Di-p-coumaroyl spermidine from bee pollen alleviates chronic non- bacterial prostatitis

Di-p-coumaroyl spermidine from bee pollen alleviates chronic non- bacterial prostatitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Di-p-coumaroyl spermidine from bee pollen alleviates chronic non- bacterial prostatitis Jie Dong, Jiawen Zhang, Jiangtao Qiao, Yu Zhang, Hequan Zhu, Eric Haubruge, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5846138/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bee pollen and its extracts have been used for decades as therapeutic agents or health food supplements to alleviate chronic non-bacterial prostatitis (CNP). However, functional compounds in bee pollen on anti-CNP remain still unclear. In this study, we evaluated the anti-CNP properties of six principal phenolamides in bee pollen. Our results provide compelling evidence that the anti-CNP property of bee pollen may be ascribed to its abundance of phenolamides. Particularly, di- p -Coumaroyl spermidine can alleviate CNP by upregulating autophagy via the AMPK/mTOR signaling pathway and regulating gut microbiota, based on the cellular and rat models. Additionally, our finding may provide a novel insight into the gut-prostate axis by regulating di- p -Coumaroyl spermidine. This is the first report that di- p -Coumaroyl spermidine in bee pollen possesses the anti-prostatitis function. This paper will be likely helpful further to develop functional foods, personalized nutraceuticals, and medicine from bee pollen. Health sciences/Health care Health sciences/Health care/Nutrition Biological sciences/Cell biology/Autophagy phenolamides rapeseed bee pollen AMPK/mTOR anti-inflammation di-p-Coumaroyl spermidine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction Pollen, comprising the male gametophytic cells of angiosperms, is a finely powdered substance characterized by microscopic grains encapsulating the plant's male cells 1 . Embedded within the realm of traditional medicine and functional food for centuries, bee pollen has been ascribed with manifold therapeutic properties, including anti-inflammatory, antioxidant, antimicrobial, and immune-enhancing attributes 2 . In recent decades, there has been a renewed surge in the attention devoted to bee pollen, driven by its pronounced effectiveness in mitigating benign prostatic hyperplasia and chronic prostatitis 3 – 5 . As the global population ages, the incidence of chronic prostatitis in men is increasing, with older age being a significant risk factor for the development of this condition. Chronic non-bacterial prostatitis (CNP) is clinically defined as a persistent inflammatory process leading to chronic pelvic pain or discomfort accompanied by urinary symptoms and sexual dysfunction 6 . It has emerged as a significant male healthcare issue, attracting considerable attention from clinicians and researchers alike 7 . Notably, a clinical research study conducted across North America, Europe, and Asia revealed that the prevalence of chronic prostatitis ranges from 2–10% among adult males at any given time, while approximately 15% of men experience prostatitis symptoms during their lifetime 7 . Furthermore, previous investigations have indicated a potential link between chronic prostatitis and an increased risk for developing benign prostatic hyperplasia and prostate cancer 8 . Fortunately, researchers have discovered that pollen or pollen extracts demonstrate notable efficacy in preventing and treating chronic prostatitis, leading to their widespread application worldwide 4 , 5 , 9 – 12 . In China, Pule'an capsules, which are made entirely from rapeseed bee pollen, have emerged as the prevailing pharmaceutical option for chronic prostatitis and prostate hyperplasia 3 . Clinical trials have shown that Pule'an capsules can effectively alleviate the symptoms of chronic prostatitis and prostate hyperplasia patients, with a symptom relief rate exceeding 90% 3 . Moreover, rapeseed bee pollen extracts have been discovered to substantially alleviate symptoms associated with chronic nonbacterial prostatitis 13 , 14 . This effect is achieved through modulating some key molecular factors, including the downregulation of mitofusin-1 (Mfn1) levels within the posterior lobes of the prostate, as well as the suppression of DHT, 5α-reductase, and Cyclooxygenase-2 (COX-2) levels 13 , 14 . In Europe and Japan, Cernitin™ (pharmaceutical brand, Cernilton®), ryegrass pollen extract, has been used to treat nonbacterial prostatitis and prostatic hyperplasia for more than 40 years with an overall success rate of 70% 15,16 . Cernitin™ can notably reduce the prostate-specific antigen levels associated with chronic prostatitis/chronic pelvic pain syndrome. In preclinical studies, Cernitin™ demonstrated significant pain relief in an induced prostatitis rat model, concomitant with a noteworthy decrease in intraprostatic levels of COX-2 and MCP-1 15–17 . Cernitin™ consists of two fractions of pollen extract: water-soluble T60 and lipid-soluble GBX 15 – 17 . The two fractions can distinctly reduce inflammation, inhibit cellular proliferation, and relax smooth muscle in animal models 11 . Evidence suggests that the anti-CNP effect of the T60 fraction is attributed to feruloyl putrescine 11 . Feruloyl putrescine is formed by ferulic acid and putrescine through an amide bond, a type of phenolamide. Phenolamides, also known as hydroxycinnamic acid amides, result from the conjugation of hydroxycinnamic acids (for example, p -coumaric, ferulic, and caffeic acid) with aliphatic or aromatic amines, such as putrescine, spermine, and spermidine. Our previous study has shown that bee pollen is a treasure trave of phenolamides 18 . Sixty-four phenolamides were identified in 20 types of monofloral bee pollen, with the highest contents among known natural products 18 . Moreover, in the last five years, multiple studies have reported the presence of abundant phenolamides in bee pollen 19 – 21 . Hence, the robust inhibitory functionality of bee pollen or extracts against prostatitis may be attributed to its abundant phenolamides. However, apart from feruloyl putrescine, the anti-CNP activities of other phenolamides from bee pollen remained unclear. The current study aimed to evaluate the anti-CNP effects and mechanisms of phenolamides in bee pollen. Firstly, we employed an LPS-induced prostate epithelial cell (RWPE-1) model to screen anti-prostatitis activities from 6 representative phenolamides in bee pollen. Subsequently, we elucidated anti-prostatitis mechanisms using a CNP rat model. 2 Materials and methods 2.1 Materials N(E), N' (E)-di- p -coumaroyl putrescine, N1(E), N10(E)-di- p -coumaroyl spermidine, N1(E), N5(E), N10(E)-tri- p -coumaroyl spermidine, N1(E), N5(E), N10(E), N14(E)-tetra- p -coumaroyl spermine and N1(E), N10(E)-di- p -coumaroyl-N14(E)- feruloyl spermine were prepared (purity ≥ 95%) according to our previous study 18 . N(E)-feruloyl putrescine (purity ≥ 95%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). All the chemical structures are shown in Fig. 1 A. Human TNF-α, IL-1β, IL-6 and Rat TNF-α, IL-1β, IL-6 Kit purchased from Solarbio (Beijing, China). NO kits (Beyotime, Nanjing, China). Anti-TNF-alpha Rabbit pAb (Servicebio, GB11188), Anti -IL-1 beta Rabbit pAb (Servicebio, GB11113), Anti -vIL-6 Rabbit pAb (Servicebio, GB11117), Cy3 conjugated Goat Anti-Rabbit IgG (H + L) (Servicebio, GB21303). Beclin-1 pAb (YT5763), LC3 A/B rabbit pAb (YT7936), GAPDH mAb (YM3029) purchased from ImmunoWay Biotechnology Company (TX,75024 USA). Rabbit anti-p62 antibody (5114), p-AMPK (2535), AMPK (5831), p-mTOR (5536) and mTOR (2983) from Cell Signaling Technology (Danvers, MA, USA). Complete Freund's adjuvant (CFA) was purchased from Sinopharm Co., Ltd (Shanghai, China). Cole's Hematoxylin solution, Eosin Y solution, Ethylene Diamine Tetraacetic Acid (EDTA) pH 8.0 were obtained from Abcam (Cambridge, MA, USA). All other experimental materials and consumables were purchased from Solarbio Life Sciences (Beijing, China). 2.2 RWPE-1 cell culture The immortalized human prostate epithelial cell line (RWPE-1) was purchased from BNCC (Henan, China). RWPE-1 cells were cultured in Keratinocyte Medium (ScienceCell, San Diego, California, USA) supplemented 5 ml of Keratinocyte Growth Supplement (KGS, Cat#2152) and 5 ml of penicillin/streptomycin solution (P/S, Cat. #0503) in a humidified 37°C incubator with 5% CO 2 . The model of chronic nonbacterial prostatitis (CNP) was induced by 10 µ/ml lipopolysaccharide (LPS) for 24 h. 2.3 Cell Viability Assays RWPE-1 cell viability was evaluated using the methyl thiazolyl tetrazolium assay (MTT, Solarbio, Beijing, China). RWPE-1 cells (20,000 cells/well) were seeded in a 96-well plate and incubated at 37°C for 24 hours. Subsequently, the culture medium was replaced respectively with 1mL fresh Keratinocyte medium containing 0.1% DMSO as a control group and 1mL various concentrations of phenolamides (0, 1, 5, 10, 20, 40, 80 µmol/L) as treatment groups. After the treatment, MTT solution (5 mg/mL) was added to each well and incubated for 4 hours. The absorbance was measured at 490 nm using a microplate reader (Biotek, USA). All experiments were performed in triplicate. Cell viability was calculated as a percentage using the following formula: cell viability = [(mean absorbance of each treatment group) / (mean absorbance of the control group)] × 100%. 2.4 Inflammatory cytokines and NO production of RWPE-1 cells Th RWPE-1 cells were incubated in 24-well culture plates and treated with different concentrations of phenolamides (0, 1, 5, 10, 20, 40, 80 µmol/L) for 2 h, and then the cells were stimulated with 5 µg/mL LPS and incubated for 24 h. TNF-α, IL-1β, and IL-6 levels were quantified using a commercially available ELISA kit from Solarbio (Beijing, China). Based on Greiss reaction, the nitric oxide (NO) concentration in the cell culture media was determined using NO kits (Beyotime, Nanjing, China). 2.5 Animal model All animal experimental procedures followed the Guidelines for the Care and Use of Laboratory Animals. Fifty-two-month-old male Sprague- Dawley (SD) rats (190– 220 g) were obtained from the Si Pei Fu (SPF biotechnology Co., Ltd., Beijing, China, licenses: 110324231104817715; animal use licenses: SYXK(JING) 2023-0004). We kept all the animals under specific pathogen-free (SPF) environmental conditions at 23 ± 2°C and relative humidity of 55 ± 5%. Besides, the rats had free access to food and water in a 12 h dark-light cycle (light cycle: 7:00 am–7:00 pm) and were given one week to adapt to the new environment before the experiment. The experimental Animal Welfare Committee of the Chinese Academy of Agricultural Sciences approved ethical requirements (permit number: IARCAAS-2019-003). Thirty-six rats were randomly divided into four groups (n = 9 rats/group), including the control group, model group, and p -Cspd low or high dose group. The rat model of CNP was established using complete Freund's adjuvant (CFA) as previously described method with slight modification 22 . Apart from the control group, the intervention involved the injection of a sterile CFA suspension (100 µL, 1% w/v) into the right and left lobes of the ventral prostate; while for the sham-operated rats (n = 9), the same volume of sterile saline was injected. The wound was then closed in layers. During surgical procedures, isoflurane inhalation anesthesia was used. Isoflurane was administered at an induction concentration of 4%-5% in 100% oxygen and maintained at 1.5%-2.5% during the procedure. The anesthetic gas mixture was delivered through a rodent-specific nose cone to ensure the animals remained unconscious and pain-free. The control group and model group were orally administered 0.5% DMSO (w/w) 1mL for 21 days. The remaining two groups of rats were orally administered 1mL di- p -coumaroyl spermidine ( p -Csp) dissolved in 0.5% DMSO (w/w), according to 5mg/kg (low dosage group, L- p -Csp) and 10 mg/kg (low dosage group, H- p -Csp) body weight for 21 days. On the final day of the experiment (day 22), all rats were humanely euthanized. The euthanasia of animals (rats) was conducted using carbon dioxide (CO₂) asphyxiation, following the AVMA Guidelines for the Euthanasia of Animals. CO₂ was introduced gradually into the chamber at a displacement rate of 30%-70% of the chamber volume per minute to minimize distress, ensuring unconsciousness prior to respiratory arrest. Subsequently, the prostate tissues were carefully harvested and promptly weighed. The prostate index was then computed as the ratio of the prostate weight to the total body weight. 2.5 Histopathology and Immunohistochemical The prostate specimens were fixed in 4% formaldehyde overnight for histopathological examination. Fixed prostate specimens were embedded in paraffin and sectioned with a microtome (5 µm) using a Leica RM2255 Microtome (Leica Biosystems, Leica, Germany). After deparaffinization with xylene and dehydration using alcohol, the sections were subjected to hematoxylin-eosin staining. Subsequently, the glandular epithelium, structure, and space were meticulously examined using a Nikon Eclipse Ci-L microscope equipped with a Nikon digital sight DS-Fi2 system (Nikon, Tokyo, Japan). Histomorphometry parameters of the normal prostate were determined based on the analysis from the control group. Slides containing 5 µm thick rat prostate sections were meticulously rinsed with phosphate-buffered saline (PBS), then permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, followed by blocking with 2% bovine serum albumin (BSA) in PBS for 30 minutes. The primary antibody was applied and left to incubate overnight at a temperature of 4°C. Subsequently, the slides were thoroughly washed with PBS and incubated with a secondary antibody at room temperature for 50 minutes. After another round of meticulous washing, the immunofluorescent images were captured using an 80i Nikon microscope. The images were captured and analyzed with Aipathwell software (Servicebio, Wuhan, China). The positive cell ratio was calculated to assay the expression of IL-6, IL-1β, and TNF-α. Positive cells ratio = the number of positive cells / total number of cells. Moreover, a NIKON Eclipse Ti confocal laser scanning microscope with DS-U3 imaging system (Nikon, Tokyo, Japan) to scan inflammatory cytokines for visualization. 2.6 Inflammatory Cytokines In this study, we determined the levels of IL-6, IL-1β, and TNF-α in both serum and prostate tissue using ELISA kit. For serum analysis, each sample was meticulously collected in serum separator tubes (BD Biosciences, Rockville, USA) and allowed to clot for two hours at room temperature. Following the clotting period, centrifugation was performed at approximately 1000 × g for 20 minutes to remove the clots and obtain the serum samples. As for the prostate tissue, a rigorous protocol was employed to ensure proper sample preparation. Each prostate tissue specimen was meticulously rinsed with ice-cold PBS (0.01M, pH = 7.4) to remove residual blood thoroughly. Subsequently, the prostate samples were weighed, and using an electric homogenizer, they were homogenized in ice-cold PBS. The homogenates were centrifuged at 5000 × g for 5 minutes to obtain the supernatants. The ELISA procedures were carried out in accordance with the manufacturer's protocols to ensure standardization and accuracy. Each ELISA assay was repeated three times. The data were analyzed meticulously, and statistical significance was established at p < 0.05. 2.7 Quantitative real-time PCR The prostate tissues were thoroughly ground in liquid nitrogen, and subsequently, total RNA was extracted utilizing the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was subjected to reverse transcription using the PrimeScript RT reagent kit (Takara, Beijing, China). Real-Time PCR was conducted on a 7500 System (Applied Biosystems, Inc., Carlsbad, CA, USA) following a two-step reaction procedure. The expression of the housekeeping gene Gapdh was used to normalize the expression levels. The primers are designed to flank introns with Primer Premier 6.0 software (Premier Biosoft, Palo Alto, CA, USA). The melting curve checked the specificity of the primers. DNA sequencing and electrophoresed on the agarose gel have also tested the PCR products. 2.8 Western blot In brief, the prostate tissues were lysed using the RIPA lysis buffer; then the proteins were quantified using the BCA kit. The protein samples were loaded and separated through SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto the polyvinylidene fluoride (PVDF) membranes, and blocked with 5% skim milk-TBST for 2 h at 20°C. Then, the blots were incubated with primary antibodies (anti-TFEB 1:500, Beclin-1 1: 5000, anti-p62/SQSTM1 1:2000, anti-LC3 1: 2000, and anti-GAPDH 1:20000, p-AMPK 1: 2000, AMPK 1: 2000, p-mTOR 1: 2000 and mTOR 1: 2000). After being washed and incubated with the secondary antibodies. Chemiluminescence, X-ray film compression, development, fixing, and data analysis using Quantity One software (Bio-Rad, CA, USA). The results represent three independent experiments. 2.9 Gut microbiota analysis Before concluding the experiment, fecal specimens were collected and promptly subjected to rapid freezing using liquid nitrogen, then stored at -80°C for subsequent microbial community analysis. The intestinal content samples from four distinct rat groups were then utilized for 16S rRNA gene sequencing, with a sample size of n = 8 in each group. Genomic DNA was isolated from fecal samples of mice in accordance with the QiAamp® Fast DNA Stool Mini Kit protocol. The amplification of 16S rRNA genes corresponding to the hypervariable region (16S V3-V4) was achieved through the utilization of a universal bacterial primer set, with Primer F = 341F (5’-CCTACGGGNGGCWGCAG-3') and Primer R = 805R (5’-GACTACHVGGGTATCTAATCC-3') 23 . Subsequently, library preparation was conducted using the NEBNext Ultra II DNA Library Prep kit (New England Biolabs E7370S/L, Ipswich, MA, USA) and then sequenced on an Illumina MiSeq PE300 platform at Beijing Allwegene Technology Co., Ltd. (Beijing, China), resulting in the generation of 250 bp paired-end reads. These reads were subsequently subjected to data processing steps, including merging, demultiplexing, and quality filtering using QIIME 2.0. Following these procedures, the reads were clustered into Operational Taxonomic Units (OTUs) at a 97% sequence identity threshold. All analytical outcomes were predicated on the characterization of OTUs. Based on OTU levels, alpha, and beta diversity analyses were executed as per our prior study 3 . To pinpoint key OTUs responsive to distinct interventions, we employed Linear Discriminant Analysis (LDA) coupled with effect size assessment. A p-value of < 0.05 determined significance and a minimum LDA score of ≥ 4.0 was deemed statistically significant. 3 Results 3.1 RWPE-1 cell viability The impacts of six phenolamides with different concentrations (Fig. 1 B) on the viability of RWPE-1 cells were evaluated using the MTT assay to determine the optimal dosage levels for subsequent investigations. As shown in Fig. 1 B, all phenolamides did not demonstrate significant growth-inhibitory activities at a concentration of 20 µmol/L ( P <0.05), except P5 and P6. When the concentrations of P5 and P6 exceed 20 µmol/L ( P <0.05), RWPE-1 cell survival was significantly decreased. Based on these results, concentration ranges (P1 to P4 are 1, 5, 10, and 20 µM, P5 and P6 are 1, 5, and 10 µM) were chosen in the subsequent experiments. 3.2 Inflammatory cytokines and NO production To assess the anti-inflammatory effects of different phenolamides, we measured the levels of TNF-α, IL-1β, IL-6, and NO production on LPS-induced RWPE-1 cells after being treated with various concentrations of phenolamides. As shown in Fig. 1 C, LPS dramatically increased the TNF-α, IL-6, IL-1β, and NO levels. Phenolamides P1, P2, P3, and P4 demonstrated a concentration-dependent reduction for the levels of inflammatory cytokines and NO, with the optimal at 20 µM. P5 and P6 exhibit anti-inflammatory activity at their maximum non-toxic concentration (10 µM). Noteworthily, P3 exhibited the most remarkable effects at 20 µM, with respectively decreasing 54.7%, 51.6%, 55.8%, and 67% in the levels of IL-6, TNF-α, IL-1β, and NO, compared to the model group. The above results indicate that among the selected six phenolamides, P3 (N1(E), N10(E)-di- p -coumaroyl spermidine, p -Csp) exhibits a better anti-inflammatory effect. Moreover, the anti-inflammatory efficacy of P3 surpasses that of feruloyl-putrescine (P1), which is the principal active constituent in the conventional pharmaceutical component known as Cernitin™. Consequently, in subsequent investigations, we employ animal models to evaluate the anti-prostatitis activity of p -Csp. 3.3 Prostate wet weight and prostate index Prostate wet weight and the prostate index can reflect the extent of prostate pathology 3 . In this study, complete Freund's adjuvant-induced model of non-bacterial prostatitis was used to evaluate the anti-prostatitis effects of p -Csp. As shown in Fig. 2 , CFA effectively induced a rat model of prostatitis, wherein the model group demonstrated a pronounced elevation in both prostate wet weight and prostate index, increasing 1.78 and 1.96-fold, respectively, compared to the control group. It is worth noting that p -Csp significantly decreased both prostate wet weight and prostate index, exhibiting a concentration-dependent response. In the high dose group (H- p -Csp), prostate wet weight and prostate index respectively reduced 31.6% and 34.4%, compared to the model group (Fig. 2 I, J). 3.4 Prostate histomorphometry Prostate histomorphology can significantly display prostatitis lesions. To further evaluate the therapeutic effect of p -Csp, prostatic histopathology was observed using the HE staining method. As shown in Fig. 2 , the model group (Fig. 2 F) reduced the number of glandular structures, along with connective tissue proliferation (black arrows), and formed numerous empty cavity structures (yellow arrows) and the infiltration of lymphocytes and granulocytes (red arrows), with a limited presence of inflammatory cells penetrating the glandular lumina (purple arrows). Multiple areas displayed signs of edema; connective tissue exhibited a loose arrangement (blue arrows). Eosinophilic material exudation was also observed (green arrows). Additionally, a significant reduction in secretions within many glandular lumina (brown arrows) and noticeable glandular expansion with thinning of glandular epithelium were noted. These evidences indicate the successful establishment of the CNP rat model (Fig. 2 F), compared to the control group (Fig. 2 E). After p -Csp administration for 21 days, the glandular cavity structure of the prostate tissues recovered; the interstitial space, inflammatory cell infiltration in the glandular cavity and interstitial space, and migration of fibroblasts and blood vessels decreased dose-dependent (Fig. 2 G, H). Specifically, the alleviation was most significant in the H- p -Csp (10mg/kg) group, presenting a remarkable decrease in inflammatory cell infiltration and necrotic foci and lacking prostatic hyperplasia, resembling normal tissue (Fig. 2 H). Additionally, a comprehensive examination of the inflammation scores pertaining to histopathological alterations in the prostatic tissue was undertaken for each experimental group to assess the extent of inflammation (Fig. 2 K). Notably, compared to the model group, the groups receiving H- p -Csp treatment exhibited a significant reduction in inflammation scores, almost 67% (Fig. 2 K). 3.5 Immunohistochemistry Immunohistochemistry was used to evaluate the expression of IL-1β, IL-6, and TNF-α in prostate tissue, and the positive cell ratio was calculated. Confocal immunofluorescence imaging was used for the real-time visualization analysis of prostate tissues. As seen in Fig. 3 , red fluorescence represented the inflammatory signal. The intensity increases of red fluorescence indicated a high expression of inflammatory cytokines, while blue fluorescence showed a normal cellular state. Based on the discernible increase in red fluorescence intensity, the model group heightened the expression of inflammatory factors. Conversely, the two groups treated with p -Csp were observed to reduce the levels of all three inflammatory factors and significantly exhibited a concentration-dependent response. By assessing the positive cell ratio, p -Csp exerted a pronounced reversal effect on the elevated expression of IL-6, IL-8, and IL-1β within the prostate tissue induced by CFA. Noteworthily, the most substantial reduction is observed in the H- p -Csp group. IL-1β notably decreased approximately 69.8%, without a statistically significant difference between the H- p -Csp and control groups. The effect of decreasing inflammatory factors in the H- p -Csp group was much better than that in the L- p -Csp group. 3.6 The expression levels of inflammatory cytokines in tissue and serum CNP and prostate carcinoma patients are associated with prolonged overproduction of inflammatory cytokines, including IL-1β, IL-6, and TNF-α. In this study, we determined the levels of IL-1β, IL-6, and TNF-α in both serum and prostate tissue using ELISA kit. Figure 4 illustrates great variations in the levels of these inflammatory cytokines among different treatment groups. In contrast to the control group, the model group greatly elevated the levels of inflammatory cytokines in both blood and tissues. Compared to the model group, two p -Csp treatment groups conspicuously reduced inflammatory cytokine levels; notably, L-6, IL-1β, and TNF-α levels in the H- p -Csp group decreased by approximately 50%. Additionally, gene expression profiles of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were subjected to analysis. In the model group, the genes responsible for encoding IL-1β, IL-6, and TNF-α were conspicuously upregulated, compared to the control group. In contrast, the L- p -Csp and H- p -Csp groups exhibited marked down-regulations of these gene expressions, compared to the model group. This further verified that p -Csp treatment can conspicuously reduce inflammatory cytokine levels in both blood and prostate tissues. 3.7 Autophagy via the AMPK/mTOR signaling pathway Autophagy plays a pivotal role in regulating inflammatory processes 24 . In recent years, multiple studies have reviewed that activating cellular autophagy presents a promising therapeutic approach for managing chronic inflammation 25 – 27 , particularly non-bacterial prostatitis 28 . The main proteins associated with autophagy include LC3 (microtubule-associated protein 1A/1B-light chain 3), Beclin-1, and p62. To investigate the relationship between p -Csp and autophagy, we detected the expression of autophagy-related proteins by Western blot analysis. As shown in Fig. 5 , The LC3- II/LC3-I ratio exhibited no notable alteration between the control and model group. However, p -Csp led to a significant increase in the LC3- II/LC3-I ratio. Moreover, the p -Csp treatment group demonstrated a significant concentration-dependent upregulation of Beclin-1. Moreover, p62 expression was decreased upon p -Csp stimulation. Together, these results suggest that p -Csp can enhance in vivo autophagy. The AMPK-mTOR pathway participates in the cellular response to energy deficiency, ultimately triggering autophagy 29 . Previous studies have offered compelling evidence supporting the notion that the activation of autophagy via the AMPK/MTOR signaling pathway holds significant potential for instigating anti-inflammatory reactions 27 , 28 , 30 . To ascertain whether the role of p -Csp in mitigating prostatitis is through activating autophagy by the modulation of AMPK/MTOR signaling pathway, we detected the expression of AMPK/MTOR by western blot analysis. As seen in Fig. 5 , the p -Csp treatment increased the ratio of p-AMPK/AMPK, whereas decreased ratio of p-mTOR / mTOR. These findings imply that p -Csp may potentiate autophagic processes through modulating AMPK and mTOR signaling pathways. 3.9 Gut microbiota Our previous research has proved that rapeseed bee pollen can mitigate CNP through regulating the gut microbiota 3 . To further investigate the potential role of p -Csp, primarily found in rapeseed bee pollen, in gut microbiota, 16S rDNA amplicon sequencing analysis of rat feces was performed. Chao1 and Shannon indexes expressed the alpha diversity. The Chao1 index estimates the total number of species in the sample, and the Shannon index describes the diversity of the microbial community. As shown in Fig. 6 A and B, alpha diversity with Chao1 and Shannon indexes on the OTU levels was significantly decreased in the model group compared to the control, suggesting that the structural diversity of gut microbiota decreased in the model group, while p -Csp treatment reinstated alpha diversity. Additionally, non-metric multidimensional scaling (NMDS) was employed as beta diversity assessment indexes to observe the structural variability of different species. As depicted in Fig. 6 C, the NMDS analysis unveiled noteworthy difference in the clustering patterns of gut microbiota species across the four experimental groups. A clear demarcation existed between the model and control groups; notably, the H- p -Csp group displayed more distinct segregation from the model group and closer to the control group. Hence, p -Csp can change the gut microbiota structure, potentially contributing to mitigating CFA-induced CNP. To enhance our understanding of the bacterial taxa subject to regulation by p -Csp, a comprehensive assessment of the gut microbiota composition was performed for the four experimental groups. The predominant bacterial phyla in all groups were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria (Fig. 6 D). In the model group, CFA decreased the relative abundance of Bacteroidetes and increased the relative abundance of Firmicutes, thus significantly increasing Firmicutes/Bacteroides (F/B) ratio. Noteworthily, p -Csp treatment reduced the Firmicutes to Bacteroidetes ratio (F/B); for example, H- p- Csp group is closer to the control group (Fig. 6 E). At the genus level (Fig. 6 F), compared to the control group, the model group decreased the relative abundance of Lactobacillus, Prevotella_9, Bifidobacterium and Bacteroides , while increased that of Lachnospiraceae, Romboutsia and Lachnospiraceae_NK4A136_group . This finding suggested that p -Csp can rectify the perturbations in gut microbial populations, with a particularly pronounced increase of Lactobacillus, Prevotella_9, Bifidobacterium , and Lachnospiraceae. Lactobacillus, Bifidobacterium, and Prevotella_9 is widely recognized as probiotic genera known to confer health benefits on host organism. Moreover, linear discriminant analysis (LDA) effect size (LEFSe) results showed that a total of 19 taxa at different OTU levels (LDA > 4) were identified with different abundances in the four groups (Fig. 6 G). Negativicutes phylum were dominant bacteria in the model group, while Firmicutes were dominant bacteria in p -Csp groups (Fig. 6 G). Moreover, Lactobacillaceae and Prevotella were obviously dominant in p -Csp treatment groups. These results suggested that p -Csp can modulate gut microbiota by increasing the abundance of beneficial bacteria in rat. 4 Discussion Bee pollen and its extracts have been employed for over 40 years as dietary supplements or medication on a global scale in the therapeutic management of chronic prostatitis. However, it remains unclear which compounds in bee pollen are responsible for its anti-chronic prostatitis effects and the underlying mechanisms, leading to product instability and lack of predictability. Recent studies have revealed that bee pollen is rich in phenolamides and is hailed as a treasure trove of phenolamides. Moreover, studies have already indicated that one of the components in the prostatitis treatment drug Cernitin™ is phenolamide. We speculated that the anti-chronic prostatitis properties of bee pollen are mainly attributed to its richness of phenolamides. In this study, we evaluated the anti-chronic prostatitis properties of six principal phenolamides in bee pollen based on cellular and rat models. Our results indicate that the six phenolamides exhibited anti-inflammatory activity in RWPE-1 cells induced by LPS. Noteworthily, p -Csp can remarkably decrease the levels of IL-6, TNF-α, IL-1β, and NO, respectively (Fig. 1 C). The anti-inflammatory efficacy of p -Csp surpasses that of feruloyl-putrescine, the principal active constituent (P1) in the conventional medicine Cernitin™. Subsequently, p -Csp also demonstrated a capacity effectively to in vivo mitigate CNP in the rat model, based on prostate wet weight, prostate index, and histomorphometry findings (Fig. 2 ). p -Csp treatments could decrease the levels and gene expression of inflammatory cytokines IL-1β, IL-6, and TNF-α in serum and prostate tissue (Fig. 2 ). These findings are consistent with the immunohistochemistry results (Fig. 3 ). Our previous research has demonstrated that p -Csp is a new found compound and is predominantly present in rapeseed bee pollen with a concentration of 10 mg/g 18 . Previous studies have consistently demonstrated that rapeseed bee pollen surpasses its counterparts in imparting anti-prostatitis efficacy 3 , 18 , 31 . Our findings provide robust evidence that the efficacy of rapeseed bee pollen on prostatitis treatment may be mainly attributable to its high level of p -Csp. Phenolamides are formed by conjugating phenolic acids such as p -coumaric acid, ferulic acid, and caffeic acid with polyamines (putrescine, spermidine, and spermine) through amide bonds. Research indicates that phenolamides present a variety of health benefits, such as anti-inflammatory, antioxidant, and anti-tyrosinase properties 18 . p -Csp is formed by two p -coumaroyl residues bound at the N1 and N14 positions of spermidine. Spermidine has been reported to exert anti-aging effects by stimulating autophagy and is hailed as a "anti-aging vitamin" 18 . As a solitary phenolamide in bee pollen, our results indicate that p -Csp can activate AMPK pathway, suppress the expression of mTOR, increase the LC3- II/LC3-I ratio, upregulate the expression of Beclin-1, and downregulate the expression of p62, thereby inducing and promoting autophagy (Fig. 5 ). Numerous studies have shown that autophagy is dependent on the regulation of intracellular AMPK-mTOR signaling pathway 24 – 26 , 32 . AMPK exerts its inhibitory effect on mTOR by activating the TSC1/TSC2 complex (Tuberous Sclerosis Complex 1/2) 29 . The TSC1/TSC2 complex, by virtue of its capacity to suppress mTOR activity, facilitates the initiation of autophagy 33 . Consequently, the activation of AMPK serves to repress mTOR, thus promoting the process of autophagy 33 . LC3, primarily in its cytosolic LC3-I form, undergoes proteolytic cleavage to LC3-II during autophagy induction 32 . Elevated Beclin-1 triggers autophagosome formation, while p62 accumulates during autophagy inhibition and decreases with active autophagy 32 . Autophagy can attenuate inflammatory processes by facilitating the catabolism of pro-inflammatory cytokines and the clearance of compromised organelles, notably mitochondria, through a selective form of autophagy known as mitophagy 26 , 30 . In recent years, studies have reviewed that activating cellular autophagy presents a promising therapeutic approach for managing chronic inflammation 24 – 26 , 32 . Autophagy diminishes the biosynthesis of pro-inflammatory cytokines, potentially resolving the persistent inflammatory state emblematic of chronic prostatitis. Our results showed that the activating autophagy of p -Csp may be responsible for alleviating CNP (Fig. 5 ). These results are consistent with the previously reported mechanism of rapamycin and IL-37 exerting anti-CNP and anti-inflammation effects by activating autophagy through the AMPK/mTOR pathway 27 , 30 . In this study, we first elucidated the mechanism against chronic prostatitis that phenolamides from bee pollen involve the activation of autophagy via the AMPK/mTOR pathway. The gut microbiota is integral to the preservation of host health 34 . Dysbiosis of the gut microbiota is associated with various inflammatory diseases, such as inflammatory bowel disease 35 , systemic inflammation in stroke patients 36 , and inflammatory conditions in diabetes patients 37 . Dysbiosis involves decreased beneficial bacteria and increased opportunistic pathogens that stimulate proinflammatory mediator synthesis via plasma membrane receptor interaction 3 . Recent studies have revealed a correlation between dysbiosis of the gut microbiota and CNP 3 , 38 . Our results indicate that significant changes occur in the gut microbiota of CNP rats, consistent with those seen in CNP patients 38 . p -Csp treatment can significantly reduce the α-diversity of the gut microbiota caused by prostatitis inflammation (Fig. 6 A and B). Some natural products have shown a similar trend in gut community diversity, including Puerarin and polysaccharides from the fruits of Lycium barbarum L 39 . Furthermore, p -Csp can inhibit pathogenic bacteria and enhance probiotics. Compared to the model group, the H- p -Csp group significantly decreased the F/B ratio by up-regulation of Bacteroidetes expression and down-regulation of Firmicutes expression (Fig. 6 D). The F/B ratio was often used as an indicator to measure the health of intestinal flora; the high F/B ratio is closely related to inflammatory disease 39 . Moreover, our results demonstrated that p -Csp treatment can enrich the abundance of Lactobacillaceae and Prevotella_9 , widely recognized as probiotic genera on host health. This is consistent with our previous report that rapeseed bee pollen can promote the growth of beneficial gut bacteria 3 . Our findings seem to provide a novel insight into modulating the gut microbiota with p -Csp for preemptive intervention to forestall prostatitis, implicating the probable presence of gut-prostate connection. 5 Conclusion Our results reveal that phenolamides in bee pollen may be responsible for anti-CNP, Specially, di- p -coumaroyl spermidine can alleviate CNP by upregulating autophagy via AMPK/mTOR signaling pathway and regulating gut microbiota. Based on our findings, we recommend that rapeseed bee pollen with the high level of di- p -coumaroyl spermidine should be selected to target prostatitis as nutraceuticals and therapeutics. Declarations Acknowledgments This research was supported by the Modern Agro-industry Technology Research System (CARS-45-KXJ19), Innovation funding from Hebei University of Engineering, the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2019-IAR) from the Ministry of Agriculture of P.R. China. The authors would also like to thank the Teaching and Research Center, Gembloux, Belgium. Conflicts of interest The authors declare that there are no conflicts of interest. Author Contributions Jiangtao Qiao and Liqiang Liu conceived the idea; Jiangtao Qiao and Jie Dong designed the experiments; Jiangtao Qiao, Jiawen Zhang and Yu Zhang performed the experiments and analyzed the data; Hequan Zhu and Jie Dong contributed reagents/materials/analysis tools; Jiangtao Qiao and Eric Haubruge wrote and reviewed the paper. A.B. and C.D. wrote the main manuscript text and Jiawen Zhang prepared figures 2 and 3, Jiangtao Qiao prepared figures1, 4,5 and 6. All authors reviewed the manuscript. References Campos, M. G. et al. Pollen composition and standardisation of analytical methods. Journal of Apicultural Research 47 , 154-161, doi:10.1080/00218839.2008.11101443 (2008). Denisow, B. & Denisow‐Pietrzyk, M. Biological and therapeutic properties of bee pollen: A review. Journal of the Science of Food and Agriculture 96 , 4303-4309, doi:10.1002/jsfa.7729 (2016). Qiao, J. et al. Rapeseed Bee Pollen Alleviates Chronic Non‐bacterial Prostatitis via Regulating Gut Microbiota. Journal of the Science of Food and Agriculture , doi:10.1002/jsfa.12878 (2023). Chabot, S. et al. Impact of Cernitin™ on induced chronic prostatitis in animal model for understanding management of lower urinary tract symptoms. Phytomedicine Plus 1 , 100057, doi:10.1016/j.phyplu.2021.100057 (2021). Algethami, J. S. et al. Bee pollen: Clinical trials and patent applications. Nutrients 14 , 2858 (2022). Zhang, J., Liang, C., Shang, X. & Li, H. Chronic prostatitis/chronic pelvic pain syndrome: a disease or symptom? Current perspectives on diagnosis, treatment, and prognosis. American journal of men's health 14 , 1557988320903200, doi:10.1177/155798832090320 (2020). Krieger, J. N., Riley, D. E., Cheah, P. Y., Liong, M. L. & Yuen, K. H. Epidemiology of prostatitis: new evidence for a world-wide problem. World journal of urology 21 , 70-74, doi:10.1007/s00345-003-0329-0 (2003). Zhang, L. et al. Correlation between prostatitis, benign prostatic hyperplasia and prostate cancer: a systematic review and meta-analysis. Journal of Cancer 11 , 177, doi:10.7150/jca.37235 (2020). Martinez-Armenta, C. et al. Therapeutic potential of bioactive compounds in honey for treating osteoarthritis. Frontiers in pharmacology 12 , 642836, doi:10.3389/fphar.2021.642836 (2021). Lazzarotto-Figueiró, J. et al. Antioxidant activity, antibacterial and inhibitory effect of intestinal disaccharidases of extracts obtained from Eugenia uniflora L. Seeds. Brazilian Journal of Biology 81 , 291-300, doi:10.1590/1519-6984.224852 (2020). NAKASE, K., KIMURA, I. & KIMURA, M. Effects of pollen-extract components, diamines and derivatives of feruloylputrescine on isolated bladder and urethral smooth muscles of mice. The Japanese Journal of Pharmacology 53 , 157-164, doi:10.1254/jjp.53.157 (1990). Münstedt, K. & Bogdanov, S. Bee products and their potential use in modern medicine. Journal of ApiProduct and ApiMedical Science 1 , 57-63, doi:10.3896/IBRA.4.01.3.01 (2009). Yang, B. C., Jin, L. L., Yang, Y. F., Li, K. & Peng, D. M. Inhibitory effect of rape pollen supercritical CO2 fluid extract against testosterone‑induced benign prostatic hyperplasia in rats. Experimental and therapeutic medicine 8 , 31-37, doi:10.3892/etm.2014.1680 (2014). Wagner, A. E. et al. DSS-induced acute colitis in C57BL/6 mice is mitigated by sulforaphane pre-treatment. The Journal of nutritional biochemistry 24 , 2085-2091, doi:10.1016/j.jnutbio.2013.07.009 (2013). Thorpe, A. & Neal, D. Benign prostatic hyperplasia. The Lancet 361 , 1359-1367, doi:10.1016/S0140-6736(03)13073-5 (2003). Chung, A., Kini, M., Hartigan, S. M., Chughtai, B. & Dmochowski, R. R. in Molecular Mechanisms of Nutritional Interventions and Supplements for the Management of Sexual Dysfunction and Benign Prostatic Hyperplasia 107-111 (Elsevier, 2021). El-Khatib, F. M., Yafi, N. R. & Yafi, F. A. in Effects of Lifestyle on Men's Health 281-300 (Elsevier, 2019). Qiao, J. et al. Phenolamide and flavonoid glycoside profiles of 20 types of monofloral bee pollen. Food Chemistry 405 , 134800, doi:10.1016/j.foodchem.2022.134800 (2023). Zhang, H. et al. Antioxidant and anti-inflammatory activities of rape bee pollen after fermentation and their correlation with chemical components by ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry-based untargeted metabolomics. Food Chemistry 409 , 135342, doi:10.1016/j.foodchem.2022.135342 (2023). Zhang, X. et al. Phenolamide extract of apricot bee pollen alleviates glucolipid metabolic disorders and modulates the gut microbiota and metabolites in high-fat diet-induced obese mice. Food & Function 14 , 4662-4680, doi:10.1039/D3FO01016C (2023). Kim, S. B. et al. Polyamine derivatives from the bee pollen of Quercus mongolica with tyrosinase inhibitory activity. Bioorganic Chemistry 81 , 127-133, doi:10.1016/j.bioorg.2018.08.014 (2018). Zhao, Q. et al. Lycopene attenuates chronic prostatitis/chronic pelvic pain syndrome by inhibiting oxidative stress and inflammation via the interaction of NF‐κB, MAPKs, and Nrf2 signaling pathways in rats. Andrology 8 , 747-755, doi:10.1111/andr.12747 (2020). Shu, Y. et al. The polysaccharides from Auricularia auricula alleviate non-alcoholic fatty liver disease via modulating gut microbiota and bile acids metabolism. International Journal of Biological Macromolecules 246 , 125662, doi:10.1016/j.ijbiomac.2023.125662 (2023). Harris, J. et al. Autophagy and inflammasomes. Molecular Immunology 86 , 10-15, doi:10.1016/j.molimm.2017.02.013 (2017). Liu, Y. et al. Autophagy alleviates ethanol-induced memory impairment in association with anti-apoptotic and anti-inflammatory pathways. Brain, Behavior, and Immunity 82 , 63-75, doi:10.1016/j.bbi.2019.07.033 (2019). Choi, A. J. & Ryter, S. W. Autophagy in inflammatory diseases. International journal of cell biology 2011 , doi:10.1155/2011/732798 (2011). Fan, X. et al. Berberine alleviates ox-LDL induced inflammatory factors by up-regulation of autophagy via AMPK/mTOR signaling pathway. Journal of translational medicine 13 , 1-11, doi:10.1186/s12967-015-0450-z (2015). Su, Y. et al. Rapamycin alleviates hormone imbalance-induced chronic nonbacterial inflammation in rat prostate through activating autophagy via the mTOR/ULK1/ATG13 signaling pathway. Inflammation 41 , 1384-1395, doi:10.1016/B978-0-12-816665-9.00015-9 (2018). Hwang, H.-Y., Shim, J. S., Kim, D. & Kwon, H. J. Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein. Autophagy 17 , 2783-2799, doi:10.1080/15548627.2020.1841953 (2021). Hou, T. et al. IL-37 ameliorating allergic inflammation in atopic dermatitis through regulating microbiota and AMPK-mTOR signaling pathway-modulated autophagy mechanism. Frontiers in Immunology 11 , 752, doi:10.3389/fimmu.2020.00752 (2020). Kujawski, R. et al. Diet based on oil of seeds of Brassica napus. Implications for the prevention and treatment of prostate diseases. Herba Polonica 60 , 77-88, doi: 10.2478/hepo-2014-0018 (2015). Cao, W., Li, J., Yang, K. & Cao, D. An overview of autophagy: Mechanism, regulation and research progress. Bulletin du cancer 108 , 304-322, doi:10.1016/j.bulcan.2020.11.004 (2021). Hindupur, S. K., González, A. & Hall, M. N. The opposing actions of target of rapamycin and AMP-activated protein kinase in cell growth control. Cold Spring Harbor perspectives in biology 7 , a019141, doi:10.1101/cshperspect.a019141 (2015). Sekirov, I., Russell, S. L., Antunes, L. C. M. & Finlay, B. B. Gut microbiota in health and disease. Physiological reviews , doi:10.1152/physrev.00045.2009 (2010). Sultan, S. et al. Metabolic influences of gut microbiota dysbiosis on inflammatory bowel disease. Frontiers in physiology 12 , 1489, doi:10.3389/fphys.2021.715506 (2021). Yamashiro, K. et al. Gut dysbiosis is associated with metabolism and systemic inflammation in patients with ischemic stroke. PloS one 12 , e0171521, doi:10.1371/journal.pone.0171521 (2017). Wang, G. et al. Lactic acid bacteria reduce diabetes symptoms in mice by alleviating gut microbiota dysbiosis and inflammation in different manners. Food & function 11 , 5898-5914, doi:10.1039/C9FO02761K (2020). White, B. et al. Microbiota of chronic prostatitis/chronic pelvic pain syndrome are distinct from interstitial cystitis/bladder pain syndrome. medRxiv , 2021.2003. 2004.21252926, doi:10.1101/2021.03.04.21252926 (2021). Yang, W., Gao, B., Qin, L. & Wang, X. Puerarin improves skeletal muscle strength by regulating gut microbiota in young adult rats. Journal of Orthopaedic Translation 35 , 87-98, doi:10.1016/j.jot.2022.08.009 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5846138","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":409212852,"identity":"be2bd671-f730-41f0-9a3f-9b48fa3711ce","order_by":0,"name":"Jie Dong","email":"","orcid":"","institution":"Hebei University of Engineering","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Dong","suffix":""},{"id":409212853,"identity":"83869a70-2bc0-406c-9323-7ac6661bf772","order_by":1,"name":"Jiawen Zhang","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiawen","middleName":"","lastName":"Zhang","suffix":""},{"id":409212854,"identity":"e0a8e2d1-0e52-44e7-b14f-b109492e6f4b","order_by":2,"name":"Jiangtao Qiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACxmYEs4GBoYJoLQkwLWeItisBZkAbEYqZ25mfPfz6w06OgT258cPHedayDeyHj27A7zA2c2OZhGRjBp6HzZIzt6UbN/Ckpd3Ar4XBTFoigTmxQSKxQZp322Egg8eMgBb2b0At9SAtzb955xClhcdM8kMCSGVimzRvA3FayqQZ0o4bs/E8bLOccSzduI2QXwz7j2+T/GFTLcfPnv74xocaa9l+9sPH8GtpAAY0D5DBBokcZsJRIw9y3A8wE6qlgZCWUTAKRsEoGHEAABK0RpdA1cGNAAAAAElFTkSuQmCC","orcid":"","institution":"Hebei University of Engineering","correspondingAuthor":true,"prefix":"","firstName":"Jiangtao","middleName":"","lastName":"Qiao","suffix":""},{"id":409212855,"identity":"fb74f90a-0868-4a31-a100-3001d56bcb90","order_by":3,"name":"Yu Zhang","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":409212856,"identity":"2a48ebed-5ba2-4d78-adb6-9dcb8acedfd9","order_by":4,"name":"Hequan Zhu","email":"","orcid":"","institution":"Chinese Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hequan","middleName":"","lastName":"Zhu","suffix":""},{"id":409212857,"identity":"31b43bd7-b6de-415d-a34d-edee1a7f659d","order_by":5,"name":"Eric Haubruge","email":"","orcid":"","institution":"University of Liege","correspondingAuthor":false,"prefix":"","firstName":"Eric","middleName":"","lastName":"Haubruge","suffix":""},{"id":409212858,"identity":"abd31b55-69f5-4c05-ab8a-ded84fafb939","order_by":6,"name":"Liqiang Liu","email":"","orcid":"","institution":"Hebei University of Engineering","correspondingAuthor":false,"prefix":"","firstName":"Liqiang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-01-17 05:23:10","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5846138/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5846138/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75141424,"identity":"e60ad0f7-d85e-491d-8832-9a858ca4c240","added_by":"auto","created_at":"2025-01-31 05:02:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1360683,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe six phenolamides in bee pollen exert inhibitory effects on LPS-induced inflammation in RWPE-1 cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eA, six phenolamides in bee pollen (P1, N(E)-feruloyl putrescine; P2, N(E), N' (E)-di-\u003cem\u003ep\u003c/em\u003e-coumaroyl putrescine; P3, N1(E), N10(E)-di-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine; P4, N1(E), N5(E), N10(E)-tri-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine; P5, N1(E), N5(E), N10(E), N14(E)-tetra- \u003cem\u003ep\u003c/em\u003e-coumaroyl spermine; P6, N1(E), N10(E)-di-p-coumaroyl-N14(E)- feruloyl spermine. B, Effect of six phenolamides on cellular viability by MTT assay. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. C, Effect of six phenolamides on pro-inflammatory cytokines and NO production in LPS-induced RWPE-1 cells. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e Sham group, ###\u003cem\u003e p\u003c/em\u003e\u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e LPS.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5846138/v1/1399bd96b14ed6815de3d0b5.png"},{"id":75141407,"identity":"47e238eb-a83f-4b9f-b1f9-a44f8b2b5eee","added_by":"auto","created_at":"2025-01-31 05:02:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8988650,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of di-p-coumaroyl spermidine on prostate wet weight, prostate index, and histomorphometry in rats induced by CAF\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eA-D, the morphology of prostate tissues; E-F, the sections of rat prostates are stained with H\u0026amp;E (× 100 and × 1000 magnification); I, Prostate wet weight (g); G, Prostate index (g/kg); H, Inflammation score. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5846138/v1/3793672fb5a23701a61d204d.png"},{"id":75141411,"identity":"377a87cb-e7ff-47db-9f6f-ead59964888b","added_by":"auto","created_at":"2025-01-31 05:02:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5864332,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression analysis of pro-inflammatory cytokines using immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eA, Immunochemical staining of IL-1β, IL-6, and TNF-α in rat prostate tissue; B-D, Positive cells rate of IL-1β, IL-6, and TNF-α in rat prostate tissue. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; E, Confocal immunofluorescence imaging.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5846138/v1/09894777b1efdc7aa9210144.png"},{"id":75141406,"identity":"fc6017eb-7c06-43e1-8ee1-63a5c843b24f","added_by":"auto","created_at":"2025-01-31 05:02:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1037942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression levels of pro-inflammatory cytokines.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eA-C, the expression levels of IL-1β, IL-6, and TNF-α in rat serum; D-F, the expression levels of IL-1β, IL-6, and TNF-α in rat prostate tissue; G-I, the expression levels of IL-1β, IL-6, and TNF-α gene. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5846138/v1/8bfadd27450a085fd6f68bd1.png"},{"id":75142127,"identity":"47da052e-1f75-4fbf-9703-aa9ae19a1006","added_by":"auto","created_at":"2025-01-31 05:10:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":773612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003edi-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Coumaroyl spermidine can upregulate autophagy via the AMPK/mTOR signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote: \u003c/strong\u003eDifferent letters (a, b, c and d) mean significant differences in other groups.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5846138/v1/a55c31cfefc226306e9ff356.png"},{"id":75141422,"identity":"d431767e-9f24-4546-8f7d-48373114e311","added_by":"auto","created_at":"2025-01-31 05:02:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1151178,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of di-\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Coumaroyl spermidine on gut microbiota in CNP rat\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNote:\u003c/strong\u003e A, the Chao1 index; B, shannon index; C, non-metric multidimensional scaling (NMDS); D, phylum level of gut microbiota; E, genus level of gut microbiota; F, the ratio of Firmicutes to Bacteroidetes; G, linear discriminant analysis with LDA score greater than 4. Data are presented as mean ± SD (n=8), **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5846138/v1/db6602c655e261f821e7e11e.png"},{"id":77267705,"identity":"571f4c1a-74df-4ff1-a141-86ebfa8e3f17","added_by":"auto","created_at":"2025-02-26 23:16:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19853027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5846138/v1/1658f319-2b57-43f9-ae44-e72996de1175.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Di-p-coumaroyl spermidine from bee pollen alleviates chronic non- bacterial prostatitis","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePollen, comprising the male gametophytic cells of angiosperms, is a finely powdered substance characterized by microscopic grains encapsulating the plant's male cells \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Embedded within the realm of traditional medicine and functional food for centuries, bee pollen has been ascribed with manifold therapeutic properties, including anti-inflammatory, antioxidant, antimicrobial, and immune-enhancing attributes \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. In recent decades, there has been a renewed surge in the attention devoted to bee pollen, driven by its pronounced effectiveness in mitigating benign prostatic hyperplasia and chronic prostatitis \u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs the global population ages, the incidence of chronic prostatitis in men is increasing, with older age being a significant risk factor for the development of this condition. Chronic non-bacterial prostatitis (CNP) is clinically defined as a persistent inflammatory process leading to chronic pelvic pain or discomfort accompanied by urinary symptoms and sexual dysfunction \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. It has emerged as a significant male healthcare issue, attracting considerable attention from clinicians and researchers alike \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Notably, a clinical research study conducted across North America, Europe, and Asia revealed that the prevalence of chronic prostatitis ranges from 2\u0026ndash;10% among adult males at any given time, while approximately 15% of men experience prostatitis symptoms during their lifetime \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, previous investigations have indicated a potential link between chronic prostatitis and an increased risk for developing benign prostatic hyperplasia and prostate cancer \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Fortunately, researchers have discovered that pollen or pollen extracts demonstrate notable efficacy in preventing and treating chronic prostatitis, leading to their widespread application worldwide \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In China, Pule'an capsules, which are made entirely from rapeseed bee pollen, have emerged as the prevailing pharmaceutical option for chronic prostatitis and prostate hyperplasia \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Clinical trials have shown that Pule'an capsules can effectively alleviate the symptoms of chronic prostatitis and prostate hyperplasia patients, with a symptom relief rate exceeding 90% \u003csup\u003e3\u003c/sup\u003e. Moreover, rapeseed bee pollen extracts have been discovered to substantially alleviate symptoms associated with chronic nonbacterial prostatitis \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This effect is achieved through modulating some key molecular factors, including the downregulation of mitofusin-1 (Mfn1) levels within the posterior lobes of the prostate, as well as the suppression of DHT, 5α-reductase, and Cyclooxygenase-2 (COX-2) levels \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In Europe and Japan, Cernitin\u0026trade; (pharmaceutical brand, Cernilton\u0026reg;), ryegrass pollen extract, has been used to treat nonbacterial prostatitis and prostatic hyperplasia for more than 40 years with an overall success rate of 70% \u003csup\u003e15,16\u003c/sup\u003e. Cernitin\u0026trade; can notably reduce the prostate-specific antigen levels associated with chronic prostatitis/chronic pelvic pain syndrome. In preclinical studies, Cernitin\u0026trade; demonstrated significant pain relief in an induced prostatitis rat model, concomitant with a noteworthy decrease in intraprostatic levels of COX-2 and MCP-1 \u003csup\u003e15\u0026ndash;17\u003c/sup\u003e. Cernitin\u0026trade; consists of two fractions of pollen extract: water-soluble T60 and lipid-soluble GBX \u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The two fractions can distinctly reduce inflammation, inhibit cellular proliferation, and relax smooth muscle in animal models \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Evidence suggests that the anti-CNP effect of the T60 fraction is attributed to feruloyl putrescine \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFeruloyl putrescine is formed by ferulic acid and putrescine through an amide bond, a type of phenolamide. Phenolamides, also known as hydroxycinnamic acid amides, result from the conjugation of hydroxycinnamic acids (for example, \u003cem\u003ep\u003c/em\u003e-coumaric, ferulic, and caffeic acid) with aliphatic or aromatic amines, such as putrescine, spermine, and spermidine. Our previous study has shown that bee pollen is a treasure trave of phenolamides \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Sixty-four phenolamides were identified in 20 types of monofloral bee pollen, with the highest contents among known natural products \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Moreover, in the last five years, multiple studies have reported the presence of abundant phenolamides in bee pollen \u003csup\u003e\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Hence, the robust inhibitory functionality of bee pollen or extracts against prostatitis may be attributed to its abundant phenolamides. However, apart from feruloyl putrescine, the anti-CNP activities of other phenolamides from bee pollen remained unclear.\u003c/p\u003e \u003cp\u003eThe current study aimed to evaluate the anti-CNP effects and mechanisms of phenolamides in bee pollen. Firstly, we employed an LPS-induced prostate epithelial cell (RWPE-1) model to screen anti-prostatitis activities from 6 representative phenolamides in bee pollen. Subsequently, we elucidated anti-prostatitis mechanisms using a CNP rat model.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eN(E), N' (E)-di-\u003cem\u003ep\u003c/em\u003e-coumaroyl putrescine, N1(E), N10(E)-di-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine, N1(E), N5(E), N10(E)-tri-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine, N1(E), N5(E), N10(E), N14(E)-tetra- \u003cem\u003ep\u003c/em\u003e-coumaroyl spermine and N1(E), N10(E)-di-\u003cem\u003ep\u003c/em\u003e-coumaroyl-N14(E)- feruloyl spermine were prepared (purity\u0026thinsp;\u0026ge;\u0026thinsp;95%) according to our previous study \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. N(E)-feruloyl putrescine (purity\u0026thinsp;\u0026ge;\u0026thinsp;95%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd (Shanghai, China). All the chemical structures are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHuman TNF-α, IL-1β, IL-6 and Rat TNF-α, IL-1β, IL-6 Kit purchased from Solarbio (Beijing, China). NO kits (Beyotime, Nanjing, China). Anti-TNF-alpha Rabbit pAb (Servicebio, GB11188), Anti -IL-1 beta Rabbit pAb (Servicebio, GB11113), Anti -vIL-6 Rabbit pAb (Servicebio, GB11117), Cy3 conjugated Goat Anti-Rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Servicebio, GB21303). Beclin-1 pAb (YT5763), LC3 A/B rabbit pAb (YT7936), GAPDH mAb (YM3029) purchased from ImmunoWay Biotechnology Company (TX,75024 USA). Rabbit anti-p62 antibody (5114), p-AMPK (2535), AMPK (5831), p-mTOR (5536) and mTOR (2983) from Cell Signaling Technology (Danvers, MA, USA). Complete Freund's adjuvant (CFA) was purchased from Sinopharm Co., Ltd (Shanghai, China). Cole's Hematoxylin solution, Eosin Y solution, Ethylene Diamine Tetraacetic Acid (EDTA) pH 8.0 were obtained from Abcam (Cambridge, MA, USA). All other experimental materials and consumables were purchased from Solarbio Life Sciences (Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 RWPE-1 cell culture\u003c/h2\u003e \u003cp\u003eThe immortalized human prostate epithelial cell line (RWPE-1) was purchased from BNCC (Henan, China). RWPE-1 cells were cultured in Keratinocyte Medium (ScienceCell, San Diego, California, USA) supplemented 5 ml of Keratinocyte Growth Supplement (KGS, Cat#2152) and 5 ml of penicillin/streptomycin solution (P/S, Cat. #0503) in a humidified 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. The model of chronic nonbacterial prostatitis (CNP) was induced by 10 \u0026micro;/ml lipopolysaccharide (LPS) for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell Viability Assays\u003c/h2\u003e \u003cp\u003eRWPE-1 cell viability was evaluated using the methyl thiazolyl tetrazolium assay (MTT, Solarbio, Beijing, China). RWPE-1 cells (20,000 cells/well) were seeded in a 96-well plate and incubated at 37\u0026deg;C for 24 hours. Subsequently, the culture medium was replaced respectively with 1mL fresh Keratinocyte medium containing 0.1% DMSO as a control group and 1mL various concentrations of phenolamides (0, 1, 5, 10, 20, 40, 80 \u0026micro;mol/L) as treatment groups. After the treatment, MTT solution (5 mg/mL) was added to each well and incubated for 4 hours. The absorbance was measured at 490 nm using a microplate reader (Biotek, USA). All experiments were performed in triplicate. Cell viability was calculated as a percentage using the following formula: cell viability = [(mean absorbance of each treatment group) / (mean absorbance of the control group)] \u0026times; 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Inflammatory cytokines and NO production of RWPE-1 cells\u003c/h2\u003e \u003cp\u003eTh RWPE-1 cells were incubated in 24-well culture plates and treated with different concentrations of phenolamides (0, 1, 5, 10, 20, 40, 80 \u0026micro;mol/L) for 2 h, and then the cells were stimulated with 5 \u0026micro;g/mL LPS and incubated for 24 h. TNF-α, IL-1β, and IL-6 levels were quantified using a commercially available ELISA kit from Solarbio (Beijing, China). Based on Greiss reaction, the nitric oxide (NO) concentration in the cell culture media was determined using NO kits (Beyotime, Nanjing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Animal model\u003c/h2\u003e \u003cp\u003eAll animal experimental procedures followed the Guidelines for the Care and Use of Laboratory Animals. Fifty-two-month-old male Sprague- Dawley (SD) rats (190\u0026ndash; 220 g) were obtained from the Si Pei Fu (SPF biotechnology Co., Ltd., Beijing, China, licenses: 110324231104817715; animal use licenses: SYXK(JING) 2023-0004). We kept all the animals under specific pathogen-free (SPF) environmental conditions at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;5%. Besides, the rats had free access to food and water in a 12 h dark-light cycle (light cycle: 7:00 am\u0026ndash;7:00 pm) and were given one week to adapt to the new environment before the experiment. The experimental Animal Welfare Committee of the Chinese Academy of Agricultural Sciences approved ethical requirements (permit number: IARCAAS-2019-003). Thirty-six rats were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;9 rats/group), including the control group, model group, and \u003cem\u003ep\u003c/em\u003e-Cspd low or high dose group.\u003c/p\u003e \u003cp\u003eThe rat model of CNP was established using complete Freund's adjuvant (CFA) as previously described method with slight modification \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Apart from the control group, the intervention involved the injection of a sterile CFA suspension (100 \u0026micro;L, 1% w/v) into the right and left lobes of the ventral prostate; while for the sham-operated rats (n\u0026thinsp;=\u0026thinsp;9), the same volume of sterile saline was injected. The wound was then closed in layers. During surgical procedures, isoflurane inhalation anesthesia was used. Isoflurane was administered at an induction concentration of 4%-5% in 100% oxygen and maintained at 1.5%-2.5% during the procedure. The anesthetic gas mixture was delivered through a rodent-specific nose cone to ensure the animals remained unconscious and pain-free. The control group and model group were orally administered 0.5% DMSO (w/w) 1mL for 21 days. The remaining two groups of rats were orally administered 1mL di-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine (\u003cem\u003ep\u003c/em\u003e-Csp) dissolved in 0.5% DMSO (w/w), according to 5mg/kg (low dosage group, L- \u003cem\u003ep\u003c/em\u003e-Csp) and 10 mg/kg (low dosage group, H- \u003cem\u003ep\u003c/em\u003e-Csp) body weight for 21 days. On the final day of the experiment (day 22), all rats were humanely euthanized. The euthanasia of animals (rats) was conducted using carbon dioxide (CO₂) asphyxiation, following the AVMA Guidelines for the Euthanasia of Animals. CO₂ was introduced gradually into the chamber at a displacement rate of 30%-70% of the chamber volume per minute to minimize distress, ensuring unconsciousness prior to respiratory arrest. Subsequently, the prostate tissues were carefully harvested and promptly weighed. The prostate index was then computed as the ratio of the prostate weight to the total body weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Histopathology and Immunohistochemical\u003c/h2\u003e \u003cp\u003eThe prostate specimens were fixed in 4% formaldehyde overnight for histopathological examination. Fixed prostate specimens were embedded in paraffin and sectioned with a microtome (5 \u0026micro;m) using a Leica RM2255 Microtome (Leica Biosystems, Leica, Germany). After deparaffinization with xylene and dehydration using alcohol, the sections were subjected to hematoxylin-eosin staining. Subsequently, the glandular epithelium, structure, and space were meticulously examined using a Nikon Eclipse Ci-L microscope equipped with a Nikon digital sight DS-Fi2 system (Nikon, Tokyo, Japan). Histomorphometry parameters of the normal prostate were determined based on the analysis from the control group.\u003c/p\u003e \u003cp\u003eSlides containing 5 \u0026micro;m thick rat prostate sections were meticulously rinsed with phosphate-buffered saline (PBS), then permeabilized with 0.1% Triton X-100 in PBS for 10 minutes, followed by blocking with 2% bovine serum albumin (BSA) in PBS for 30 minutes. The primary antibody was applied and left to incubate overnight at a temperature of 4\u0026deg;C. Subsequently, the slides were thoroughly washed with PBS and incubated with a secondary antibody at room temperature for 50 minutes. After another round of meticulous washing, the immunofluorescent images were captured using an 80i Nikon microscope. The images were captured and analyzed with Aipathwell software (Servicebio, Wuhan, China). The positive cell ratio was calculated to assay the expression of IL-6, IL-1β, and TNF-α. Positive cells ratio\u0026thinsp;=\u0026thinsp;the number of positive cells / total number of cells. Moreover, a NIKON Eclipse Ti confocal laser scanning microscope with DS-U3 imaging system (Nikon, Tokyo, Japan) to scan inflammatory cytokines for visualization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Inflammatory Cytokines\u003c/h2\u003e \u003cp\u003eIn this study, we determined the levels of IL-6, IL-1β, and TNF-α in both serum and prostate tissue using ELISA kit. For serum analysis, each sample was meticulously collected in serum separator tubes (BD Biosciences, Rockville, USA) and allowed to clot for two hours at room temperature. Following the clotting period, centrifugation was performed at approximately 1000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 20 minutes to remove the clots and obtain the serum samples. As for the prostate tissue, a rigorous protocol was employed to ensure proper sample preparation. Each prostate tissue specimen was meticulously rinsed with ice-cold PBS (0.01M, pH\u0026thinsp;=\u0026thinsp;7.4) to remove residual blood thoroughly. Subsequently, the prostate samples were weighed, and using an electric homogenizer, they were homogenized in ice-cold PBS. The homogenates were centrifuged at 5000 \u0026times; g for 5 minutes to obtain the supernatants. The ELISA procedures were carried out in accordance with the manufacturer's protocols to ensure standardization and accuracy. Each ELISA assay was repeated three times. The data were analyzed meticulously, and statistical significance was established at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eThe prostate tissues were thoroughly ground in liquid nitrogen, and subsequently, total RNA was extracted utilizing the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was subjected to reverse transcription using the PrimeScript RT reagent kit (Takara, Beijing, China). Real-Time PCR was conducted on a 7500 System (Applied Biosystems, Inc., Carlsbad, CA, USA) following a two-step reaction procedure.\u003c/p\u003e \u003cp\u003eThe expression of the housekeeping gene Gapdh was used to normalize the expression levels. The primers are designed to flank introns with Primer Premier 6.0 software (Premier Biosoft, Palo Alto, CA, USA). The melting curve checked the specificity of the primers. DNA sequencing and electrophoresed on the agarose gel have also tested the PCR products.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot\u003c/h2\u003e \u003cp\u003eIn brief, the prostate tissues were lysed using the RIPA lysis buffer; then the proteins were quantified using the BCA kit. The protein samples were loaded and separated through SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto the polyvinylidene fluoride (PVDF) membranes, and blocked with 5% skim milk-TBST for 2 h at 20\u0026deg;C. Then, the blots were incubated with primary antibodies (anti-TFEB 1:500, Beclin-1 1: 5000, anti-p62/SQSTM1 1:2000, anti-LC3 1: 2000, and anti-GAPDH 1:20000, p-AMPK 1: 2000, AMPK 1: 2000, p-mTOR 1: 2000 and mTOR 1: 2000). After being washed and incubated with the secondary antibodies. Chemiluminescence, X-ray film compression, development, fixing, and data analysis using Quantity One software (Bio-Rad, CA, USA). The results represent three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Gut microbiota analysis\u003c/h2\u003e \u003cp\u003eBefore concluding the experiment, fecal specimens were collected and promptly subjected to rapid freezing using liquid nitrogen, then stored at -80\u0026deg;C for subsequent microbial community analysis. The intestinal content samples from four distinct rat groups were then utilized for 16S rRNA gene sequencing, with a sample size of n\u0026thinsp;=\u0026thinsp;8 in each group. Genomic DNA was isolated from fecal samples of mice in accordance with the QiAamp\u0026reg; Fast DNA Stool Mini Kit protocol. The amplification of 16S rRNA genes corresponding to the hypervariable region (16S V3-V4) was achieved through the utilization of a universal bacterial primer set, with Primer F\u0026thinsp;=\u0026thinsp;341F (5\u0026rsquo;-CCTACGGGNGGCWGCAG-3') and Primer R\u0026thinsp;=\u0026thinsp;805R (5\u0026rsquo;-GACTACHVGGGTATCTAATCC-3') \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Subsequently, library preparation was conducted using the NEBNext Ultra II DNA Library Prep kit (New England Biolabs E7370S/L, Ipswich, MA, USA) and then sequenced on an Illumina MiSeq PE300 platform at Beijing Allwegene Technology Co., Ltd. (Beijing, China), resulting in the generation of 250 bp paired-end reads. These reads were subsequently subjected to data processing steps, including merging, demultiplexing, and quality filtering using QIIME 2.0. Following these procedures, the reads were clustered into Operational Taxonomic Units (OTUs) at a 97% sequence identity threshold. All analytical outcomes were predicated on the characterization of OTUs. Based on OTU levels, alpha, and beta diversity analyses were executed as per our prior study \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To pinpoint key OTUs responsive to distinct interventions, we employed Linear Discriminant Analysis (LDA) coupled with effect size assessment. A p-value of \u0026lt;\u0026thinsp;0.05 determined significance and a minimum LDA score of \u0026ge;\u0026thinsp;4.0 was deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1 RWPE-1 cell viability\u003c/h2\u003e \u003cp\u003eThe impacts of six phenolamides with different concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) on the viability of RWPE-1 cells were evaluated using the MTT assay to determine the optimal dosage levels for subsequent investigations. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, all phenolamides did not demonstrate significant growth-inhibitory activities at a concentration of 20 \u0026micro;mol/L (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), except P5 and P6. When the concentrations of P5 and P6 exceed 20 \u0026micro;mol/L (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), RWPE-1 cell survival was significantly decreased. Based on these results, concentration ranges (P1 to P4 are 1, 5, 10, and 20 \u0026micro;M, P5 and P6 are 1, 5, and 10 \u0026micro;M) were chosen in the subsequent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Inflammatory cytokines and NO production\u003c/h2\u003e \u003cp\u003eTo assess the anti-inflammatory effects of different phenolamides, we measured the levels of TNF-α, IL-1β, IL-6, and NO production on LPS-induced RWPE-1 cells after being treated with various concentrations of phenolamides. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, LPS dramatically increased the TNF-α, IL-6, IL-1β, and NO levels. Phenolamides P1, P2, P3, and P4 demonstrated a concentration-dependent reduction for the levels of inflammatory cytokines and NO, with the optimal at 20 \u0026micro;M. P5 and P6 exhibit anti-inflammatory activity at their maximum non-toxic concentration (10 \u0026micro;M). Noteworthily, P3 exhibited the most remarkable effects at 20 \u0026micro;M, with respectively decreasing 54.7%, 51.6%, 55.8%, and 67% in the levels of IL-6, TNF-α, IL-1β, and NO, compared to the model group. The above results indicate that among the selected six phenolamides, P3 (N1(E), N10(E)-di-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine, \u003cem\u003ep\u003c/em\u003e-Csp) exhibits a better anti-inflammatory effect. Moreover, the anti-inflammatory efficacy of P3 surpasses that of feruloyl-putrescine (P1), which is the principal active constituent in the conventional pharmaceutical component known as Cernitin\u0026trade;. Consequently, in subsequent investigations, we employ animal models to evaluate the anti-prostatitis activity of \u003cem\u003ep\u003c/em\u003e-Csp.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Prostate wet weight and prostate index\u003c/h2\u003e \u003cp\u003eProstate wet weight and the prostate index can reflect the extent of prostate pathology \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. In this study, complete Freund's adjuvant-induced model of non-bacterial prostatitis was used to evaluate the anti-prostatitis effects of \u003cem\u003ep\u003c/em\u003e-Csp. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, CFA effectively induced a rat model of prostatitis, wherein the model group demonstrated a pronounced elevation in both prostate wet weight and prostate index, increasing 1.78 and 1.96-fold, respectively, compared to the control group. It is worth noting that \u003cem\u003ep\u003c/em\u003e-Csp significantly decreased both prostate wet weight and prostate index, exhibiting a concentration-dependent response. In the high dose group (H-\u003cem\u003ep\u003c/em\u003e-Csp), prostate wet weight and prostate index respectively reduced 31.6% and 34.4%, compared to the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Prostate histomorphometry\u003c/h2\u003e \u003cp\u003eProstate histomorphology can significantly display prostatitis lesions. To further evaluate the therapeutic effect of \u003cem\u003ep\u003c/em\u003e-Csp, prostatic histopathology was observed using the HE staining method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF) reduced the number of glandular structures, along with connective tissue proliferation (black arrows), and formed numerous empty cavity structures (yellow arrows) and the infiltration of lymphocytes and granulocytes (red arrows), with a limited presence of inflammatory cells penetrating the glandular lumina (purple arrows). Multiple areas displayed signs of edema; connective tissue exhibited a loose arrangement (blue arrows). Eosinophilic material exudation was also observed (green arrows). Additionally, a significant reduction in secretions within many glandular lumina (brown arrows) and noticeable glandular expansion with thinning of glandular epithelium were noted. These evidences indicate the successful establishment of the CNP rat model (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). After \u003cem\u003ep\u003c/em\u003e-Csp administration for 21 days, the glandular cavity structure of the prostate tissues recovered; the interstitial space, inflammatory cell infiltration in the glandular cavity and interstitial space, and migration of fibroblasts and blood vessels decreased dose-dependent (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H). Specifically, the alleviation was most significant in the H-\u003cem\u003ep\u003c/em\u003e-Csp (10mg/kg) group, presenting a remarkable decrease in inflammatory cell infiltration and necrotic foci and lacking prostatic hyperplasia, resembling normal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Additionally, a comprehensive examination of the inflammation scores pertaining to histopathological alterations in the prostatic tissue was undertaken for each experimental group to assess the extent of inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK). Notably, compared to the model group, the groups receiving H-\u003cem\u003ep\u003c/em\u003e-Csp treatment exhibited a significant reduction in inflammation scores, almost 67% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eImmunohistochemistry was used to evaluate the expression of IL-1β, IL-6, and TNF-α in prostate tissue, and the positive cell ratio was calculated. Confocal immunofluorescence imaging was used for the real-time visualization analysis of prostate tissues. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, red fluorescence represented the inflammatory signal. The intensity increases of red fluorescence indicated a high expression of inflammatory cytokines, while blue fluorescence showed a normal cellular state. Based on the discernible increase in red fluorescence intensity, the model group heightened the expression of inflammatory factors. Conversely, the two groups treated with \u003cem\u003ep\u003c/em\u003e-Csp were observed to reduce the levels of all three inflammatory factors and significantly exhibited a concentration-dependent response. By assessing the positive cell ratio, \u003cem\u003ep\u003c/em\u003e-Csp exerted a pronounced reversal effect on the elevated expression of IL-6, IL-8, and IL-1β within the prostate tissue induced by CFA. Noteworthily, the most substantial reduction is observed in the H-\u003cem\u003ep\u003c/em\u003e-Csp group. IL-1β notably decreased approximately 69.8%, without a statistically significant difference between the H-\u003cem\u003ep\u003c/em\u003e-Csp and control groups. The effect of decreasing inflammatory factors in the H-\u003cem\u003ep\u003c/em\u003e-Csp group was much better than that in the L-\u003cem\u003ep\u003c/em\u003e-Csp group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.6 The expression levels of\u003c/b\u003e inflammatory cytokines \u003cb\u003ein tissue and serum\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eCNP and prostate carcinoma patients are associated with prolonged overproduction of inflammatory cytokines, including IL-1β, IL-6, and TNF-α. In this study, we determined the levels of IL-1β, IL-6, and TNF-α in both serum and prostate tissue using ELISA kit. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates great variations in the levels of these inflammatory cytokines among different treatment groups. In contrast to the control group, the model group greatly elevated the levels of inflammatory cytokines in both blood and tissues. Compared to the model group, two \u003cem\u003ep\u003c/em\u003e-Csp treatment groups conspicuously reduced inflammatory cytokine levels; notably, L-6, IL-1β, and TNF-α levels in the H-\u003cem\u003ep\u003c/em\u003e-Csp group decreased by approximately 50%. Additionally, gene expression profiles of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were subjected to analysis. In the model group, the genes responsible for encoding IL-1β, IL-6, and TNF-α were conspicuously upregulated, compared to the control group. In contrast, the L-\u003cem\u003ep\u003c/em\u003e-Csp and H-\u003cem\u003ep\u003c/em\u003e-Csp groups exhibited marked down-regulations of these gene expressions, compared to the model group. This further verified that \u003cem\u003ep\u003c/em\u003e-Csp treatment can conspicuously reduce inflammatory cytokine levels in both blood and prostate tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Autophagy via the AMPK/mTOR signaling pathway\u003c/h2\u003e \u003cp\u003eAutophagy plays a pivotal role in regulating inflammatory processes \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. In recent years, multiple studies have reviewed that activating cellular autophagy presents a promising therapeutic approach for managing chronic inflammation \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, particularly non-bacterial prostatitis \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. The main proteins associated with autophagy include LC3 (microtubule-associated protein 1A/1B-light chain 3), Beclin-1, and p62. To investigate the relationship between \u003cem\u003ep\u003c/em\u003e-Csp and autophagy, we detected the expression of autophagy-related proteins by Western blot analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, The LC3- II/LC3-I ratio exhibited no notable alteration between the control and model group. However, \u003cem\u003ep\u003c/em\u003e-Csp led to a significant increase in the LC3- II/LC3-I ratio. Moreover, the \u003cem\u003ep\u003c/em\u003e-Csp treatment group demonstrated a significant concentration-dependent upregulation of Beclin-1. Moreover, p62 expression was decreased upon \u003cem\u003ep\u003c/em\u003e-Csp stimulation. Together, these results suggest that \u003cem\u003ep\u003c/em\u003e-Csp can enhance \u003cem\u003ein vivo\u003c/em\u003e autophagy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe AMPK-mTOR pathway participates in the cellular response to energy deficiency, ultimately triggering autophagy \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Previous studies have offered compelling evidence supporting the notion that the activation of autophagy via the AMPK/MTOR signaling pathway holds significant potential for instigating anti-inflammatory reactions \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To ascertain whether the role of \u003cem\u003ep\u003c/em\u003e-Csp in mitigating prostatitis is through activating autophagy by the modulation of AMPK/MTOR signaling pathway, we detected the expression of AMPK/MTOR by western blot analysis. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, the \u003cem\u003ep\u003c/em\u003e-Csp treatment increased the ratio of p-AMPK/AMPK, whereas decreased ratio of p-mTOR / mTOR. These findings imply that \u003cem\u003ep\u003c/em\u003e-Csp may potentiate autophagic processes through modulating AMPK and mTOR signaling pathways.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Gut microbiota\u003c/h2\u003e \u003cp\u003eOur previous research has proved that rapeseed bee pollen can mitigate CNP through regulating the gut microbiota \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. To further investigate the potential role of \u003cem\u003ep\u003c/em\u003e-Csp, primarily found in rapeseed bee pollen, in gut microbiota, 16S rDNA amplicon sequencing analysis of rat feces was performed.\u003c/p\u003e \u003cp\u003eChao1 and Shannon indexes expressed the alpha diversity. The Chao1 index estimates the total number of species in the sample, and the Shannon index describes the diversity of the microbial community. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B, alpha diversity with Chao1 and Shannon indexes on the OTU levels was significantly decreased in the model group compared to the control, suggesting that the structural diversity of gut microbiota decreased in the model group, while \u003cem\u003ep\u003c/em\u003e-Csp treatment reinstated alpha diversity. Additionally, non-metric multidimensional scaling (NMDS) was employed as beta diversity assessment indexes to observe the structural variability of different species. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, the NMDS analysis unveiled noteworthy difference in the clustering patterns of gut microbiota species across the four experimental groups. A clear demarcation existed between the model and control groups; notably, the H-\u003cem\u003ep\u003c/em\u003e-Csp group displayed more distinct segregation from the model group and closer to the control group. Hence, \u003cem\u003ep\u003c/em\u003e-Csp can change the gut microbiota structure, potentially contributing to mitigating CFA-induced CNP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo enhance our understanding of the bacterial taxa subject to regulation by \u003cem\u003ep\u003c/em\u003e-Csp, a comprehensive assessment of the gut microbiota composition was performed for the four experimental groups. The predominant bacterial phyla in all groups were Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). In the model group, CFA decreased the relative abundance of Bacteroidetes and increased the relative abundance of Firmicutes, thus significantly increasing Firmicutes/Bacteroides (F/B) ratio. Noteworthily, \u003cem\u003ep\u003c/em\u003e-Csp treatment reduced the Firmicutes to Bacteroidetes ratio (F/B); for example, H-\u003cem\u003ep-\u003c/em\u003eCsp group is closer to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). At the genus level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), compared to the control group, the model group decreased the relative abundance of \u003cem\u003eLactobacillus, Prevotella_9, Bifidobacterium\u003c/em\u003e and \u003cem\u003eBacteroides\u003c/em\u003e, while increased that of \u003cem\u003eLachnospiraceae, Romboutsia\u003c/em\u003e and \u003cem\u003eLachnospiraceae_NK4A136_group\u003c/em\u003e. This finding suggested that \u003cem\u003ep\u003c/em\u003e-Csp can rectify the perturbations in gut microbial populations, with a particularly pronounced increase of \u003cem\u003eLactobacillus, Prevotella_9, Bifidobacterium\u003c/em\u003e, and \u003cem\u003eLachnospiraceae. Lactobacillus, Bifidobacterium, and Prevotella_9\u003c/em\u003e is widely recognized as probiotic genera known to confer health benefits on host organism. Moreover, linear discriminant analysis (LDA) effect size (LEFSe) results showed that a total of 19 taxa at different OTU levels (LDA\u0026thinsp;\u0026gt;\u0026thinsp;4) were identified with different abundances in the four groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Negativicutes phylum were dominant bacteria in the model group, while Firmicutes were dominant bacteria in \u003cem\u003ep\u003c/em\u003e-Csp groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Moreover, \u003cem\u003eLactobacillaceae\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e were obviously dominant in \u003cem\u003ep\u003c/em\u003e-Csp treatment groups. These results suggested that \u003cem\u003ep\u003c/em\u003e-Csp can modulate gut microbiota by increasing the abundance of beneficial bacteria in rat.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eBee pollen and its extracts have been employed for over 40 years as dietary supplements or medication on a global scale in the therapeutic management of chronic prostatitis. However, it remains unclear which compounds in bee pollen are responsible for its anti-chronic prostatitis effects and the underlying mechanisms, leading to product instability and lack of predictability. Recent studies have revealed that bee pollen is rich in phenolamides and is hailed as a treasure trove of phenolamides. Moreover, studies have already indicated that one of the components in the prostatitis treatment drug Cernitin\u0026trade; is phenolamide. We speculated that the anti-chronic prostatitis properties of bee pollen are mainly attributed to its richness of phenolamides. In this study, we evaluated the anti-chronic prostatitis properties of six principal phenolamides in bee pollen based on cellular and rat models.\u003c/p\u003e \u003cp\u003eOur results indicate that the six phenolamides exhibited anti-inflammatory activity in RWPE-1 cells induced by LPS. Noteworthily, \u003cem\u003ep\u003c/em\u003e-Csp can remarkably decrease the levels of IL-6, TNF-α, IL-1β, and NO, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The anti-inflammatory efficacy of \u003cem\u003ep\u003c/em\u003e-Csp surpasses that of feruloyl-putrescine, the principal active constituent (P1) in the conventional medicine Cernitin\u0026trade;. Subsequently, \u003cem\u003ep\u003c/em\u003e-Csp also demonstrated a capacity effectively to \u003cem\u003ein vivo\u003c/em\u003e mitigate CNP in the rat model, based on prostate wet weight, prostate index, and histomorphometry findings (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003ep\u003c/em\u003e-Csp treatments could decrease the levels and gene expression of inflammatory cytokines IL-1β, IL-6, and TNF-α in serum and prostate tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings are consistent with the immunohistochemistry results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Our previous research has demonstrated that \u003cem\u003ep\u003c/em\u003e-Csp is a new found compound and is predominantly present in rapeseed bee pollen with a concentration of 10 mg/g \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Previous studies have consistently demonstrated that rapeseed bee pollen surpasses its counterparts in imparting anti-prostatitis efficacy \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our findings provide robust evidence that the efficacy of rapeseed bee pollen on prostatitis treatment may be mainly attributable to its high level of \u003cem\u003ep\u003c/em\u003e-Csp.\u003c/p\u003e \u003cp\u003ePhenolamides are formed by conjugating phenolic acids such as \u003cem\u003ep\u003c/em\u003e-coumaric acid, ferulic acid, and caffeic acid with polyamines (putrescine, spermidine, and spermine) through amide bonds. Research indicates that phenolamides present a variety of health benefits, such as anti-inflammatory, antioxidant, and anti-tyrosinase properties \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ep\u003c/em\u003e-Csp is formed by two \u003cem\u003ep\u003c/em\u003e-coumaroyl residues bound at the N1 and N14 positions of spermidine. Spermidine has been reported to exert anti-aging effects by stimulating autophagy and is hailed as a \"anti-aging vitamin\" \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. As a solitary phenolamide in bee pollen, our results indicate that \u003cem\u003ep\u003c/em\u003e-Csp can activate AMPK pathway, suppress the expression of mTOR, increase the LC3- II/LC3-I ratio, upregulate the expression of Beclin-1, and downregulate the expression of p62, thereby inducing and promoting autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Numerous studies have shown that autophagy is dependent on the regulation of intracellular AMPK-mTOR signaling pathway \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. AMPK exerts its inhibitory effect on mTOR by activating the TSC1/TSC2 complex (Tuberous Sclerosis Complex 1/2) \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The TSC1/TSC2 complex, by virtue of its capacity to suppress mTOR activity, facilitates the initiation of autophagy \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Consequently, the activation of AMPK serves to repress mTOR, thus promoting the process of autophagy \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. LC3, primarily in its cytosolic LC3-I form, undergoes proteolytic cleavage to LC3-II during autophagy induction \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Elevated Beclin-1 triggers autophagosome formation, while p62 accumulates during autophagy inhibition and decreases with active autophagy \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Autophagy can attenuate inflammatory processes by facilitating the catabolism of pro-inflammatory cytokines and the clearance of compromised organelles, notably mitochondria, through a selective form of autophagy known as mitophagy \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In recent years, studies have reviewed that activating cellular autophagy presents a promising therapeutic approach for managing chronic inflammation \u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Autophagy diminishes the biosynthesis of pro-inflammatory cytokines, potentially resolving the persistent inflammatory state emblematic of chronic prostatitis. Our results showed that the activating autophagy of \u003cem\u003ep\u003c/em\u003e-Csp may be responsible for alleviating CNP (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results are consistent with the previously reported mechanism of rapamycin and IL-37 exerting anti-CNP and anti-inflammation effects by activating autophagy through the AMPK/mTOR pathway \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. In this study, we first elucidated the mechanism against chronic prostatitis that phenolamides from bee pollen involve the activation of autophagy via the AMPK/mTOR pathway.\u003c/p\u003e \u003cp\u003eThe gut microbiota is integral to the preservation of host health \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Dysbiosis of the gut microbiota is associated with various inflammatory diseases, such as inflammatory bowel disease \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, systemic inflammation in stroke patients \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, and inflammatory conditions in diabetes patients \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Dysbiosis involves decreased beneficial bacteria and increased opportunistic pathogens that stimulate proinflammatory mediator synthesis via plasma membrane receptor interaction \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Recent studies have revealed a correlation between dysbiosis of the gut microbiota and CNP \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Our results indicate that significant changes occur in the gut microbiota of CNP rats, consistent with those seen in CNP patients \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ep\u003c/em\u003e-Csp treatment can significantly reduce the α-diversity of the gut microbiota caused by prostatitis inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and B). Some natural products have shown a similar trend in gut community diversity, including Puerarin and polysaccharides from the fruits of \u003cem\u003eLycium barbarum\u003c/em\u003e L\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Furthermore, \u003cem\u003ep\u003c/em\u003e-Csp can inhibit pathogenic bacteria and enhance probiotics. Compared to the model group, the H-\u003cem\u003ep\u003c/em\u003e-Csp group significantly decreased the F/B ratio by up-regulation of Bacteroidetes expression and down-regulation of Firmicutes expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). The F/B ratio was often used as an indicator to measure the health of intestinal flora; the high F/B ratio is closely related to inflammatory disease \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Moreover, our results demonstrated that \u003cem\u003ep\u003c/em\u003e-Csp treatment can enrich the abundance of \u003cem\u003eLactobacillaceae\u003c/em\u003e and \u003cem\u003ePrevotella_9\u003c/em\u003e, widely recognized as probiotic genera on host health. This is consistent with our previous report that rapeseed bee pollen can promote the growth of beneficial gut bacteria \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Our findings seem to provide a novel insight into modulating the gut microbiota with \u003cem\u003ep\u003c/em\u003e-Csp for preemptive intervention to forestall prostatitis, implicating the probable presence of gut-prostate connection.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eOur results reveal that phenolamides in bee pollen may be responsible for anti-CNP, Specially, di-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine can alleviate CNP by upregulating autophagy via AMPK/mTOR signaling pathway and regulating gut microbiota. Based on our findings, we recommend that rapeseed bee pollen with the high level of di-\u003cem\u003ep\u003c/em\u003e-coumaroyl spermidine should be selected to target prostatitis as nutraceuticals and therapeutics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Modern Agro-industry Technology Research System (CARS-45-KXJ19), Innovation funding from Hebei University of Engineering, the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2019-IAR) from the Ministry of Agriculture of P.R. China. The authors would also like to thank the Teaching and Research Center, Gembloux, Belgium.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiangtao Qiao and Liqiang Liu conceived the idea; Jiangtao Qiao and Jie Dong designed the experiments; Jiangtao Qiao, Jiawen Zhang and Yu Zhang performed the experiments and analyzed the data; Hequan Zhu and Jie Dong contributed reagents/materials/analysis tools; Jiangtao Qiao and Eric Haubruge wrote and reviewed the paper. A.B. and C.D. wrote the main manuscript text and Jiawen Zhang prepared figures 2 and 3, Jiangtao Qiao prepared figures1, 4,5 and 6. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCampos, M. G.\u003cem\u003e et al.\u003c/em\u003e Pollen composition and standardisation of analytical methods. \u003cem\u003eJournal of Apicultural Research\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 154-161, doi:10.1080/00218839.2008.11101443 (2008).\u003c/li\u003e\n\u003cli\u003eDenisow, B. \u0026amp; Denisow‐Pietrzyk, M. Biological and therapeutic properties of bee pollen: A review. \u003cem\u003eJournal of the Science of Food and Agriculture\u003c/em\u003e \u003cstrong\u003e96\u003c/strong\u003e, 4303-4309, doi:10.1002/jsfa.7729 (2016).\u003c/li\u003e\n\u003cli\u003eQiao, J.\u003cem\u003e et al.\u003c/em\u003e Rapeseed Bee Pollen Alleviates Chronic Non‐bacterial Prostatitis via Regulating Gut Microbiota. \u003cem\u003eJournal of the Science of Food and Agriculture\u003c/em\u003e, doi:10.1002/jsfa.12878 (2023).\u003c/li\u003e\n\u003cli\u003eChabot, S.\u003cem\u003e et al.\u003c/em\u003e Impact of Cernitin\u0026trade; on induced chronic prostatitis in animal model for understanding management of lower urinary tract symptoms. \u003cem\u003ePhytomedicine Plus\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 100057, doi:10.1016/j.phyplu.2021.100057 (2021).\u003c/li\u003e\n\u003cli\u003eAlgethami, J. S.\u003cem\u003e et al.\u003c/em\u003e Bee pollen: Clinical trials and patent applications. \u003cem\u003eNutrients\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 2858 (2022).\u003c/li\u003e\n\u003cli\u003eZhang, J., Liang, C., Shang, X. \u0026amp; Li, H. Chronic prostatitis/chronic pelvic pain syndrome: a disease or symptom? Current perspectives on diagnosis, treatment, and prognosis. \u003cem\u003eAmerican journal of men\u0026apos;s health\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 1557988320903200, doi:10.1177/155798832090320 (2020).\u003c/li\u003e\n\u003cli\u003eKrieger, J. N., Riley, D. E., Cheah, P. Y., Liong, M. L. \u0026amp; Yuen, K. H. Epidemiology of prostatitis: new evidence for a world-wide problem. \u003cem\u003eWorld journal of urology\u003c/em\u003e \u003cstrong\u003e21\u003c/strong\u003e, 70-74, doi:10.1007/s00345-003-0329-0 (2003).\u003c/li\u003e\n\u003cli\u003eZhang, L.\u003cem\u003e et al.\u003c/em\u003e Correlation between prostatitis, benign prostatic hyperplasia and prostate cancer: a systematic review and meta-analysis. \u003cem\u003eJournal of Cancer\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 177, doi:10.7150/jca.37235 (2020).\u003c/li\u003e\n\u003cli\u003eMartinez-Armenta, C.\u003cem\u003e et al.\u003c/em\u003e Therapeutic potential of bioactive compounds in honey for treating osteoarthritis. \u003cem\u003eFrontiers in pharmacology\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 642836, doi:10.3389/fphar.2021.642836 (2021).\u003c/li\u003e\n\u003cli\u003eLazzarotto-Figueir\u0026oacute;, J.\u003cem\u003e et al.\u003c/em\u003e Antioxidant activity, antibacterial and inhibitory effect of intestinal disaccharidases of extracts obtained from Eugenia uniflora L. Seeds. \u003cem\u003eBrazilian Journal of Biology\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 291-300, doi:10.1590/1519-6984.224852 (2020).\u003c/li\u003e\n\u003cli\u003eNAKASE, K., KIMURA, I. \u0026amp; KIMURA, M. Effects of pollen-extract components, diamines and derivatives of feruloylputrescine on isolated bladder and urethral smooth muscles of mice. \u003cem\u003eThe Japanese Journal of Pharmacology\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 157-164, doi:10.1254/jjp.53.157 (1990).\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;nstedt, K. \u0026amp; Bogdanov, S. Bee products and their potential use in modern medicine. \u003cem\u003eJournal of ApiProduct and ApiMedical Science\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 57-63, doi:10.3896/IBRA.4.01.3.01 (2009).\u003c/li\u003e\n\u003cli\u003eYang, B. C., Jin, L. L., Yang, Y. F., Li, K. \u0026amp; Peng, D. M. Inhibitory effect of rape pollen supercritical CO2 fluid extract against testosterone‑induced benign prostatic hyperplasia in rats. \u003cem\u003eExperimental and therapeutic medicine\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 31-37, doi:10.3892/etm.2014.1680 (2014).\u003c/li\u003e\n\u003cli\u003eWagner, A. E.\u003cem\u003e et al.\u003c/em\u003e DSS-induced acute colitis in C57BL/6 mice is mitigated by sulforaphane pre-treatment. \u003cem\u003eThe Journal of nutritional biochemistry\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 2085-2091, doi:10.1016/j.jnutbio.2013.07.009 (2013).\u003c/li\u003e\n\u003cli\u003eThorpe, A. \u0026amp; Neal, D. Benign prostatic hyperplasia. \u003cem\u003eThe Lancet\u003c/em\u003e \u003cstrong\u003e361\u003c/strong\u003e, 1359-1367, doi:10.1016/S0140-6736(03)13073-5 (2003).\u003c/li\u003e\n\u003cli\u003eChung, A., Kini, M., Hartigan, S. M., Chughtai, B. \u0026amp; Dmochowski, R. R. in \u003cem\u003eMolecular Mechanisms of Nutritional Interventions and Supplements for the Management of Sexual Dysfunction and Benign Prostatic Hyperplasia\u003c/em\u003e 107-111 (Elsevier, 2021).\u003c/li\u003e\n\u003cli\u003eEl-Khatib, F. M., Yafi, N. R. \u0026amp; Yafi, F. A. in \u003cem\u003eEffects of Lifestyle on Men\u0026apos;s Health\u003c/em\u003e 281-300 (Elsevier, 2019).\u003c/li\u003e\n\u003cli\u003eQiao, J.\u003cem\u003e et al.\u003c/em\u003e Phenolamide and flavonoid glycoside profiles of 20 types of monofloral bee pollen. \u003cem\u003eFood Chemistry\u003c/em\u003e \u003cstrong\u003e405\u003c/strong\u003e, 134800, doi:10.1016/j.foodchem.2022.134800 (2023).\u003c/li\u003e\n\u003cli\u003eZhang, H.\u003cem\u003e et al.\u003c/em\u003e Antioxidant and anti-inflammatory activities of rape bee pollen after fermentation and their correlation with chemical components by ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry-based untargeted metabolomics. \u003cem\u003eFood Chemistry\u003c/em\u003e \u003cstrong\u003e409\u003c/strong\u003e, 135342, doi:10.1016/j.foodchem.2022.135342 (2023).\u003c/li\u003e\n\u003cli\u003eZhang, X.\u003cem\u003e et al.\u003c/em\u003e Phenolamide extract of apricot bee pollen alleviates glucolipid metabolic disorders and modulates the gut microbiota and metabolites in high-fat diet-induced obese mice. \u003cem\u003eFood \u0026amp; Function\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 4662-4680, doi:10.1039/D3FO01016C (2023).\u003c/li\u003e\n\u003cli\u003eKim, S. B.\u003cem\u003e et al.\u003c/em\u003e Polyamine derivatives from the bee pollen of Quercus mongolica with tyrosinase inhibitory activity. \u003cem\u003eBioorganic Chemistry\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 127-133, doi:10.1016/j.bioorg.2018.08.014 (2018).\u003c/li\u003e\n\u003cli\u003eZhao, Q.\u003cem\u003e et al.\u003c/em\u003e Lycopene attenuates chronic prostatitis/chronic pelvic pain syndrome by inhibiting oxidative stress and inflammation via the interaction of NF‐\u0026kappa;B, MAPKs, and Nrf2 signaling pathways in rats. \u003cem\u003eAndrology\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 747-755, doi:10.1111/andr.12747 (2020).\u003c/li\u003e\n\u003cli\u003eShu, Y.\u003cem\u003e et al.\u003c/em\u003e The polysaccharides from Auricularia auricula alleviate non-alcoholic fatty liver disease via modulating gut microbiota and bile acids metabolism. \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e \u003cstrong\u003e246\u003c/strong\u003e, 125662, doi:10.1016/j.ijbiomac.2023.125662 (2023).\u003c/li\u003e\n\u003cli\u003eHarris, J.\u003cem\u003e et al.\u003c/em\u003e Autophagy and inflammasomes. \u003cem\u003eMolecular Immunology\u003c/em\u003e \u003cstrong\u003e86\u003c/strong\u003e, 10-15, doi:10.1016/j.molimm.2017.02.013 (2017).\u003c/li\u003e\n\u003cli\u003eLiu, Y.\u003cem\u003e et al.\u003c/em\u003e Autophagy alleviates ethanol-induced memory impairment in association with anti-apoptotic and anti-inflammatory pathways. \u003cem\u003eBrain, Behavior, and Immunity\u003c/em\u003e \u003cstrong\u003e82\u003c/strong\u003e, 63-75, doi:10.1016/j.bbi.2019.07.033 (2019).\u003c/li\u003e\n\u003cli\u003eChoi, A. J. \u0026amp; Ryter, S. W. Autophagy in inflammatory diseases. \u003cem\u003eInternational journal of cell biology\u003c/em\u003e \u003cstrong\u003e2011\u003c/strong\u003e, doi:10.1155/2011/732798 (2011).\u003c/li\u003e\n\u003cli\u003eFan, X.\u003cem\u003e et al.\u003c/em\u003e Berberine alleviates ox-LDL induced inflammatory factors by up-regulation of autophagy via AMPK/mTOR signaling pathway. \u003cem\u003eJournal of translational medicine\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 1-11, doi:10.1186/s12967-015-0450-z (2015).\u003c/li\u003e\n\u003cli\u003eSu, Y.\u003cem\u003e et al.\u003c/em\u003e Rapamycin alleviates hormone imbalance-induced chronic nonbacterial inflammation in rat prostate through activating autophagy via the mTOR/ULK1/ATG13 signaling pathway. \u003cem\u003eInflammation\u003c/em\u003e \u003cstrong\u003e41\u003c/strong\u003e, 1384-1395, doi:10.1016/B978-0-12-816665-9.00015-9 (2018).\u003c/li\u003e\n\u003cli\u003eHwang, H.-Y., Shim, J. S., Kim, D. \u0026amp; Kwon, H. J. Antidepressant drug sertraline modulates AMPK-MTOR signaling-mediated autophagy via targeting mitochondrial VDAC1 protein. \u003cem\u003eAutophagy\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 2783-2799, doi:10.1080/15548627.2020.1841953 (2021).\u003c/li\u003e\n\u003cli\u003eHou, T.\u003cem\u003e et al.\u003c/em\u003e IL-37 ameliorating allergic inflammation in atopic dermatitis through regulating microbiota and AMPK-mTOR signaling pathway-modulated autophagy mechanism. \u003cem\u003eFrontiers in Immunology\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 752, doi:10.3389/fimmu.2020.00752 (2020).\u003c/li\u003e\n\u003cli\u003eKujawski, R.\u003cem\u003e et al.\u003c/em\u003e Diet based on oil of seeds of Brassica napus. Implications for the prevention and treatment of prostate diseases. \u003cem\u003eHerba Polonica\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 77-88, doi: 10.2478/hepo-2014-0018 (2015).\u003c/li\u003e\n\u003cli\u003eCao, W., Li, J., Yang, K. \u0026amp; Cao, D. An overview of autophagy: Mechanism, regulation and research progress. \u003cem\u003eBulletin du cancer\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 304-322, doi:10.1016/j.bulcan.2020.11.004 (2021).\u003c/li\u003e\n\u003cli\u003eHindupur, S. K., Gonz\u0026aacute;lez, A. \u0026amp; Hall, M. N. The opposing actions of target of rapamycin and AMP-activated protein kinase in cell growth control. \u003cem\u003eCold Spring Harbor perspectives in biology\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, a019141, doi:10.1101/cshperspect.a019141 (2015).\u003c/li\u003e\n\u003cli\u003eSekirov, I., Russell, S. L., Antunes, L. C. M. \u0026amp; Finlay, B. B. Gut microbiota in health and disease. \u003cem\u003ePhysiological reviews\u003c/em\u003e, doi:10.1152/physrev.00045.2009 (2010).\u003c/li\u003e\n\u003cli\u003eSultan, S.\u003cem\u003e et al.\u003c/em\u003e Metabolic influences of gut microbiota dysbiosis on inflammatory bowel disease. \u003cem\u003eFrontiers in physiology\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1489, doi:10.3389/fphys.2021.715506 (2021).\u003c/li\u003e\n\u003cli\u003eYamashiro, K.\u003cem\u003e et al.\u003c/em\u003e Gut dysbiosis is associated with metabolism and systemic inflammation in patients with ischemic stroke. \u003cem\u003ePloS one\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, e0171521, doi:10.1371/journal.pone.0171521 (2017).\u003c/li\u003e\n\u003cli\u003eWang, G.\u003cem\u003e et al.\u003c/em\u003e Lactic acid bacteria reduce diabetes symptoms in mice by alleviating gut microbiota dysbiosis and inflammation in different manners. \u003cem\u003eFood \u0026amp; function\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 5898-5914, doi:10.1039/C9FO02761K (2020).\u003c/li\u003e\n\u003cli\u003eWhite, B.\u003cem\u003e et al.\u003c/em\u003e Microbiota of chronic prostatitis/chronic pelvic pain syndrome are distinct from interstitial cystitis/bladder pain syndrome. \u003cem\u003emedRxiv\u003c/em\u003e, 2021.2003. 2004.21252926, doi:10.1101/2021.03.04.21252926 (2021).\u003c/li\u003e\n\u003cli\u003eYang, W., Gao, B., Qin, L. \u0026amp; Wang, X. Puerarin improves skeletal muscle strength by regulating gut microbiota in young adult rats. \u003cem\u003eJournal of Orthopaedic Translation\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 87-98, doi:10.1016/j.jot.2022.08.009 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"phenolamides, rapeseed bee pollen, AMPK/mTOR, anti-inflammation, di-p-Coumaroyl spermidine","lastPublishedDoi":"10.21203/rs.3.rs-5846138/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5846138/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBee pollen and its extracts have been used for decades as therapeutic agents or health food supplements to alleviate chronic non-bacterial prostatitis (CNP). However, functional compounds in bee pollen on anti-CNP remain still unclear. In this study, we evaluated the anti-CNP properties of six principal phenolamides in bee pollen. Our results provide compelling evidence that the anti-CNP property of bee pollen may be ascribed to its abundance of phenolamides. Particularly, di-\u003cem\u003ep\u003c/em\u003e-Coumaroyl spermidine can alleviate CNP by upregulating autophagy via the AMPK/mTOR signaling pathway and regulating gut microbiota, based on the cellular and rat models. Additionally, our finding may provide a novel insight into the gut-prostate axis by regulating di-\u003cem\u003ep\u003c/em\u003e-Coumaroyl spermidine. This is the first report that di-\u003cem\u003ep\u003c/em\u003e-Coumaroyl spermidine in bee pollen possesses the anti-prostatitis function. This paper will be likely helpful further to develop functional foods, personalized nutraceuticals, and medicine from bee pollen.\u003c/p\u003e","manuscriptTitle":"Di-p-coumaroyl spermidine from bee pollen alleviates chronic non- bacterial prostatitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-31 05:02:15","doi":"10.21203/rs.3.rs-5846138/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"54f204a7-739b-4fdb-8023-9de4d72005ee","owner":[],"postedDate":"January 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":43636999,"name":"Health sciences/Health care"},{"id":43637000,"name":"Health sciences/Health care/Nutrition"},{"id":43637001,"name":"Biological sciences/Cell biology/Autophagy"}],"tags":[],"updatedAt":"2025-02-26T23:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-31 05:02:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5846138","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5846138","identity":"rs-5846138","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}