Ganoderma lucidum polysaccharide ameliorated diabetes mellitus-induced erectile dysfunction in rats by regulating fibrosis and the NOS/ERK/JNK pathway

Abstract

Background: Diabetes mellitus-induced erectile dysfunction (DMED) is a frequent complication of diabetes mellitus (DM), with limited therapy at present. This study aimed to explore the role and mechanism of Ganoderma lucidum polysaccharide (GLP) on DMED.

Methods: DMED was induced in the experimental rats [male 12-week-old Sprague-Dawley (SD) rats] by treatment with streptozotocin (60 mg/kg) and apomorphine (APO). Next, rats in the GLP low dose (GLP-L)/GLP high dose (GLP-H) groups were treated with GLP (100 or 400 mg/kg/d, respectively) for 8 weeks. Subsequently, erectile function was assessed by APO and electrostimulation of the cavernous nerve (CN). Serum or penile testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and cyclic guanosine monophosphate (cGMP) contents were evaluated by enzyme-linked immunosorbent assay (ELISA). The levels of oxidative stress indicators in the corpus cavernosum (CC) were measured by corresponding kits, and histological changes in the CC were observed by hematoxylin-eosin (HE) and Masson staining. Additionally, the apoptosis index, caspase-3, caspase-9, and eNOS expression, and mitochondrial membrane potential (MMP) were also detected. Furthermore, quantitative polymerase chain reaction (qPCR) and western blot assays were conducted to determine the NOS, TGF-β1 mRNA expression, ERK1/2, eNOS, JNK phosphorylation, and arginase II protein expression.

Results: The erectile function test revealed that erectile dysfunction (ED) was alleviated in the DMED rats following treatment with GLP. Moreover, GLP upregulated the T and cGMP content, improved the oxidative stress and histological injuries of CC, and also inhibited the apoptosis and MMP loss of penile tissues in DMED rats. Furthermore, GLP treatment enhanced the mRNA expression of NOS and TGF-β1 and suppressed the phosphorylation of ERK1/2, eNOS, and JNK, as well as the protein expression of arginase II in DMED rats.

Conclusions: GLP ameliorated DMED by repairing the CC pathological damage and upregulating NOS expression and ERK/JNK phosphorylation, indicating that GLP may be a candidate drug for DMED therapy.

Keywords: Ganoderma lucidum polysaccharide (GLP); NOS/ERK/JNK pathway; corpus cavernosum (CC); diabetes mellitus-induced erectile dysfunction (DMED); fibrosis.

Figure 1

GLP upregulated the body weight and downregulated blood glucose in DMED rats. The body weight and blood glucose level of the DMED rats were recorded during GLP treatment. ^@@P<0.01 vs. NC; *P<0.05, and **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; DMED, diabetes mellitus-induced erectile dysfunction.

Figure 2

GLP improved ED in DMED rats. (A) The number and latency of erections after GLP treatment. (B) The representative waves of ICP and MAP as well as the value of ICP/MAP after GLP treatment. ^@@P<0.01 vs. NC; **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; ICP, intracavernous pressure; MAP, mean arterial pressure; ED, erectile dysfunction; DMED, diabetes mellitus-induced erectile dysfunction.

Figure 3

GLP increased the T and cGMP levels in DMED rats. ELISA results showing the levels of T, LH, and FSH in the serum and cGMP in the penile tissue after GLP treatment. ^@@P<0.01 vs. NC; **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; T, testosterone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; cGMP, cyclic guanosine monophosphate; DMED, diabetes mellitus-induced erectile dysfunction; ELISA, enzyme-linked immunosorbent assay.

Figure 4

GLP attenuated the oxidative stress of the CC in DMED rats. The results showing the levels of MDA, GSH and SOD in the CC tissues after GLP treatment. ^@@P<0.01 vs. NC; *P<0.05 and **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; MDA, Malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; CC, corpus cavernosum; DMED, diabetes mellitus-induced erectile dysfunction.

Figure 5

GLP alleviated the histological changes of the CC in DMED rats. (A) Representative image of HE staining after GLP treatment (×200 and ×400). (B) Representative image of Masson staining and the value of CF/MF in CC after GLP treatment (×200 and ×400). ^@@P<0.01 vs. NC; **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; CF/MF, collagen fiber/muscle fiber; CC, corpus cavernosum; DMED, diabetes mellitus-induced erectile dysfunction; HE, hematoxylin-eosin.

Figure 6

GLP prevented the increase in cell apoptosis in the penile tissue of DMED rats. (A) Representative image of TUNEL assay and quantification of the apoptosis index after GLP treatment (×400). (B) Representative images of the caspase-3 and caspase-9 proteins assessed by immunohistochemistry (×400). ^@@P<0.01 vs. NC; **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4',6-diamidino-2-phenylindole; DMED, diabetes mellitus-induced erectile dysfunction.

Figure 7

GLP ameliorated the loss of MMP in the penile tissue of DMED rats. ^@@P<0.01 vs. NC; **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; MMP, mitochondrial membrane potential; DMED, diabetes mellitus-induced erectile dysfunction.

Figure 8

GLP enhanced the expressions of NOS and TGF-β1 in the penile and CC tissues of DMED rats. (A) The mRNA expressions of NOS and TGF-β1 were measured by qPCR. (B) The representative images of eNOS protein expression were assessed by immunohistochemistry (×400). (C) The expressions of eNOS and p-eNOS were assessed by western blot. ^@@P<0.01 vs. NC; **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; CC, corpus cavernosum; DMED, diabetes mellitus-induced erectile dysfunction; qPCR, quantitative polymerase chain reaction.

Figure 9

GLP suppressed the phosphorylation of ERK1/2 and JNK as well as the protein expression of arginase II in the cavernosum tissue of DMED rats. ^@@P<0.01 vs. NC; *P<0.05, and **P<0.01 vs. MC. The results were presented as the mean ± standard deviation. n=6. NC, normal control; MC, model control; GLP-L, GLP low dose; GLP-H, GLP high dose; GLP, Ganoderma lucidum polysaccharide; DMED, diabetes mellitus-induced erectile dysfunction.