Dihydroartemisinin ameliorated the inflammatory response and regulated miRNA-mRNA expression profile of chronic nonbacterial prostatitis

Background. Chronic nonbacterial prostatitis (CNP) is a chronic inflammatory disease. Patients often have trouble urinating, experience painful and frequent urination, and pelvic floor pain, which seriously affects their quality of life. Dihydroartemisinin (DHA) is the most important artemisinin derivative with good anti-inflammatory effects. However, the mechanism of DHA for CNP has not been fully elucidated. Objectives. To examine the protective effect of DHA on CNP in mice model and to explore the potential mechanisms from the perspective of microRNAs (miRNAs). Materials and methods


Background
Chronic prostatitis is a chronic inflammation that affects approx.8-11.5% of men worldwide, 1,2 and more than 90% of chronic prostatitis are caused by chronic nonbacterial prostatitis (CNP). 3,4Patients often experience difficulty urinating, painful and frequent urination, and pelvic floor pain, which seriously affects their quality of life. 5Due to the multifactorial and complex pathogenesis of CNP, its mechanism has long been debated.However, at present, it remains not fully elucidated.Most researchers believe it is caused by a combination of etiological infection, inflammation, abnormal neuromuscular activity in the pelvic floor, and immune abnormalities. 6][9][10] Antibiotics, botanicals, alpha-blockers, and non-steroidal anti-inflammatory analgesics are common treatment methods for CNP. 11However, chemicals are not appropriate as permanent therapeutics because they are prone to adverse reactions.Antibiotics are unlikely to help unless combined with alpha-blockers, and they are prone to dependence. 12Surprisingly, the therapeutic role of traditional Chinese medicine (TCM) and some botanical ingredients in the treatment of chronic prostatitis has received increasing attention in recent years. 13,14Therefore, the search for a safe, efficient and well-absorbed botanical component is of great importance for the treatment of CNP.
Recently, there has been remarkable advancement in TCM research.Some natural products have significant anti-inflammatory activity and promising applications for treating prostate diseases.Artemisinin is a special sesquiterpene lactone containing a peroxide bond extracted from the annual Compositae family member Artemisia annua L., which is an effective antimalarial component. 15,16ihydroartemisinin (DHA) is the most important artemisinin derivative, created by the oxidation of artemisinin with sodium tetrahydroborate. 17As the first-generation derivative of artemisinin, DHA has better water solubility and stability, high efficiency, low toxicity, and strong activity. 180][21] Moreover, studies have reported that DHA improved the symptoms of systemic lupus erythematosus in mice and regulated the secretion of the pro-inflammatory mediator tumor necrosis factor-α (TNF-α).It was thought that the possible mechanism was via the inhibition of NF-κB-induced inflammatory cascade by affecting NF-κB activation and translocation to the nucleus. 22Previously, we investigated the protective effect of DHA on hyperglycemia-induced vascular smooth muscle cell (VSMC) proliferation and associated inflammation.Dihydroartemisinin dramatically lowered the mRNA levels of interleukin-1β (IL-1β) and TNF-α, and this mechanism may play a protective role in VSMC proliferation and inflammation via suppressing the miR-376b-3p/KLF15 axis. 23Previous research has demonstrated that DHA can lower the generation of inflammatory factors in prostatitis tissues and alleviate prostatitis and the inflammatory response by inhibiting the E2F7/HIF1α pathway. 24owever, little attention has been paid to gene regulation.MicroRNAs (miRNAs) have been extensively studied in many diseases, and their expression can be modulated by many different compounds. 25To identify CNP-specific differentially expressed miRNAs (DEMs) and mRNAs, we used a bioinformatics approach to investigate candidate miRNA-mRNA expression profiles of DHA involved in the CNP pathogenesis.

Objectives
In this study, we investigated the therapeutic impact of DHA on CNP mice from pharmacogenetic point of view and examined the underlying mechanisms responsible for this effect.The effect of DHA on miRNA-mRNA expression profile in the prostate tissue of CNP model mice was investigated by small RNA sequencing, which laid the foundation for the study on CNP.

Animals and drugs
Male nonobese diabetic (NOD) mice (6-8 weeks old, 20-22 g), supplied from Beijing Zhongke Zesheng Biotechnology Co., Ltd.(Beijing, China), were used in all experiments.Before experimental manipulation, all animals were exposed to 12 h of light and dark cycle and constantly supplied with food and drink for at least 7 days.The mice were fed a general experimental animal diet provided by Chengdu Dossy Experimental Animal Co., Ltd.(Chengdu, China).The composition of the animal diet was as follows: corn, soybeans, bone flour, wheat, fish flour, sodium chloride, and vitamin complex.All animal procedures were approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University (approval No. IACUC-Hebmu-P2021075).Dihydroartemisinin was purchased from Shanghai Yuanye Biotechnology Co., Ltd (batch No. B21182, purity ≥98%).

Experimental autoimmune prostatitis
Prostate tissue was harvested from Sprague-Dawley rats under aseptic conditions, washed with saline, and added to a saline solution containing 0.5% Triton X-100.The prostate tissue was homogenized on ice prior to centrifugation to obtain the supernatant.The bicinchoninic acid (BCA) protein concentration assay kit was used to determine the protein concentration in the supernatant, which was adjusted to 20 mg/mL.Subsequently, the rat prostate protein purification solution was combined with an equivalent volume of complete Freund's adjuvant via ultrasound to make a suspension.
Nonobese diabetic mice (n = 24) were randomized into control, model, DHA (150 mg/kg), and positive drug (aspirin tablets, 150 mg/kg) groups (n = 6).Each mouse in the control group was injected subcutaneously with phosphate buffer saline (PBS) + complete Freund's adjuvant (0.1 mL).In the experimental autoimmune prostatitis (EAP) model group, DHA group and positive drug group, each mouse was injected subcutaneously with 0.1 mL of purified prostate protein solution and complete Freund's adjuvant.A second immunization was performed after 14 days to establish a mouse model of chronic nonbacterial prostatitis, EAP.The DHA and positive drug groups were administered by gavage starting with the second immunization and repeated every 2 weeks until 28 days of harvesting.The pain thresholds were measured using von Frey fiber filaments on day 28.All mice were sedated with ip.pentobarbital (5%, 50 mg/kg), and the prostate tissue was immediately isolated and weighed to calculate the prostate index.Prostate index = prostate weight (mg)/ body weight (g).The tissues were stored in a -80°C environment for later analysis.The detailed experimental procedure is shown in Fig. 1A.

Behavioral nociception measurement
The pain threshold of NOD mice was determined using von Frey filaments, and the skin abnormalities were assessed on day 28 after immunization. 26The test was performed in a transparent plastic chamber with a stainless-steel grid floor.Mice were placed in the experimental room for at least 30 min to acclimatize to the environment before the behavioral tests began.Von Frey filaments were then used to measure the tactile abnormal discomfort and hyperalgesia in each mouse at forces of 0, 0.4, 1.0, and 4.0 g, respectively.Von Frey filaments were administered on the abdomen for 1-2 s, 10 times, with 2 min between each stimulation.To prevent desensitization, stimulation was limited to the lower abdomen and varied areas within this area were stimulated.Positive reactions to filament stimulation were classified into 3 types, namely 1) abrupt abdominal retraction; 2) quick licking or scratching; or 3) jumping. 27The percentage reaction frequency was calculated as [(the number of positive responses/10 trials) × 100 = percentage response frequency].

Hematoxylin and eosin staining
The prostate tissue was fixed, dehydrated and embedded in paraffin before being cut into sections and stained as follows. 28The prostate tissue sections were first dewaxed, and then stained with hematoxylin for 20 min.Next, after being separated by HCl/95% alcohol for 10 s, they were put into a weak ammonia solution at 50°C to return to a blue color.The slides were then stained with eosin for 5 min.Sections then underwent a soaking process in graded concentrations of alcohol, followed by treatment with xylene to achieve transparency.They were then sealed with neutral gum and examined under a microscope (Zeiss AxioVision, Oberkochen, Germany).

TUNEL staining
TUNEL staining was performed in the previous study. 29o prepare the tissue sections, they were first deparaffinized and rehydrated, and then soaked in water for 5 min.Antigen retrieval was performed with a microwave, heating the samples for 8 min, followed by 3 washes with PBS (5 min per wash).Next, a TUNEL reaction mixture consisting of 50 μL TDT and 450 μL fluorescein-labeled dUTP solution was added to each slice, which was then incubated at 37°C for 1 h.Then, the sections were rinsed with PBS for 5 min × 3 times.DAPI nuclear staining was performed for 5 min, followed by rinsing with PBS and mounting the sections with 50% glycerol.The samples were stored at -20°C for microscopic examination (Nikon, Tokyo, Japan).The TUNEL analysis was assessed as 10 visual fields per mouse, and the apoptosis rate of each mouse was calculated.

Western blotting
The tissues were first frozen and subsequently ground to a powder.The resulting powder was then lysed using RIPA lysate while kept on ice for 15 min.The lysed samples were then centrifuged at 12,000 rpm at 4°C for 10 min, and the resulting supernatant was collected.The protein concentration of the supernatant was determined using a BCA protein assay kit.To denature proteins, they were heated at 95°C for 5 min before being stored at a low temperature (-80°C) for subsequent analysis.Next, the samples were separated on 8-10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA).The membranes were blocked with 5% skim milk and left in a shaker for 2 h at 37°C.Then, primary antibodies (anti-IL-1β, No. A1112, 1:2000; anti-IL-6, No. A0286, 1:2000; anti-TNF-α, No. A11534, 1:2000; MCP-1, No. A22744, 1:2000; β-actin, No. AC026, 1:100,000; all from Abclonal, Woburn, USA) were added and incubated overnight at 4°C.After washing with PBS, they were incubated with secondary antibodies (goat anti-rabbit IgG (H + L), No. s0001, 1:5000; Affinity, Changzhou, China) for 2 h, then washed with Tris-buffered saline with Tween (TBST) 3 times and colored by enhanced chemiluminescence (ECL) (Bio-Rad, Foster City, USA).Finally, the images were scanned and analyzed by a gel image analysis system.

Small RNA sequencing
The total RNA extraction reagent Trizol was used to obtain the total RNA.An Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, USA) was used to evaluate sample concentration and integrity, which served as a reference for the library building and bioinformatic analysis.Polyacrylamide gel electrophoresis (PAGE) was used to separate the RNA fragments, and short RNAs with lengths of 18-30 nt were obtained.The Illumina TruSeq Small RNA Sample Prep kit (New England Biolabs, Inc., Ipswich, USA) was used to create small RNA libraries.Then, enriched libraries were amplified by polymerase chain reaction (PCR) to enrich cDNA with both 3' and 5' connectors.After cDNA amplification, the small RNA libraries were further purified by electrophoresis.The quality of the library was identified using an Agilent 2100 Bioanalyzer and an Agilent High Sensitivity DNA Kit (Agilent Technologies, Foster City, USA), with the criterion of the qualified libraries showing only a single peak and no junction.The libraries were then measured using the Quan-iT PicoGreen dsDNA assay kit (Thermo Fisher Scientific, Waltham, USA), and the Illumina platform was used for RNA sequencing.Single-stranded cDNA was used as a template for PCR amplification and sequencing by annealing a sequencing primer.The target genes of these miRNAs were classified after the identification of DEMs using sequencing.Finally, the miRNA targets were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.

Data analysis and statistical methods
GraphPad Prism 8.0 (GraphPad Software, San Diego, USA) and IBM SPSS 25.0 statistical software (IBM SPSS, Armonk, USA) were used for statistical analysis.The Shapiro-Wilk method was first used to test for normal distribution, and these were expressed as mean ± standard deviation (SD), and non-normally distributed data were expressed as median and percentiles (25 th percentile (P25) and 75 th percentile (P75)).For data obeying normal distribution, one-way ANOVA was used for comparison between multiple groups, Levene's test was used for homogeneity of variance analysis, the LSD test for data with homogeneity of variance, and Tamhane's T2 test for data with unequal variance.Repeated measures were analyzed using a two-way repeated measures ANOVA followed by Bonferroni's multiple comparison test.Comparisons of non-normally distributed data were analyzed by nonparametric tests using the Kruskal-Wallis method.All data are presented as the means and 95% confidence interval whiskers.The value of p < 0.05 was considered statistically significant.In addition, Spearman's correlation analysis was utilized to compare 2 genes, with p < 0.05 representing statistical significance.

DHA improved nociceptive sensitivity in the CNP model mice
The pain threshold of NOD mice was evaluated by von Frey filaments on day 28 after immunization to evaluate the effect of DHA on chronic pelvic pain in the CNP mice.The response frequency to tactile hypersensitivity was considerably greater in the model group compared with the control group (Kruskal-Wallis, F = 20.205,p < 0.001), and the response frequency was higher after the second vaccination compared to the first, indicating that CNP can directly produce pain in mice.The response frequency was lower in the DHA and positive drug groups compared with the model group under the forces of 0.4 g, 1.0 g and 4.0 g, respectively (Fig. 1B, Supplementary Table 1).To varying degrees, continuous daily doses of DHA and aspirin reduced the frequency of CNP-induced responses, suggesting that DHA has a pain-relieving effect on CNP mice.
Four weeks later, the average body weight of mice treated with rat prostate protein + Freund's adjuvant decreased sharply (F (3.9) = 144.847,p < 0.001).However, body weight increased after DHA administration, although it remained significantly lower than in the control group (Fig. 1C, Supplementary Table 1).We also investigated how DHA affected benign prostatic hyperplasia (BPH).The prostate index, which is the ratio of prostate weight to body weight, can be used to indicate BPH.Our results revealed that the prostate index was raised in the model group (one-way ANOVA, F = 21.002,p < 0.001) but was lowered by DHA treatment (one-way ANOVA, F = 21.002,p = 0.006) (Fig. 1D, Supplementary Table 1).The difference in prostate index between the DHA and model group may be due to DHA reducing the inflammatory congestion and edema of the prostate tissue in mice.

DHA ameliorated histopathological damage and apoptosis of prostate tissue in the CNP model mice
Next, we evaluated the effect of DHA on prostate tissue using pathological staining.Hematoxylin and eosin staining of prostate tissues showed that the control group contained normal structures without histopathological changes.The prostate tissue framework in the control group was integrated and transparent and exhibited an enlarged gland cavity with many folds.Moreover, the prostate gland exhibited a monolayer of flattened, cuboidal, columnar, or pseudostratified columnar glandular epithelium, which was neatly arranged and without showing obvious degeneration and necrosis.The model group's prostate tissue structure was considerably deteriorated as compared to the control group.It was obvious that some of the glandular ducts in the tissue were obscured, epithelial cells displayed degeneration and necrosis, were relatively flat with surrounding interstitial edema and inflammatory cell infiltration, and a large number of lymphocytes were observed.Surprisingly, the prostate tissue improved markedly after DHA therapy.However, edema with inflammatory cell infiltration, lymphocytes, plasma cells, neutrophils and mast cells could still be observed in the interstitium surrounding the glandular duct (Fig. 2A).
The principle of the TUNEL technique is that apoptosis is detected by labeling the 3'-OH site of DNA with fluorescein-conjugated nucleotides in the presence of deoxyribonuclease (TdT).Nuclei were stained with DAPI, and yellow cells were considered apoptotic.The results of TU-NEL staining showed that DHA ameliorated cell apoptosis in the prostate tissue (Fig. 2B).Freund's adjuvant stimulation significantly increased apoptosis in prostate cells (one-way ANOVA, F = 271.622,p < 0.001), although the significant increase of apoptosis level in the model group was suppressed by DHA (one-way ANOVA, F = 271.622,p < 0.001) (Fig. 2C, Supplementary Table 2).

Data filtering and small RNA sorting
Small RNA sequencing was performed on the control group, model group and DHA group, producing a total of 201,337,093 raw reads and 197,003,650 clean reads (≥18 nt) from 9 samples (3 samples per group).We discarded sequences with average sequencing quality below 20 and then filtered them for subsequent analysis.The average clean rate was 97.83%, which indicated that the quality control of sequencing data was reliable (Table 1).We then summarized the annotation of all small RNAs compared to various types of RNAs (Fig. 3A).

Screening and analysis of differentially expressed miRNAs
We conducted a Pearson correlation coefficient analysis to find any correlation between miRNA expressions (Fig. 3B).The Pearson correlation coefficient was 0.99, indicating that the samples were extremely strongly correlated with each other.Next, we analyzed the differences in miRNA expression levels by DESeq (the criteria are based on |log2(fold-change)| ≥1 and p < 0.05).The findings revealed that 13 miRNAs were significantly modified in the model group (p < 0.05), with 7 miRNAs upregulated and 6 miRNAs downregulated (Fig. 3C).A total of 30 miR-NAs were notably expressed in the DHA group (p < 0.05), with 20 miRNAs being upregulated and 10 miRNAs being downregulated (Fig. 3D).Next, we performed bidirectional clustering analysis of all miRNAs and samples, which revealed differences in 48 differentially expressed genes in each sample (Fig. 3E).Four miRNAs (miR-467a-5p, miR-467b-5p, miR-335-3p, miR-669c-5p) were downregulated in the CNP mice and upregulated after DHA treatment, while 2 miRNAs (miR-490-3p, miR-672-5p) were upregulated in the CNP mice and downregulated after DHA treatment (Table 2).Subsequently, we detected 6 differentially expressed miRNAs in prostatitis tissues using real-time quantitative PCR (Fig. 4A-F, Supplementary Table 3), and statistical analysis was performed using oneway ANOVA and Kruskal-Wallis test.Although there was no significant difference in the expression of miR-669c-5p (one-way ANOVA, F = 12.930, p = 0.134), miR-467a-5p, miR-467b-5p and miR-335-3p displayed reduced expression in the model group compared to the control group (F = 6.006, p = 0.017; F = 25.987,p < 0.001; F = 6.030, p = 0.015), and the expression levels of miR-467b-5p and  miR-669c-5p were higher after DHA treatment compared to the model group before treatment (F = 25.987,p = 0.021; F = 12.930, p = 0.006).In contrast, miR-490-3p and miR-672-5p levels were higher in the model group when compared to the control group (F = 6.489, p = 0.011; F = 6.489, p = 0.011), while miR-490-3p and miR-672-5p levels were lower in the DHA group relative to the model group.However, this final result did not reach statistical significance, which was consistent with the sequencing data.

miRNA target gene prediction and bioinformatics analysis
We performed target gene prediction on differentially expressed miRNA sequences.A total of 48 miRNAs were analyzed, resulting in 15,689 predicted target genes, and 73,083 predicted target sites.To investigate the function and mechanisms of miRNA target genes, GO and KEGG pathway enrichment studies were carried out.Gene Ontology enrichment analysis of the control compareed to the model group showed that there were 1887 statistically significant GO term entries in the total differential genome (p < 0.05).The top 20 enriched GO terms of control vs model groups are shown in Fig. 5A.The biological processing (BP) contained "regulation of cellular process", "regulation of biological process", "signaling", etc.For the cellular components (CC), terms related to "membrane" and "cell periphery" were enriched, while the molecular function (MF) included olfactory receptor activity.In the control vs model group, there were 2,631 statistically significant GO term entries in the total differential genome (p < 0.05).The top 20 enriched GO terms of model vs DHA groups were involved in BP (cellular process, biological regulation, regulation biological process, regulation of the cellular process, etc.) and CC (cell periphery, membrane, intracellular, etc.) (Fig. 5B).
Further KEGG pathway analysis of the control vs model group showed that 27 pathways were enriched (p < 0.05), including 5 major categories, namely "organismal systems", "human diseases", "environmental information processing", "cellular processes", and "metabolism".Correspondingly, pathways were also involved in cancer (breast cancer, microRNAs in cancer), glycan biosynthesis and metabolism, signal transduction (phosphatidylinositol signaling system, Wnt, MAPK, Rap1, and sphingolipid signaling pathway), lipid metabolism (biosynthesis of unsaturated fatty acids), and infectious disease: bacterial (yersinia infection), among others.Detailed information is shown in Fig. 5C.In the model vs DHA group, there were 94 statistically significant KEGG pathways in the total differential genome (p < 0.05).The top 20 signaling pathways were divided into 4 major categories, "cellular processes", "human diseases", "organismal systems", and "environmental information processing", all of which were also involved in cancer (microRNAs in cancer, choline metabolism in cancer, gastric cancer, breast cancer), signal transduction (mTOR, Hippo, sphingolipid, and phosphatidylinositol signaling), immune system (T cell receptor signaling pathway), etc. Detailed information is shown in Fig. 5D.

Discussion
Prostatitis seriously affects men's health, and the malignant development of prostate tissue cells may further transform into prostate cancer.Even though it is one of the most prevalent prostate disorders in the world, CNP is still debatable in terms of its genesis.1][32] However, accumulated evidence has indicated that TCM has amassed rich experience in clinical practice and achieved certain curative effects in the treatment of prostate diseases.Due to the disadvantages of poor water solubility and low bioavailability of artemisinin, DHA, an important derivative of artemisinin, exhibits better bioavailability and increased antimalarial effects compared to artemisinin.In addition, DHA also has anti-inflammatory and anti-tumor activity, among other pharmacological effects.Herein, we examined how DHA affected mice with prostatitis caused by the administration of prostate tissue and complete Freund's adjuvant.The EAP model is straightforward to construct and has a high success rate.The modeled Previous investigations have demonstrated that cytokines and inflammation are key factors in the development of CNP. 33When the prostate is stimulated, it produces a wide range of host defense and inflammatory factors. 34he IL-1β is a critical pro-inflammatory factor that leads to the occurrence and persistence of inflammation.The release of caspase-1 induced by NOD-like receptor protein 3 (NLRP3) inflammasome and the downstream cytokine IL-1β may be one of the pathogenic mechanisms of prostatitis. 35,36The IL-6 is an important proinflammatory interleukin, and the level of IL-6 in CNP has been strongly correlated to the intensity of patients' clinical symptoms. 37The IL-6 is a pleiotropic cytokine that regulates T cell migration and chronic immune responses and is strongly linked to prostate cell growth and death. 38In addition, IL-6 may regulate the occurrence and development of CNP by regulating cellular autophagy. 39he TNF-α is a major pro-inflammatory cytokine that is involved in the induction of inflammatory responses as well as the regulation of immune responses. 33Furthermore, TNF-α can increase the production of cytokines, including IL-1, IL-6 and IL-8, by neutrophils and mononuclear macrophages, which stimulates the inflammatory cascade and exacerbates the inflammatory response. 40n addition to inflammatory factors, MCP-1 is used as an inflammatory biomarker to identify the response of CNP patients to treatment. 38Research has revealed that MCP-1 activates monocytes and macrophages and is involved in chemotaxis. 41It promoted the production of pro-inflammatory cytokines, which in turn boosted the inflammatory response in the CNP pathological conditions.Our previous clinical study showed that reducing prostate inflammatory levels was effective in relieving urinary tract infections in prostate cancer patients. 42n the current investigation, CNP had elevated levels of IL-1β, TNF-α and IL-6, while DHA markedly decreased these, as well as suppressed MCP-1 levels in prostate tissue.Therefore, DHA may be beneficial for preventing the development of CNP by downregulating inflammatory factors and inflammatory mediators.
Changes in miRNA expression profiles are currently the subject of substantial research in conditions like prostate cancer, but studies in CNP are still relatively sparse.We used small RNA sequencing and real-time PCR to evaluate and validate the abnormally expressed miRNAs in order to further clarify the mechanism underlying DHA's impact on the miRNA expression profile in CNP mice.Small RNA is a key regulator of biological processes, including the control of gene expression, biological maturation, metabolism, and the emergence of illness, and CNP pathogenesis is accompanied by abnormal miRNA expression. 43e found 48 target gene miRNAs, indicating that DHA has a powerful role in altering prostate miRNA expression.The RNA sequencing results showed that 4 miRNAs (miR-467a-5p, miR-467b-5p, miR-335-3p, miR-669c-5p) were dramatically diminished in the model group and substantially elevated after DHA treatment.Meanwhile, 2 miRNAs (miR-490-3p, miR-672-5p) were elevated in the CNP mice and depressed after DHA treatment.Previous investigations have reported that miR-467a-5p reduced inflammation and elevated blood glucose and insulin levels to prevent insulin resistance. 446][47] The miR-669c-5p has been reported to be differentially expressed in mouse spermatocyte-derived GC-2 cells and may affect male reproductive function. 48he miR-490-3-p was elevated in the reproductive organs of male rats induced by a high cholesterol diet, 49 while miR-672-5p was shown to be altered in the kidney of urolithiasis rats. 50Therefore, these genes have the potential to become novel biomarkers and therapeutic targets for CNP.In this study, we performed a preliminary investigation of DEMs in CNP, and the relationship of these miRNAs with inflammatory, immune, metabolic and other signaling pathways and their mechanisms need to be further examined.
The KEGG pathway analysis of miRNA expression profile in the CNP mice revealed several signaling pathways related to the pathogenesis of CNP.These included the phosphatidylinositol signaling system, Wnt, MAPK, Rap1, and sphingolipid signaling pathway, and there is a clear correlation between some signaling pathways.However, the role of miRNAs and related signal transduction pathways in CNP occurrence and development needs further investigation.Of note, the development of DHA therapy for CNP involves several signaling pathways, including the mTOR, Hippo, sphingolipid, phosphatidylinositol, and T cell receptor signaling pathways.As expected, we found that all these pathways are involved in inflammatory and immune responses, suggesting a key regulatory role for inflammation and the immune system in initiating and progressing CNP.For example, Akt-mTOR could modulate the immune response and prevent Th17 cell over-activation in order to improve chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS). 51Jiedu Huoxue decoction was found to inhibit apoptosis and activate the Wnt/ GSK-3β/β-catenin signaling pathway to treat CP/CPPS. 52n addition, inflammatory factors could reduce CP/CPPS by downregulating NF-κB and JNK/MAPK pathway to alleviate prostatitis pain. 53In this study, DHA has demonstrated a powerful therapeutic effect for CNP.However, the role of DHA in the CNP by regulating miRNA profiles acting on inflammatory, immune and apoptotic pathways deserves further exploration.

Limitations
In this study, we screened some DEMs by transcriptomics, but the role of these miRNAs in prostatitis mice remains to be further investigated.Meanwhile, we found that inflammation and the immune system play a key regulatory role in the CNP, but the exact mechanism still needs to be examined.

Conclusions
This study explored the effectiveness and mechanism of DHA in the treatment of CNP.We found considerable improvement in the pathogenic condition following DHA administration in a CNP mouse model.The development of biomarkers associated with CNP will be based on the detection of miRNA expression profiles in prostate tissues, which will aid in the early detection, diagnosis and treatment of CNP diseases.This work added to the body of knowledge on the mechanism of DHA in the treatment of CNP and may offer a fresh approach to its prevention and management.

Fig. 1 .
Fig. 1.Dihydroartemisinin improved nociceptive sensitivity in chronic nonbacterial prostatitis (CNP) mice. A. The experimental method is depicted in a timeline diagram; B. Nonobese diabetic (NOD) mice responded to mechanical abdominal stimulation using von Frey filaments at forces of 0, 0.4, 1.0, and 4.0 g.Response frequency was analyzed by the Kruskal-Wallis test (n = 6); C. Body weight changes of mice; p-value calculated for the difference between groups by two-way repeated measures analysis of variance (ANOVA) (n = 6); D. DHA significantly decreased the prostate index compared with the model group.The prostate index was analyzed by one-way ANOVA (n = 4).Data are presented as the means and 95% confidence interval (95% CI) whiskers ## p < 0.01; ### p < 0.001 vs control group; *p < 0.05; **p < 0.01 compared with the model group; DHA -dihydroartemisinin group.

Fig. 2 .
Fig. 2. Dihydroartemisinin ameliorated histopathological damage, apoptosis and inflammatory response in prostate tissue of chronic nonbacterial prostatitis (CNP) mice. A. Hematoxylin and eosin (H&E) staining of prostate tissues.All images are exhibited at the same magnification, ×400, scale bar = 50 μm; B. Representative TUNEL images show that DHA ameliorated apoptosis in prostate cells.Blue represents nuclear staining (red arrow), and green represents TUNEL staining (yellow arrow), scale bar = 20 μm; C. The apoptosis rate of prostate cells; D. The protein expression of IL-1β, IL-6, TNF-α, and MCP-1 in the prostate tissues was detected using western blotting; E. Densitometric analysis of IL-1β, IL-6, TNF-α, and MCP-1 protein expression normalized to β-actin content (n = 3).Data are presented as the means and 95% confidence interval (95% CI) whiskers.Statistical analysis of the data was performed using one-way analysis of variance (ANOVA) ### p < 0.001compared with the control group; **p < 0.01; ***p < 0.001compared with the model group; DHA -dihydroartemisinin group.

Fig. 3 .
Fig. 3. Dihydroartemisinin affected miRNA-mRNA expression profile of prostate tissue in the chronic nonbacterial prostatitis (CNP) mice. A. Small RNA classification statistics chart.The horizontal coordinate is the sample name, and the vertical coordinate is the proportion of de-duplicated sequences annotated to various RNAs to the total de-duplicated sequences; B. Pearson correlation coefficient for a sample test.The left and the top are the sample clustering cases, the right and the bottom are the sample names, and the squares of different colors represent the high or low correlation between the 2 samples.Volcano map of differentially expressed genes (DEGs) in the control vs model group (C) and the model vs DHA group (D).Red dots represent upregulated genes, blue dots represent downregulated genes, and gray dots represent non-significant DEGs (fold change ≥2, p ≤ 0.05); E. Heatmap of DEMs in each sample based on hierarchical clustering analysis.The distance was calculated using the Euclidean method, and hierarchical clustering was applied.MiRNA with the most similar expression patterns are grouped together.Clustering was accomplished through the usage of complete linkage.Green denotes low expression, whereas red denotes strong expression C -control group; M -model group; DHA -dihydroartemisinin group.

Fig. 5 .
Fig. 5. Function and pathway analysis of the target genes of differentially expressed miRNAs (DEMs).A. Gene Ontology (GO) enrichment analysis between the control and model groups; B. GO enrichment analysis between the model and dihydroartemisinin (DHA) group; C. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis between the control and model group; D. KEGG enrichment analysis between the model and DHA group.The false discovery rate (FDR) value ranges from 0 to 1, with the values closer to 0 indicating a more substantial enrichment

Table 1 .
Sequencing data volume statistics for 9 samples

Table 2 .
Differentially expressed miRNAs responding to dihydroartemisinin (DHA) treatment CNP, which has been widely used, may cause a significant inflammatory response in the prostate gland with strong pathological specificity, prolonged model duration, and pathological changes similar to clinical manifestations.In this study, the results showed that DHA significantly improved CNP.Dihydroartemisinin reduced the frequency of pain responses, improved the epithelial cells and glandular ducts of the prostate tissue, reduced inflammatory infiltration, and decreased the prostate index.Simultaneously, DHA significantly reduced apoptosis and the inf lammatory response in prostate tissue.