Application of sarcodon aspratus exosome in preparation of medicine for treating interstitial cystitis

Abstract

The invention belongs to the technical field of exosome application, and discloses application of a sarcodon aspratus exosome in preparation of a medicine for treating interstitial cystitis. The prepared sarcodon aspratus exosome is perfused into the bladder of a rat with interstitial cystitis, and it is found that the sarcodon aspratus exosome can significantly improve urination disorder of the model rat and relieve symptoms of interstitial cystitis. In addition, the invention also finds that (1) the sarcodon aspratus exosome can obviously reduce the expression of IL-1beta, IL-6, MCP-1, CXCL-1, ICAM-1 and BAX in the bladder tissue; (2) the sarcodon aspratus exosome can significantly reduce the activation number of mast cells and inhibit degranulation of the mast cells; and (3) the sarcodon aspratus exosome can significantly improve epithelial cell apoptosis. The research shows that the sarcodon aspratus exosome can be used for treating interstitial cystitis. The research provides a new idea for the treatment of interstitial cystitis.

Description

Technical Field

[0001] This invention belongs to the field of exosome application technology, and more specifically, relates to the application of *Phyllostachys nigra* exosomes in the preparation of drugs for treating interstitial cystitis. Background Technology

[0002] Interstitial cystitis is a complex chronic disease characterized by chronic inflammation and structural changes in the bladder wall. A normal bladder wall has a protective mucosal layer that prevents irritants in urine from penetrating into the bladder muscle tissue. In patients with interstitial cystitis, this protective mechanism is abnormal, leading to increased bladder wall permeability. Urine components continuously irritate the bladder tissue, triggering a series of pathological changes. Microscopically, numerous mast cells and nerve fiber proliferations are visible in the bladder wall interstitium; these changes are closely related to persistent pain signal transmission. As the disease progresses, some patients develop characteristic "ulcerative lesions" (Hunner's ulcers) in the bladder wall, and the bladder capacity gradually decreases, losing its normal urine storage function.

[0003] The clinical presentation of this disease is highly characteristic yet highly variable. The most common symptoms include chronic pain or pressure in the pelvic region, which often worsens when the bladder is full and is temporarily relieved after urination. Urinary frequency and urgency are another core symptom; patients may urinate as many as 40-60 times a day, and in severe cases, they may need to urinate every 10-15 minutes. Nocturia (nighttime urination) is particularly troublesome, with many patients needing to get up 5-8 times a night to urinate, leading to severe sleep deprivation. The severity of symptoms often fluctuates, and may be significantly aggravated at specific stages of the menstrual cycle, during periods of increased stress, or after consuming certain foods or liquids. Approximately 90% of patients are women, and this significant gender difference suggests that hormonal factors may play an important role in the development and progression of the disease.

[0004] The etiology of interstitial cystitis remains incompletely understood. Current medical research considers it a multifactorial disease, with possible mechanisms including bladder epithelial barrier defects, abnormal autoimmune responses, neurogenic inflammation, and mast cell activation. Recent studies have also found that many patients with interstitial cystitis also suffer from other chronic pain syndromes, such as fibromyalgia, irritable bowel syndrome, and chronic fatigue syndrome, suggesting a possible shared pathophysiological basis. Genetic factors are also believed to be involved, with approximately 15-20% of patients having a family history. Notably, a significant proportion of patients recall symptoms beginning with a specific bladder infection, suggesting that infection may be one of the triggering factors, but the condition subsequently develops into a chronic inflammatory state independent of infection.

[0005] Exosomes, primarily found in body fluids, are extracellular vesicles with a diameter of 40-160 nm, playing crucial roles in maintaining cellular homeostasis, clearing cellular debris, and promoting intercellular and organ-to-cell communication. As key regulators of cell signaling, exosomes coordinate various autocrine and paracrine functions to regulate the growth and progression of the tumor microenvironment. Recent studies have found that, compared to animal-derived exosomes, plant-derived exosome-like nanovesicles have advantages such as easy availability, low immunogenicity, and good stability. Some fungal-derived exosomes exhibit functions similar to those derived from traditional Chinese medicine; for example, Ganoderma lucidum exosomes show significant inhibitory effects on non-small cell lung cancer, and exosome-like nanovesicles derived from Lentinus shiitake mushrooms demonstrate protective effects against d-galactosamine and lipopolysaccharide-induced acute liver injury in mice.

[0006] Sarcodon imbricatus, also known as the black tiger paw fungus, is a well-known edible and medicinal fungus with a tender texture and delicious flavor. Its wide distribution has attracted considerable attention from scholars both domestically and internationally. The fruiting bodies and mycelia of Sarcodon imbricatus are rich in polysaccharides, proteins, minerals, amino acids, trace elements, sterols, and other nutrients, exhibiting pharmacological activities such as antitumor, antioxidant, and antibacterial effects, showing broad application prospects. However, pharmacological studies on exosomes derived from Sarcodon imbricatus have not yet been disclosed. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing exosomes of *Pleurotus eryngii*.

[0008] A second objective of this invention is to provide the application of *Phyllostachys nigra* exosomes in the treatment of interstitial cystitis.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] This invention first provides the application of *Pleurotus eryngii* exosomes in the preparation of drugs for treating and / or alleviating interstitial cystitis. This invention uses *Pleurotus eryngii* exosomes to construct an animal model of interstitial cystitis by bladder instillation of protamine sulfate combined with lipopolysaccharide, and finds that it can significantly improve urination disorders in rats and alleviate the symptoms of interstitial cystitis.

[0011] Subsequent mechanistic studies revealed that *Pleurotus eryngii* exosomes significantly reduced the expression of IL-1β, IL-6, MCP-1, CXCL-1, ICAM-1, and BAX in bladder tissue; indicating that *Pleurotus eryngii* alleviates interstitial cystitis in the following ways:

[0012] (1) Inhibits pro-inflammatory responses;

[0013] (2) Reduce mast cell activation and degranulation;

[0014] (3) Reduce apoptosis of urothelial cells;

[0015] (4) Inhibit the adhesion reaction between inflammatory cells and epithelial cells, thereby relieving inflammation.

[0016] Therefore, the present invention also provides the use of *Phyllostachys nigra* exosomes in the preparation of medicaments for relieving and / or inhibiting the inflammatory response of interstitial cystitis.

[0017] Preferably, the exosomes of *Pleurotus eryngii* inhibit the expression of pro-inflammatory factors IL-1β and IL-6 in the inflammatory response.

[0018] Therefore, the present invention also provides the use of *Pteris vittata* exosomes in the preparation of drugs that inhibit urothelial cell apoptosis.

[0019] As a specific implementation method, the above-mentioned exosomes of *Pterocarya stenoptera* were prepared by differential centrifugation.

[0020] As a specific implementation scheme, the preparation method of the exosomes of *Pleurotus eryngii* according to the present invention is as follows: clean *Pleurotus eryngii*, cut off the root, cut the remaining part into pieces, add 2-3 times the volume of 1×PBS buffer as the extraction solvent, break the cell wall, and obtain the filtrate after filtration;

[0021] The filtrate was centrifuged at 1000×g for 20 min; 2000×g for 20 min; 4000×g for 40 min; and 10000×g for 60 min, respectively. After each centrifugation, the precipitate was removed, the supernatant was collected, and the last filtrate was filtered. Subsequently, the filtrate was centrifuged at 150000×g for 100 min, and the supernatant was discarded. The obtained precipitate was the exosomes derived from *Pleurotus eryngii*.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention extracts *Pleurotus eryngii* exosomes from fresh *Pleurotus eryngii*. The prepared *Pleurotus eryngii* exosomes were perfused into the bladders of rats with interstitial cystitis, and it was found that they significantly improved urination disorders and alleviated the symptoms of interstitial cystitis in the model rats. Furthermore, this invention also found, mechanistically, that: (1) *Pleurotus eryngii* exosomes significantly reduced the expression of IL-1β, IL-6, MCP-1, CXCL-1, ICAM-1, and BAX in bladder tissue; (2) *Pleurotus eryngii* exosomes significantly reduced the number of activated mast cells and inhibited mast cell degranulation; (3) *Pleurotus eryngii* exosomes significantly improved epithelial cell apoptosis.

[0024] The above research indicates that *Pleurotus eryngii* exosomes can be used to treat interstitial cystitis. This study provides a new approach to the treatment of interstitial cystitis. Attached Figure Description

[0025] Figure 1 Transmission electron microscopy image of exosomes derived from *Pleurotus eryngii*; scale bar is 400 nm.

[0026] Figure 2 NTA diagram of exosomes derived from *Pleurotus eryngii*;

[0027] Figure 3 For behavioral assessment of rats;

[0028] Figure 4 Detection of cytokine expression levels in rat bladder tissue;

[0029] Figure 5 Toluidine blue staining of rat bladder tissue;

[0030] Figure 6 TUNEL staining of rat bladder tissue. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0032] Example 1: Preparation of exosomes from *Pleurotus eryngii*

[0033] The fresh raw material of Black Tiger Paw Fungus came from Chuxiong, Yunnan. The preparation of Black Tiger Paw Fungus exosomes was as follows: after gently washing with deionized water, the roots were cut off, the remaining part was cut into pieces, and 1×PBS buffer with 3 times the volume of the fruiting body was added as the extraction solvent. The mixture was then broken up by a cell wall breaker, and then filtered through qualitative filter paper with the assistance of a vacuum pump to obtain the filtrate.

[0034] The obtained filtrate was further centrifuged at differential speeds of 1000×g for 20 min; 2000×g for 20 min; 4000×g for 40 min; and 10000×g for 60 min. After each centrifugation, the precipitate was removed, the supernatant was collected, and the last filtrate was filtered using 0.2 μm pore size filter paper with vacuum pump assistance. The filtrate was then centrifuged at 150000×g for 100 min, and the supernatant was discarded. The obtained precipitate was the exosomes derived from *Pleurotus eryngii*.

[0035] Figure 1 The image shows a transmission electron microscope image of exosomes derived from *Cymbidium goeringii*, revealing that the exosomes have a cup-shaped structure similar to exosomes. Figure 2 The hydrated particle size of the exosomes derived from *Pleurotus eryngii* was approximately 125.31 nm ± 11.42 nm. The protein concentration determined by BCA method was 5.37 ± 0.41 μg / μL.

[0036] Example 2 Animal Experiment

[0037] An animal model of interstitial cystitis was constructed using protamine sulfate combined with lipopolysaccharide, as detailed below:

[0038] Forty SPF-grade female SD rats were anesthetized by intraperitoneal injection of 0.3-0.4 ml / 100g of 3% sodium pentobarbital solution. After anesthesia, the rats were fixed, and the urethral orifice was disinfected twice with povidone-iodine. A sterile PE-50 catheter was slowly inserted into the bladder. After emptying the bladder, bladder irrigation was performed. Different treatments were administered according to the following groups.

[0039] A. In the control group, 1 ml of 0.9% sodium chloride solution was instilled into the urethra and bladder, retained for 45 min, and the bladder was emptied. After that, the bladder was flushed 3 times with phosphate buffer solution (PBS). No other special treatment was given. The instillation was repeated every 2 days for 2 consecutive weeks.

[0040] B. In the model group, 1 ml of 10 mg / ml protamine sulfate was instilled into the urethra and bladder, retained for 45 min, and after the drug was expelled, the bladder was rinsed 3 times with PBS. Then, 1 ml of 750 μg / ml lipopolysaccharide (LPS) was instilled, retained for 30 min, and after the drug was expelled, the bladder was rinsed 3 times with PBS. The catheter was then removed. Instillation was performed once every 2 days for 2 consecutive weeks.

[0041] C. The exosome group was formed by instilling *Pleurotus eryngii* exosomes into the bladder in addition to the model group. The procedure was to instill 100 μL of *Pleurotus eryngii* exosomes into the bladder 1 hour after the lipopolysaccharide instillation in the model group, retain it for 30 minutes, and after the drug was expelled, rinse it 3 times with PBS and remove the catheter. After the first instillation of *Pleurotus eryngii* exosomes into the bladder, the instillation of *Pleurotus eryngii* exosomes into the bladder was performed every 2 days for 2 consecutive weeks.

[0042] I. Metabolic cage monitoring

[0043] Animals were housed individually in metabolic cages 3-7 days in advance, and provided with the same diet and water as in the formal experiment during the acclimatization period.

[0044] The metabolic cage has a mesh or funnel-shaped bottom to separate urine from feces, with the urine flowing into a collection tube below (such as an EP tube or a graduated container).

[0045] The three groups of animals were kept at a constant temperature (22±1℃), constant humidity (50±10%), and a 12-hour light-dark cycle in metabolic cages (to avoid strong light interfering with the diurnal rhythm).

[0046] Urination frequency: The number of urine drops is recorded manually or by electronic sensors (such as a drop counter).

[0047] Relative volume of urine: Weigh the collection tube using a microbalance (1 mg ≈ 1 μL), or read the volume of the graduated tube directly.

[0048] The results are as follows Figure 3 As shown, compared with the blank group, the average number of urinations in the model group rats increased significantly, but the area of ​​urination per urination decreased significantly, proving that the rat IC model constructed in this invention was successful, and the rats in the model group showed bladder dysfunction; while compared with the model group, the number of urinations in the exosome group rats decreased significantly, and the area of ​​urination per urination increased. It can be seen that *Heterodon spp.* exosomes can significantly improve urination disorders in rats and alleviate the symptoms of interstitial cystitis.

[0049] Two weeks later, the rats were sacrificed, and the bladder tissue was divided into three equal parts for toluidine blue staining, TUNEL staining, and cytokine detection.

[0050] II. Detection of Cytokine Expression Levels in Bladder Tissue

[0051] ICAM-1 assay in bladder tissue: The reagents from the ICAM-1 ELISA kit were placed at room temperature. Two copies of the sample and standard were made, and after centrifugation, the resulting tissue supernatant and standard were thoroughly mixed at room temperature. The concentrated washing buffer from the ELISA kit was diluted with distilled water approximately 10-fold. The number of wells required for the assay was determined, and each well was labeled (one well was reserved as a control). The supernatant, control serum, and 100 µl of standard were added sequentially to each labeled well. After covering, the plate was incubated at 37°C for 30 min. The solution in the microplate was then discarded, and the plate was rinsed 5 times with approximately 300 µl of rinsing buffer per well. After thorough rinsing, the microplate was inverted on dry absorbent paper to remove any remaining liquid. Next, add enzyme-labeled reagent to the microplate, mix thoroughly, and react at 37°C for 30 min. Discard the liquid in the microplate, rinse five times with rinsing buffer, and then add approximately 100 µl of chromogenic substrates A and B to each well. Incubate at 37°C in the dark for 10 min, and terminate the reaction by adding 0.05 ml of 2 mol / L H₂SO₄ stop solution. Within 15 min, measure the absorbance (OD value) at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Plot a standard curve on logarithmic graph paper with the concentration of ICAM-1 standard as the x-axis and the sample absorbance divided by the absorbance at the "0" point of the standard as the y-axis. Calculate the ICAM-1 value in the sample from the standard curve.

[0052] The detection procedures for IL-1β, IL-6, MCP-1, CXCL-1, and BAX in bladder tissue are similar to those for ICAM-1.

[0053] Cytokine IL-1β exacerbates bladder pain, urinary frequency, and mucosal damage by promoting inflammatory responses, neurosensitization, and tissue fibrosis. Figure 4 As can be seen, the expression level of IL-1β was significantly increased in the model group compared with the control group, while the expression level of IL-1β in the exosome group was significantly decreased. This indicates that *Heterodon spp.* exosomes can significantly reduce the expression of the pro-inflammatory factor IL-1β.

[0054] The cytokine IL-6 plays a dual role (pro-inflammatory and pro-repair) in the pathogenesis of interstitial cystitis (IC), but its chronic overexpression mainly leads to persistent inflammation, neurosensitization and tissue fibrosis. IL-6 promotes the release of other pro-inflammatory factors (such as IL-1β and TNF-α) by activating the JAK / STAT3 signaling pathway, forming a positive feedback loop of inflammation. Figure 4 As can be seen, the expression level of IL-6 was significantly increased in the model group compared with the blank group, while the expression level of IL-6 decreased in the exosome group.

[0055] The cytokine MCP-1 is involved in activating various inflammatory cell functions, suggesting that MCP-1 may also play a key role in mast cell activation and degranulation during the development of intraepithelial neoplasia (IC). Furthermore, the release of various bioactive mediators and factors from mast cell activation and degranulation can promote MCP-1 production, further propelling the inflammatory response. Figure 4 As can be seen, compared with the control group, the expression level of MCP-1 in the model group was significantly increased, while the expression level of MCP-1 in the exosome group was significantly decreased, basically returning to a level similar to that of the control group. This indicates that *Heterodon spp.* exosomes can significantly reduce the expression of MCP-1, thereby alleviating the inflammatory response of mesenchymal cystitis.

[0056] Cytokine CXCL-1 can stimulate bladder epithelial cells and immune cells (such as macrophages) to secrete inflammatory factors such as IL-6 and IL-1β, forming a positive feedback loop and aggravating chronic inflammation. Figure 4 As can be seen, compared with the control group, the expression level of CXCL-1 in the model group was significantly increased, while the expression level of CXCL-1 in the exosome group was significantly decreased. This indicates that *Heterodon spp.* exosomes can significantly reduce the expression of CXCL-1, alleviate the positive feedback loop caused by CXCL-1, and relieve chronic inflammation.

[0057] Activation of the cytokine BAX induces apoptosis via the mitochondrial pathway, leading to the death of bladder urothelial cells, damaging the mucosal barrier (such as the loss of the glycosaminoglycan layer), and allowing urinary components (such as potassium ions) to permeate into the bladder interstitium, triggering inflammation and pain. Figure 4As can be seen, BAX expression was significantly increased in the model group compared to the control group, while BAX expression in the exosome group decreased to a level similar to that in the control group. This indicates that *Heterodon spp.* exosomes can significantly reduce BAX expression and alleviate chronic inflammation and pain.

[0058] Cytokine ICAM-1 can promote adhesion reactions between inflammatory cells at the site of inflammation and between inflammatory cells and epithelial cells, thereby aggravating the local inflammatory response. Figure 4 As can be seen, the expression level of ICAM-1 was significantly increased in the model group compared with the control group, while the expression level of ICAM-1 was significantly decreased in the exosome group. This indicates that *Heterodon spp.* exosomes can significantly reduce the expression of ICAM-1.

[0059] III. Toluidine Blue Staining

[0060] After rats were sacrificed, bladder tissue from both the experimental and control groups was marked and immediately fixed in 4% paraformaldehyde solution for paraffin section preparation. The tissues underwent routine fixation, dehydration, clearing, embedding, and sectioning, with a section thickness of 4 µm.

[0061] Toluidine blue staining procedure: ① Dewaxing: Place paraffin sections at room temperature for 60 minutes, then immerse them in xylene solution twice for routine dewaxing, 5-10 minutes each time, until the paraffin sections become transparent. ② Gradient alcohol hydration: Immerse the sections sequentially in 100% absolute ethanol I, 100% absolute ethanol II, 95% ethanol, 85% ethanol, and 75% ethanol solutions for 5 minutes each, then transfer them to water to wash away the alcohol for about 2 minutes, and finally transfer them to distilled water for about 2 minutes. ③ Toluidine blue staining: Immerse the sections in toluidine blue solution for 30 minutes. Rinse with tap water for 2 minutes. ④ Dehydration: After the sections have dried, immerse them sequentially in 95% ethanol for 1 minute and 100% absolute ethanol for 2 minutes twice. ⑤ Clearing and mounting: Clear the sections with xylene solution for 10 minutes. Then, apply a drop of neutral resin to a glass slide for mounting. Gently cover with a coverslip to avoid air bubbles. Allow the sections to air dry before microscopic examination.

[0062] The pathogenesis of interstitial cystitis includes mast cell activation; therefore, the number of activated mast cells can be used as one of the diagnostic criteria for interstitial cystitis. Mast cells stained with toluidine blue show purplish-black cytoplasm, dark blue nuclei, indistinct borders, and punctate granules around the cells; these are degranulated mast cells. Figure 5 As shown, a small number of mast cells were observed in the blank group, with no obvious degranulated mast cells. In the model group, mast cell infiltration in the bladder tissue was significantly increased, with a large number of degranulated mast cells. Compared to the model group, mast cell infiltration was significantly reduced in the exosome group, with only a small number of degranulated mast cells visible.

[0063] Depend on Figure 5The results clearly show that *Heterophyte niger* exosomes can significantly reduce the number of activated mast cells, thereby achieving the purpose of relieving / treating interstitial cystitis.

[0064] IV. TUNEL staining

[0065] The paraffin sections obtained above were processed using the one-step TUNEL apoptosis detection kit (green fluorescence), and then the apoptosis status of the three groups of paraffin sections was detected using the same kit.

[0066] The processing of paraffin sections includes the following steps: a. Dewaxing: Place the paraffin sections in xylene at room temperature for 5-10 minutes, repeating twice. b. Hydration: Immerse in anhydrous ethanol for 5 minutes, repeating twice. Then immerse in a series of ethanol solutions (90%, 80%, 70%) for 2 minutes each. c. Washing: Wash twice with PBS for 5 minutes each time. Gently discard the PBS and blot away excess liquid with filter paper. d. Permeabilization: Add an appropriate amount of proteinase K solution and incubate at room temperature for 15-30 minutes. e. Washing: Wash twice with PBS for 5 minutes each time to remove proteinase K, and blot away excess liquid with filter paper.

[0067] Studies have shown that abnormal regulation of apoptosis may play an important role in the pathogenesis of interstitial cystitis. Increased apoptosis of urothelial cells leads to impaired barrier function, allowing urinary components to infiltrate the bladder interstitium, activating mast cells and nerve fibers, resulting in inflammation and pain. Elevated levels of the apoptosis-associated protein BAX lead to decreased expression of the anti-apoptotic protein Bcl-2, thereby exacerbating epithelial cell apoptosis. Trypsin-like proteins released by mast cells can activate protease-activating receptors (PARs), further inducing urothelial cell apoptosis. To verify the above, this example investigated epithelial cell apoptosis.

[0068] TUNEL staining results are as follows Figure 6 As shown, Figure 6 The results showed that, compared with the control group, the model group had more significant epithelial cell apoptosis, while the exosome group had significantly fewer epithelial cell apoptosis compared with the model group. This indicates that *Pleurotus eryngii* exosomes can significantly improve epithelial cell apoptosis, thereby alleviating barrier damage and persistent inflammation, and thus treating interstitial cystitis.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. The use of *Pleurotus erythrorhizon* exosomes in the preparation of drugs for the treatment and / or relief of interstitial cystitis.

2. Application of *Phyllostachys nigra* exosomes in the preparation of drugs to relieve and / or inhibit the inflammatory response of interstitial cystitis.

3. The application according to claim 2, characterized in that, The exosomes of *Pleurotus eryngii* inhibit the expression of pro-inflammatory factors IL-1β and IL-6 in the inflammatory response.

4. Application of *Pteris vittata* exosomes in the preparation of drugs that inhibit apoptosis of urothelial cells.

5. The application according to any one of claims 1 to 4, characterized in that, The exosomes of *Pleurotus eryngii* were prepared by differential centrifugation.

6. The application according to claim 5, characterized in that, The preparation method of exosomes of Black Tiger Paw Fungus is as follows: clean the Black Tiger Paw Fungus, cut off the root, cut the remaining part into pieces, add 2-3 times the volume of 1×PBS buffer as the extraction solvent, break the cell wall, and obtain the filtrate after filtration. The filtrate was centrifuged at 1000×g for 20 min; 2000×g for 20 min; 4000×g for 40 min; and 10000×g for 60 min, respectively. After each centrifugation, the precipitate was removed, the supernatant was collected, and the last filtrate was filtered. Subsequently, the filtrate was centrifuged at 150000×g for 100 min, and the supernatant was discarded. The obtained precipitate was the exosomes derived from *Pleurotus eryngii*.

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