High-Fat Diet Induced Gut Microbiota Alterations Associating With Ghrelin/Jak2/Stat3 Up-Regulation to Promote Benign Prostatic Hyperplasia Development

Abstract

The role of high-fat diet (HFD) induced gut microbiota alteration and Ghrelin as well as their correlation in benign prostatic hyperplasia (BPH) were explored in our study. The gut microbiota was analyzed by 16s rRNA sequencing. Ghrelin levels in serum, along with Ghrelin and Ghrelin receptor in prostate tissue of mice and patients with BPH were measured. The effect of Ghrelin on cell proliferation, apoptosis, and induction of BPH in mice was explored. Our results indicated that BPH mice have the highest ratio of Firmicutes and Bacteroidetes induced by HFD, as well as Ghrelin level in serum and prostate tissue was significantly increased compared with control. Elevated Ghrelin content in the serum and prostate tissue of BPH patients was also observed. Ghrelin promotes cell proliferation while inhibiting cell apoptosis of prostate cells. The effect of Ghrelin on enlargement of the prostate was found almost equivalent to that of testosterone propionate (TP) which may be attenuated by Ghrelin receptor antagonist YIL-781. Ghrelin could up-regulate Jak2/pJak2/Stat3/pStat3 expression in vitro and in vivo. Our results suggested that Gut microbiota may associate with Ghrelin which plays an important role in activation of Jak2/Stat3 in BPH development. Gut microbiota and Ghrelin might be pathogenic factors for BPH and could be used as a target for mediation.

Keywords: Ghrelin; Jak2/Stat3; benign prostatic hyperplasia; gut microbiota; metabolic syndrome.

FIGURE 1

Gut microbiota associated ghrelin to induce BPH in mice. Normal, mice fed with regular chow diet for 12 weeks; Control: mice fed with high-fat diet (HFD) for 12 weeks; BPH group: mice fed with HFD for 12 weeks and received testosterone propionate (TP) (7.5 mg/kg body weight, s.c.) daily for 2 weeks; BPH + NGM: BPH mice planted with gut microbiota from normal mice group. n = 6. Gut microbiota was analyzed by 16s rRNA sequencing. (A,B) The relative abundance of gut microbiota changes in Normal, Control, and BPH animals. (C) The ratio of Firmicutes and Bacteroidetes in Normal, Control and BPH animals was showed. n = 6. (D) Ghrelin level in serum of animals was measured by Elisa kit. n = 6. (E) The prostatic index (PI, prostate weight mg/100 g body weight) of mice in Normal, Control, and BPH group. n = 6. (F) Ghrelin and Ghrelin receptor expression of prostate tissue detected by immunohistochemistry. Normal, mice fed with regular chow diet for 12 weeks; HFD: mice fed with HFD for 12 weeks; BPH group: mice fed with HFD for 12 weeks and received testosterone propionate (TP) (7.5 mg/kg body weight, s.c.) daily for 2 weeks; Normal + HFD_GM: Normal mice planted with gut microbiota from HFD mice group; BPH + YIL-781: BPH mice injected with YIL-781 (5 mg/kg body weight, i.p.) daily for a week. n = 5. (G) The ratio of Firmicutes and Bacteroidetes in the animals. (H) The prostatic index (PI, prostate weight mg/100 g body weight). n = 5. (I) Ghrelin level in serum of animals was measured by Elisa kit. n = 5. *p < 0.05, **p < 0.01 compared with normal.

FIGURE 2

Ghrelin or/and Ghrelin receptor measured in the serum and prostate tissue in the BPH patients. Compared with normal (n = 10), Ghrelin levels in the BPH patients (n = 30) are significantly increased (p < 0.01). (A) Ghrelin levels in the BPH patients’ serum might be positively correlated with prostate volume. (B) Ghrelin expression analyzed by immunohistochemistry (C) and western blotting (D–F) was significantly increased in prostate tissue of all BPH patients (n = 7) compared with normal prostate tissue while Ghrelin receptor increased in parts of samples of BPH tissue in our study (**p < 0.01 compared with normal prostate tissue).

FIGURE 3

Ghrelin treatment enhanced cell proliferation induces protein Jak2 and Stat3 expression of both prostate epithelial cells (RWPE-1) and stromal cells (WPMY-1). Ghrelin (1 μmol/L) increases the proliferation observed using CCK-8 kit (A,B) and 3D cell culture (C) which could be attenuated by Jak2/STAT3 inhibitor WP1066 (1 μM). Jak2 and STAT3 expression was significantly up-regulated in Ghrelin treated cells measured by western blotting showed in (D) RWPE-1 and (E) WPMY-1. (n = 5) *p < 0.05, **p < 0.01 compared with Control (Ghrelin(-)); #p < 0.05, ##p < 0.01 compared with Control (Ghrelin + WP1066).

FIGURE 4

Ghrelin treatment inhibited cell apoptosis of both prostate epithelial cells (RWPE-1) and stromal cells (WPMY-1). Ghrelin (1 μmol/L) reduces the apoptosis of the RWPE and WPMY cell lines (A–C) which could be attenuated by Jak2/STAT3 inhibitor WP1066 (1 μM). Bcl2 and BAX expression measured by western blotting confirmed the effect of Ghrelin on apoptosis (D,E). (n = 5) **p < 0.01 compared with Control (Ghrelin(-)).

FIGURE 5

Ghrelin treatment enlarges prostate glands in mice. (A) The HE stains of prostatic tissue sections. Increased epithelial cells hyperplasia and enhanced connective tissue with hyperplastic dilated glands were observed in Ghrelin treated mice (after castration, 0.15 mg/kg body weight, i.p., daily for 1 week, n = 5). (B) The effect of Ghrelin was equivalent to that of Testosterone propionate (TP, 7.5 mg/kg body weight, s.c., daily for 2 weeks, n = 5) on the prostatic index (PI) (prostate weight mg/100 g body weight). The expression of Ghrelin and its receptor was increased in Ghrelin treated mice and TP also tended to induce enhanced Ghrelin and Ghrelin receptor expression measured by immunohistochemistry (C) and western blotting (D–F). **p < 0.01 compared with Control (n = 5, Normal mice with placebo). ##p < 0.01 compared with Model (Mice treated with Testosterone propionate, 7.5 mg/kg body weight, s.c., daily for 2 weeks, n = 5). The samples for detection in panel D are the same as that in Figure 6D and GAPDH blots was the same.

FIGURE 6

Ghrelin affects BPH development through Jak2/Stat3 signaling pathway in vivo. Mice after castration treated with Ghrelin (0.15 mg/kg body weight, i.p., daily for 1 week, n = 5) showed increased Jak2/Stat3 analyzed by immunohistochemistry (A). Compared with control group (Normal mice with placebo) and Testosterone propionate (TP) (7.5 mg/kg body weight, s.c., daily for 2 weeks, n = 5) treated group, not only Jak2/Stat3 mRNA detected by qPCR (B,C) but also protein expression including pJak2/pStat3 measured by western blotting (D–H) was significantly increased in Ghrelin treated group. *p < 0.05, **p < 0.01 compared with Control (n = 5, Normal mice with placebo). ##p < 0.01 compared with Model (Mice treated with Testosterone propionate, 7.5 mg/kg body weight, s.c., daily for 2 weeks, n = 5). The samples for detection in panel D are the same as that in Figure 5D and GAPDH blots was the same.