Search

To verify the effect of Buyang Huanwu Decoction (BHD) in ameliorating erectile dysfunction (ED) after radical prostatectomy (RP).The composition of BHD was verified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) analysis. Bilateral cavernous nerve crush injury (BCNI) in rats was used to mimic the neurovascular injury occurring after RP. By the envelope method, forty rats were randomly divided into 4 groups as follows: sham (cavernous nerves exposed only), model (BCNI), low-dosage BHD [LBHD, 12.8 g/(kg·d)], and high-dosage BHD [HBHD, 51.2 g/(kg·d)] groups, 10 rats in each group, feeding for 3 weeks respectively. Erectile function was evaluated by measuring intracavernosal pressure (ICP). Changes in the histopathology of corpus cavernosum (CC) were examined by hematoxylin-eosin staining. Meanwhile, the fibrosis of CC was measured by Masson's trichrome staining and Western blot was used to detect the expressions of collagen I, transforming growth factor beta 1 (TGF- β 1) and α-smooth muscle actin (α-SMA). Apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and Western blot for determining the expressions of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax). The oxidative stress in the CC were assessed by the superoxide dismutase (SOD), malondialdehyde (MDA) and reactive oxygen species (ROS) levels. The proteins expression of c-Jun N-terminal kinase (JNK) and c-Jun were detected by Western blot. In addition, the expression of α-SMA and p-c-Jun in the CC was observed by double immunofluorescence staining.The UPLC-QTOF-MS/MS analysis showed that BHD contained calycosin-7-O- β -D-glucoside, ononin, calycosin and formononetin. Compared with the model group, LBHD and HBHD treatment improved the ICP and the circumference, area, and weight of CC (P0.05 or P0.01). Furthermore, LBHD and HBHD treatments increased CC smooth muscle content and decreased apoptosis index (P0.05 or P0.01). LBHD and HBHD also elevated SOD and expression level of α -SMA and Bcl-2, and reduced MDA and ROS levels, as well as expression of TGF- β 1, collagen I, Bax, p-c-JNK, p-JNK in the CC compared with the model group (P0.05 or P0.01). The double immunofluorescence staining showed that the fluorescence degree of p-c-Jun in both LBHD and HBHD treatment groups was significantly reduced, whereas the α -SMA expression increased (P0.05 or P0.01).BHD can improve ED of rats with BCNI, which is related to inhibiting fibrosis, apoptosis, and oxidative stress of CC. The ROS/JNK/c-Jun signaling pathway may play an important role in the process.

Supplementary Material 1

Additional file 1: Fig. S1. Schematic diagram of Pelage DPCs isolation. Fig. S2. Morphology, adhesion and cell migration of dermal papilla cells. Whisker (A) and Pelage (B) follicles morphology observed under stereoscope, no blood sinus was found in pelage follicle, the number of pelage follicles was much more than whisker follicles. Hair shafts and hair bulb located in the same suspensions after enzyme digestion and scarping (C). After two-steps isolation, several Pelage DP spheres can be seen under microscope (D-F, Red arrow). Whisker DP spheres were observed under microscope (G, white arrow). Pelage DPCs started emigration in 24 h (H, white arrow showed DPCs started emigrated out of the DP sphere) and proliferation, most of pelage DPCs finished emigration in 4 days (I, white arrow showed plenty of DPCs emigrated out of the DP sphere), while whisker DPCs started emigrate in 72 h and finished in 7 days (J). Pelage DPCs (K) and whisker DPCs (L) showed similar cell morphology and aggregation growth pattern. Fig. S3. Differential expression of Sox2 between whisker and pelage DPCs. Sox2 expression was detected in primary DP sphere and cultured DPCs. Whisker DP showed high Sox2 expression both in DP sphere and DPCs, while Pelage DP showed almost no expression on Sox2 whether in primary DP sphere or cultured DPCs. Fig. S4. Isolation and identification of Pelage HF-MSC and whisker HF-MSC exosomes. The morphology of exosomes was observed by electron microscope; Western Blotting verification specific markers CD9 and CD81; The detection particle size was between 30 and 100 nm. Fig. S5. Effect and distribution of Whisker HF-MSC treatment on dorsal hair growth. 3-week-old C57BL/6 mice were divided into Pelage HF-MSC group and control group. Pelage HF-MSC group enter into anagen period earlier than control group, which was similar to Pelage HF-MSC group. Labeling whisker HF-MSC with DIO (green), showed that whisker HF-MSC located around the host hair follicle (White arrow). Fig. S6. Effect and distribution of whisker HF-MSC Exosomes treatment on dorsal hair growth. 7-week-old C57BL/6 mice were divided into whisker HF-MSC exosomes group and control group (The same as the control group in Fig. 6). Exosomes were injected every other day, a total of three times injection were performed. On day 8, whisker HF-MSC exosomes group enter into anagen period earlier than control group. Injected whisker HF-MSC exosomes were labeled with DIO (green) and located around host hair follicle (white arrow). Fig. S7. Pictorial diagram to show the location of MSCs and exosomes injection protocol. The cell/exosomes suspension was injected along the midline of the back of the mouse, and the amount of cell suspension was 300 µl. HF-MSCs suspension injection was performed once, exosomes suspension injection was performed.

To explore the effects of Hongjing-1 Recipe (HJ-1) on erectile function and the expression of the gap junction protein Connexin43 (Cx43) in the penile tissue in male rats with bilateral cavernous nerve injury (BCNI).Fifty male SD rats were randomly divided into five groups of an equal number: sham operation, BCNI model control, and low-, medium- and high-dose HJ-1. The BCNI model was made in the latter four groups by clamping the bilateral cavernous nerves with hemostatic forceps. Three days after modeling, the rats in the sham operation and BCNI model control groups were treated intragastrically with pure water and those in the low-, medium- and high-dose HJ-1 groups with HJ-1 at 2.835, 5.67 and 11.34 g/kg/d, respectively, all for 28 successive days. Then, the animals were subjected to intracavernous pressure (ICP) measurement for evaluation of their erectile function and immunofluorescence staining and Western blot for determination of the Cx43 level in the penile tissue.The BCNI model controls, compared with the rats in the sham operation group, showed a dramatically decreased ratio of maximum ICP to mean arterial pressure (mICP/MAP) (0.40 ± 0.04 vs 0.83 ± 0.10, P0.01) and that of total ICP to MAP (tICP/MAP) (21.89 ± 2.16 vs 50.27 ± 4.45, P0.01), as well as a down-regulated expression of Cx43 in the penile tissue (P0.01). In comparison with the rats in the BCNI model control group, those in the medium- and high-dose HJ-1 groups exhibited significantly increased ratios of mICP/MAP (0.54 ± 0.05, P0.05; 0.61 ± 0.06, P0.01) and tICP/MAP (31.20 ± 3.85, P0.01; 37.82 ± 4.17, P0.01) and up-regulated expression of Cx43 (P0.05 and P0.01).Hongjing-1 Recipe can effectively improve ED in rats with bilateral cavernous nerve injury, which may be attributed to its effect of maintaining the expression level of the gap junction protein Cx43 in the penile tissue.