Gandou Bushen Decoction improves spermatogenesis and promotes spermatogenic cell proliferation in Wilson disease TX mice by activating testicular ERK signaling pathway

doi:10.12122/j.issn.1673-4254.2024.11.02

Abstract

Abstract:

Objective To investigate the therapeutic mechanism of Gandou Bushen Decoction (GDBSD) for improving reproductive disorders in male mouse models of Wilson disease (WD). Methods Sixty male homozygous TX mice were randomized equally into 4 groups and treated with daily gavage of saline (WD model group), penicillamine (0.09 g/kg), or GDBSD (0.2 mL/10 g), or with intraperitoneal injection of U0126 (20 mg/kg) in addition to GDBSD gavage, with 15 male DL mice as control. After 4 weeks of treatment, copper content in testicular tissue of the mice was detected, and histopathology of the testes and epididymis was examined using HE staining and electron microscopy. TUNEL staining was used to identify apoptotic cells in the testes. The protein expressions of Bcl-2, Cytc, caspase-3, ERK, and p-ERK in the testicular tissue were evaluated with Western blotting, and BrdU-positive cells were detected with immunohistochemical labeling. Sperm density, viability, malformation rate and fertility levels of male mice were studied. Results Treatment with penicillamine and GDBSD obviously improved pathological changes of the testis, increased sperm density and motility, lowered sperm abnormality rate,fertility levels and increased testicular JOHNSEN score of TK mice, but the therapeutic effect of GDBSD was blocked by U0126. GDBSD treatment significantly lowered Cytc and caspase-3 expressions and increased Bcl-2 expression in the testicular tissue of TX mice (P<0.05), while U0126 treatment significantly lowered testicular Bcl-2 expression level. No significant differences were found in total protein expression levels of ERK1/2 among the 5 groups, but p-ERK protein expression was significantly reduced in WD and U0126 groups and increased in penicillamine and GDBSD groups. Conclusion GDBSD can improve spermatogenesis and enhance fertility of male TX mice with WD possibly by activating the ERK signaling pathway to enhance proliferation and reduce apoptosis of the spermatogenic cells.

Key words: Wilson disease, Gandou Bushen Decoction, copper deposition, ERK signaling pathway, spermatogenesis

Miaozhu YIN, Kuiyu CHEN, Limin WU, Pengyu JIANG, Zhihui JI, Nian ZHANG, Huan ZHOU, Hui HAN. Gandou Bushen Decoction improves spermatogenesis and promotes spermatogenic cell proliferation in Wilson disease TX mice by activating testicular ERK signaling pathway[J]. Journal of Southern Medical University, 2024, 44(11): 2063-2073.

Figures/Tables 12

Tab.1 Comparison of testicular copper levels among the 5 groups (μg/g, Mean±SD, n=5)

Group	Testicular copper level (μg/g)
Control	2.772±0.306
WD	9.355±0.425*
Penicillamine	4.129±0.550^#
GDBSD	4.952±0.264 *^#
GDBSD+U0126	5.597±0.678^#

Fig.1 Western blotting for detecting expressions of ERK signaling pathway proteins in mouse testicular tissue. A: ERK. B: p-ERK (Mean±SD, n=5). C: Original protein bands. *P<0.05 vs Control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

Fig.2 HE staining of testicular tissues of the mice. A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group. F: Johnsen scores of the germinal tubules in the testicular tissue. *P<0.05 vs control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

Fig.3 HE staining of mouse epididymal tissues. A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group.

Tab.2 Comparison of sperm quality of the mice (Mean±SD, n=5)

Group	Sperm density (10⁷/mL)	Sperm survival rate (%)	Sperm malformation rate (%)
Control	4.73±0.17	71.51±1.65	14.97±2.35
WD	2.20±0.22*	20.59±1.81*	79.62±4.44*
Penicillamine	3.50±0.16*^#	61.72±1.12*^#	26.96±3.60*^#
GDBSD	3.43±0.50*^#	66.53±1.72*^#	24.84±1.67*^#
GDBSD+U0126	2.67±0.12^&	52.50±2.25 ^&	77.74±3.39^&

Fig.4 Representative images of HE staining of mouse spermatozoa. A: Control group. B: WD group. C: penicillamine group. D: GDBSD group. E: GDBSD+U0126 group. Red arrows: Sperms with tail curl; Orange arrows: Sperms with a head without a tail; Green arrows: Sperms with head deformity.

Fig. 5 Ultrastructural changes of mouse spermatozoa in each group observed with transmission electron microscope (scale bar=500 nm). A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group.

Fig.6 Ultrastructure of mouse spermatocytes in each group observed with transmission electron microscope. A, B: Control group. C, D: WD group. E, F: Penicillamine group. G, H: GDBSD group. I, J: GDBSD+U0126 group. Black arrows indicate mitochondrial structures.

Fig. 7 TUNEL staining of mouse testicular tissue. A-C: Control group. D-F: WD group. G-I: Penicillamine group. J-L: GDBSD group. M-O: GDBSD+U0126 group. P: Apoptosis rate of testicular tissue in each group. Apoptotic cells are stained green and nuclei are stained blue (Mean±SD,n=5). *P<0.05 vs Control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

Fig. 8 Western blotting for detecting expressions of apoptosis-related proteins in mouse testis. A: Caspase-3. B: Cytc. C: Bcl-2. D: Original protein bands. *P<0.05 vs Control group, #P<0.05 vs WD group, &P<0.05 vs GDBSD group.

Fig.9 Immunohistochemically labeled BrdU-positive cells in mouse testicular tissue. A: Control group. B: WD group. C: Penicillamine group. D: GDBSD group. E: GDBSD+U0126 group. F: Percentages of BrdU-positive cells in each group. *P<0.05 vs Control group; #P<0.05 vs WD group; &P<0.05 vs GDBSD group.

Tab.3 Comparison of fertility of male mice among the 5 groups (Mean±SD, n=5)

Group	Fertility rate (%)	Pups per litter
Control	80.00	10.11±1.73
WD	37.50	4.00±0.82*
Penicillamine	62.50	7.40±1.04^#
GDBSD	66.67	8.14±1.55^#
GDBSD+U0126	42.86	5.00±0.81^&

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