates and targets of oligoasthenospermia owing to its complex mechanism. In this study, stem cell factor (SCF), c-kit, and transient receptor potential vanilloid 1 (TRPV1) biosensors were successfully established and applied to studying apoptosis and autophagy mechanisms. Interestingly, the detection limit reached 2.787 × 10^-15 g/L, and the quantitative limit reached 1.0 × 10^-13 g/L. Furthermore, biosensors were used to investigate the interplay between autophagy and apoptosis. Schisandrin A is an excellent candidate to form a system with c-kit similar to SCF/c-kit with a detection constant (K_D) of 5.701 × 10^-11 mol/L, whereas it had no affinity for SCF. In addition, it also inhibited autophagy in oligoasthenospermia through antagonizing TRPV1 with a K_D of up to 4.181 × 10^-10 mol/L. In addition, in vivo and in vitro experiments were highly consistent with the biosensor. In summary, high-potency schisandrin A and two potential targets were identified, through which schisandrin A could reverse the apoptosis caused by excessive autophagy during oligoasthenospermia. Our study provides promising insights into the discovery of effective compounds and potential targets via a well-established in vitro-in vivo strategy.

Keywords: Apoptosis; Autophagy; Biosensor; Male infertility; Oligoasthenospermia; Schisandrin A..

Graphical abstract

Figure 1

Superior efficacy of schisandrin A (CQA) on oligoasthenospermia. (A) Pathological sections of rat testis in each group magnified 200 and 400 times, respectively (black arrows pointed to the representative physiological and pathological characteristics). (a1) and (a6), Normal group; (a2) and (a7), model group by GTW; (a3) and (a8), group of levocarnitine; (a4) and (a9), group of Huangjing Zanyu capsule; (a5) and (a10), group of schisandrin A. (B) Superior efficacy of schisandrin A on testicular interstitial cells, sperm quality. (b1) Cell viability; (b2) grade A sperm (%); (b3) grade B sperm (%); (b4) sperm density; (b5) sperm mobility (%); (b6) sperm viability. Note: ^#P < 0.05 (vs normal group); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (vs control group).

Figure 2

Fabrication and performance of SCF/c-kit biosensors. (A) The fabrication protocol of the new generation of SCF and c-kit biosensors. (B) The results of protein modification. (b1) SEM of Blank HEMT; (b2) SEM of HEMT modified protein; (b3) I_DS-V_DS shift caused by protein modification. (C) The parameter optimization and performance of c-kit biosensors. (c1) Sampling interval; (c2) scanning speed; (c3) protein modification time; (c4) and (c5) sample reaction time; (c6) specificity; (c7) stability; (c8) repeatability; (c9) precision; (c10) the I_DS-V_DS caused by tested samples with increasing concentration; (c11) and (c12) the simulated data of 3000 measurements for DL and QL.

Figure 3

Discovery of apoptosis mechanism of schisandrin A on SCF/c-kit system. (A) Schematic diagram of interaction results between schisandrin A and SCF/c-kit biosensors. (B) Interaction results between schisandrin A and SCF/c-kit biosensors by CHI660E and artificial intelligence algorithm. (a1)–(d1) I_DS-V_DS decreases (ΔI) with increasing concentration (record as C) of SCF, schisandrin A, the complex of SCF added schisandrin A, the complex of schisandrin A added SCF; (a2)–(d2) The linear fitting between Lg of C and ΔI/I₀ of SCF added schisandrin A, the complex of schisandrin A added SCF; (a3)–(d3) C/ΔI versus C of SCF added schisandrin A, the complex of schisandrin A added SCF.

Figure 4

(A) TUNEL assay for testicular tissue samples in different groups. (a1)–(a5) The TUNEL assay results of normal control, GTW, WWZ-L, WWZ-M, and WWZ-H, respectively; (a6)–(a10) The DAPI results of normal control, GTW, WWZ-L, WWZ-M, and WWZ-H, respectively; (a11)–(a15) The merge charts by TUNEL and DAPI of normal control, GTW, WWZ-L, WWZ-M, and WWZ-H, respectively. (B) Western blot strip charts and RT-PCR results of SCF and c-kit expression in different groups. (b1) c-kit protein; (b2) c-kit mRNA; (b3) SCF protein; (b4) SCF mRNA. ∗P < 0.05 vs NS group; ^#P < 0.05 vs GTW group in all figures. (C) The schematic diagram of oligoasthenospermia by schisandrin A. (c1) The schematic diagram of oligoasthenia; and (c2) The schematic diagram of asthenospermia; (c3) The schematic representation of schisandrin A modulated oligoasthenospermia; (c4) The schematic diagram of oligoasthenospermia recovery by SCF/c-kit system.

Figure 5

The interplay between autophagy and apoptosis in oligoasthenospermia by schisandra A. (A) Schematic diagram of interaction between schisandrin A and TRPV1 biosensor. (a1) The TRPV1-HEMT biosensor; (a2) The enlarged view of the protein modified area; (a3) The interaction between schisandrin A and TRPV1-HEMT biosensor; (a4) The detection of interaction between schisandrin A and TRPV1 by CHI660E. (B) The results of protein modification. (b1) SEM of blank HEMT (b2) SEM of HEMT modified protein. (C) Interaction results between schisandrin A and TRPV1 by CHI660E and artificial intelligence algorithm. (c1) I_DS-V_DS regularity decreases with increasing concentration of schisandrin A; (c2) The linear fitting between Lg of schisandrin A concentration and (I–I₀)/I₀; (c3) [A_g]/ΔI versus [A_g] of schisandrin A. (D) Western blot strip charts and expression levels of autophagy- and apoptosis-related proteins. (d1) Akt; (d2) p-Akt; (d3) p-Akt/Akt; (d4) Bcl-2; (d5) MDM2; (d6) p53; (d7) LC3B-II; (d8) LC3B-II/I; (d9) Beclin1; (d10) ULK1; (d11) p-ULK1; (d12) p-ULK1/ULK1. Note: ∗P < 0.05, ∗∗P < 0.01 (E) The schematic diagram of schisandrin A regulating the interplay of autophagy and apoptosis through TRPV1 and SCF/c-kit systems.