Pseudoephedrine Nanoparticles Alleviate Adriamycin-Induced Reproductive Toxicity Through the GnRhR Signaling Pathway

Figures & data

Table 1 Primer Sequences

Target	Forward Primer (5′–3′)	Reverse Primer (5′–3′)	Accession Number	Product Size
GnRhR	TGCTCGGCCATCAACAACA	GGCAGTAGAGAGTAGGAAAAGGA	NM_001310653.1	114
LHR	CTCGCCCGACTATCTCTCAC	ACGACCTCATTAAGTCCCCTG	NM_001364898.1	77
AR	TGCCCGAATGCAAAGGTCTT	TTGGCGTAACCTCCCTTGAAA	NM_013476.4	94
GNAS	CAGAGCCTCCATTGGGGTC	GCTTCTCGCTCAACTGGGG	NM_001310085.1	141
ADCY1	TTGGCAAGTTCGATGAGTTAGC	GGCGTGATCCGTCTTAGGC	NM_009622.2	110
PKA	AGGGCAGGACATGGACATTG	CGCCTTATTGTAACCCTTGCTG	NM_001164199.1	80
DDX4	CGGATGATGCAGAGAGAGTACC	AATGATGGGCCTGAAAAAGAGTT	NM_001145885.1	157
DAZ1	ATACCTCCGGCTTATACAACTGT	GACTTCTTTTGCGGGCCATTT	NM_010021.5	128
CYCLIN-1	CAGTTTCCCCAATGCTGGTTG	CCTCTGCATACTCCGTTACGTTA	NM_001305221.1	99
STAR	GCCGGACCTCATGGAATTTGA	TCACTTCATGTGCAGAGATGATG	NM_009292.2	95
SCP3	AGCCAGTAACCAGAAAATTGAGC	CCACTGCTGCAACACATTCATA	NM_011517.2	106
REC8	GGTCATCACCTTACAGGAGGC	TCTGCGATCAGCAGTTCTAAGT	NM_001360390.1	105
SMC1B	CATGAGGGAAAACGTCAGCAG	TGACACAGATCAAGCAGTCTTC	NM_080470.1	98
Miwi	AGACCTCATTGGAAGGTGTCA	TGTTCCCCATTCCGAGTCTGA	NM_021311.3	108
TNF-α	CTGAACTTCGGGGTGATCGG	GGCTTGTCACTCGAATTTTGAGA	NM_013693.3	122
IL-1β	GAAATGCCACCTTTTGACAGTG	TGGATGCTCTCATCAGGACAG	NM_008361.4	116
Caspase 3	TGGTGATGAAGGGGTCATTTATG	TTCGGCTTTCCAGTCAGACTC	NM_009810.3	105
GAPDH	TGTGTCCGTCGTGGATCTGA	TTGCTGTTGAAGTCGCAGGAG	NM_008084.3	150

Figure 1 Characterization of PSE-NPs: (A) SEM micrographs of the PSE NPs. (B) Dynamic light scattering of pseudoephedrine nanoparticles (PSE-NPs) and (C) in vitro release of PSE-NPs.

Figure 2 Effect of pseudoephedrine nanoparticles (PSE-NPs) on percent apoptosis of GC-1 cells and expression of apoptosis-related proteins: (A and B) Effect of PSE-NPs on percent apoptosis and survival of GC-1 cells in each group. (C) Effect of PSE-NPs on expression of apoptosis-related proteins of each group, scale bar 50 μm.

Notes: Data are the mean ± SD (n = 6). **P < 0.01 vs the CON group. ^#P < 0.05 and ^##P < 0.01 vs the Model group. ^&P < 0.05 and ^&&P < 0.01 vs the PSE group.

Abbreviations: CON, control group underwent standard culture; M, model group; PLGA, empty nanoparticles; PSE, 0.5μM pseudoephedrine; PSE-PLGA, 0.25 μM pseudoephedrine-loaded PLGA nanoparticles; Bax, BCL2-Associated X; Bcl-2, B cell lymphoma-2.

Figure 3 Effects of pseudoephedrine nanoparticles (PSE-NPs) on the mitochondrial functions and glycolysis rate of GC-1 cells: (A–C) Effects of PSE-NPs on basal respiration, ATP production, and maximal respiration in each group. (D and E) Effects of PSE-NPs on basal glycolysis and compensatory glycolysis in each group.

Notes: Data are the mean ± SD (n = 6). **P < 0.01 vs the CON group. ^##P < 0.01 vs the Model group. ^&P < 0.05 vs the PSE group.

Abbreviations: CON, control group underwent standard culture; M, model group; PLGA, empty nanoparticles; PSE, 0.5μM pseudoephedrine; PSE-PLGA, 0.25 μM pseudoephedrine-loaded PLGA nanoparticles.

Figure 4 Effect of PSE-NPs on the GnRhR signaling pathway and expression of meiosis-related proteins SCP3 of GC-1 cells damaged by adriamycin: (A–E) Protein expression of GnRhR, GNAS, p-CREB and SCP3 in GC-1 cells, scale bar 50 μm.

Notes: Data are the mean ± SD (n = 6). *P < 0.05 and **P < 0.01 vs the CON group. ^##P < 0.01 vs the Model group. ^&&P < 0.01 vs the PSE group.

Abbreviations: CON, control group underwent standard culture; M, model group; PLGA, empty nanoparticles; PSE, 0.5μM pseudoephedrine; PSE-PLGA, 0.25 μM pseudoephedrine-loaded PLGA nanoparticles; GnRhR, gonadotropin-releasing hormone receptor; CREB, cyclic adenosine monophosphate (cAMP)-response element binding protein; GNAS, guanine nucleotide binding protein; SCP3, small C-terminal domain phosphatase 3.

Figure 5 Effect of PSE-NPs on testicular histology in mice. The CON group showed a normal organizational structure. The Model group and PLGA group showed cell shedding and a decrease in the spermatogenic-cell layer. Compared with the Model group, the PSE group and PSE-PLGA group had significantly improved histology, and the PSE-PLGA group had the best effect. Black arrows mark showing spermatogenic cell layers.

Abbreviations: CON, Control group; M, Model group with 30 mg/kg adriamycin, i.p.; PSE, 1.4 mg/kg PSE, i.v.; PLGA, 53.8 mg/kg PLGAs, i.v.; PSE-PLGA, 53.8 mg/kg PSE-PLGAs, i.v.

Table 2 Effects of PSE-NPs on Bodyweight, Testicular Indices, and Sperm Motility in Mice Treated with Adriamycin

	CON	Model	PLGA	PSE	PSE-PLGA
Weight, g	36.5±2.0	35.0±2.9	35.0±2.3	35.5±2.1	35.3±1.5
Testes Index	6.60±0.36	4.59±0.43**	4.48±0.46	5.14±0.22	5.89±0.68^##&
Epididymis index	1.39±0.20	0.86±0.14**	0.68±0.09	1.22±0.07^#	1.49±0.24^##
Motility, %	69.4±7.6	20.4±7.3**	26.5±9.0	49.4±8.6^##	62.2±10.3^##&&
Density, ×10^6	190.0±40.6	88.3±27.2**	83.2±12.0	101.1±22.0	132.0±15.5^##&
Dead sperm, %	17.3±3.8	65.1±4.9**	59.9±8.8	43.6±14.1^##	31.1±12.1^##&&

Notes: Data are the mean ± SD (n = 8). **P < 0.01 vs the CON group. ^#P < 0.05 and ^##P < 0.01 vs the Model group. ^&P < 0.05 and ^&&P < 0.01 vs the PSE group.

Figure 6 Effects of PSE-NPs on percent apoptosis, oxidative stress, and expression of apoptosis-related proteins in mouse testicular cells: (A) Percent apoptosis in the testicular tissue of mice in each group. (B) mRNA expression of IL-1β, caspase 3 and TNF-α in the testicular tissue of mice each group measured by RT-qPCR. (C) Protein expression of Bax and Bcl-2 in the testicular tissue of mice in each group measured by Western blotting. (D) Levels of SOD and MDA in the testicular tissue of mice in each group.

Notes: Data are the mean ± SD (n = 6). **P < 0.01 vs the CON group. ^#P < 0.05 and ^##P < 0.01 vs the Model group. ^&&P < 0.01 vs the PSE group.

Abbreviations: CON, Control group; M, Model group with 30 mg/kg adriamycin, i.p.; PSE, 1.4 mg/kg PSE, i.v.; PLGA, 53.8 mg/kg PLGAs, i.v.; PSE-PLGA, 53.8 mg/kg PSE-PLGAs, i.v.; 7-AAD, 7-amino-actinomycin D; PE, Phycoerythrin; TNF-α, tumor necrosis factor-α; IL-1β, interleukin-1β; Bax, BCL2-Associated X; Bcl-2, B cell lymphoma-2; SOD, Superoxide dismutase; MDA, Mobile device assistant.

Figure 7 Continued.

Figure 7 Continued.

Figure 7 Effects of PSE-NPs on sex-hormone levels, GnRh signaling pathway-related proteins in testes, and indices of meiosis in mice: (A) Levels of GnRh, LH and testosterone in the serum of mice in each group. (B) mRNA expression of GnRh signaling pathway-related proteins and meiosis-associated genes in the testicular tissue of mice in each group was measured by RT-qPCR. (C) Protein expression of GnRh signaling pathway and meiosis-associated proteins in the testicular tissue of mice in each group was measured by western blotting. (D) The protein expression of GnRhR and SCP3 in the testicular tissue of mice in each group was measured by Immunofluorescence, scale bar 50 μm.

Notes: Data are the mean ± SD (n = 6). *P < 0.05 and **P < 0.01 vs the CON group. ^#P < 0.05 and ^##P < 0.01 vs the Model group. ^&P < 0.05 and ^&&P < 0.01 vs the PSE group.

Abbreviations: CON, Control group; M, Model group with 30 mg/kg adriamycin, i.p.; PSE, 1.4 mg/kg PSE, i.v.; PLGA, 53.8 mg/kg PLGAs, i.v.; PSE-PLGA, 53.8 mg/kg PSE-PLGAs, i.v.; GnRh, gonadotropin-releasing hormone; LH, luteinizing hormone; GnRHR, gonadotropin-releasing hormone receptor; LHR, luteinising hormone receptor; AR, androgen receptor; GNAS, guanine nucleotide-binding proteins; ADCY1, adenylate cyclase type 1; PKA, protein kinase A; DAZ1, deleted in azoospermia protein 1; DDX4, Dead box polypeptide 4; STAR, StAR-related lipid transfer protein 8; SCP3, synaptonemal complex protein 3; REC8, meiotic recombination protein; SMC1B, structural maintenance of chromosomes protein 1B; Miwi, murine piwi gene; cAMP, Cyclic Adenosine monophosphate.