Therapeutic Effects of Acorus calamus L. Extract on Radiation-induced Skin Injury in a Rat Model

Radiation-induced skin injury is a frequent and difficult complication of both nuclear accidents and tumor radiotherapy¹. In radiotherapy, about 95% of patients develop some degree of skin damage, including erythema, desquamation, recurrent necrotic ulcers, persistent pain, and an elevated risk of malignant progression². These lesions are often refractory to treatment, substantially reducing patients' quality of life and sometimes necessitating interruption of radiotherapy, thus posing a major clinical challenge in radiation oncology³. The underlying mechanisms of radiation-induced skin damage are complex, primarily involving the production of free radicals within the skin tissues, which disrupts cellular processes and exacerbates the healing process^4,5. This results in the prolonged expression of apoptosis-related genes, dysregulated inflammatory responses, and altered signaling pathways, all of which contribute to the persistent nature of radiation-induced wounds⁶.

There is currently no universally accepted gold-standard therapy for radiation-induced skin injury. Clinical management emphasizes anti-inflammatory or antioxidant approaches⁷, notably topical corticosteroids, which reduce skin reactions via anti-inflammatory, immunosuppressive, and vasoconstrictive effects, but can cause skin thinning with prolonged use⁸. Amifostine, an FDA-approved radioprotective agent, is limited in clinical use because of significant adverse effects⁹. Growth factors accelerate wound healing by promoting cell proliferation, angiogenesis, and granulation tissue formation, yet their actions are narrowly focused, offer limited control over deep tissue injury and inflammation, and carry a risk of excessive hyperplasia⁷. Consequently, Traditional Chinese Medicine (TCM) has drawn attention for its holistic philosophy, multiple active constituents with diverse therapeutic effects, and a favorable safety profile. Previous studies have shown that natural products, such as licorice extract, aloe polysaccharides, and curcumin, alleviate radiation-induced inflammation and tissue damage by scavenging free radicals, inhibiting pro-inflammatory mediators (TNF-α, IL-6), and promoting fibroblast proliferation^10,11,12. However, most of these investigations examine single active compounds or single targets, providing inadequate insight into mechanisms of multi-component synergistic action. Acorus calamus L., a plant well-known for its anti-inflammatory, antioxidant, antibacterial, and wound-healing properties, has shown potential in various skin disorders.

Acorus calamus L., a perennial herb from the Araneae family, contains several bioactive compounds, including terpenes, phenylpropanoids, flavonoids, steroids, and alkaloids. Its multi-component composition allows simultaneous action on multiple targets, producing synergistic pharmacological effects¹³. Studies show that it inhibits inflammatory signaling pathways such as NF-κB and MAPK and dose-dependently downregulates mRNA expression of proinflammatory factors, such as TNF-α and IL-6, while increasing activities of antioxidant enzymes such as SOD and GSH-Px, thereby exerting anti-inflammatory, antioxidant, and antibacterial effects^14,15. Despite its documented benefits in treating skin conditions caused by infections and allergies, the role of Acorus calamus L. in radiation-induced skin injuries remains underexplored. Recent studies have demonstrated its protective effects against radiation-induced damage, promoting wound healing and reducing inflammation¹³. However, the specific therapeutic mechanisms of Acorus calamus L. in radiation-induced skin injuries have not been fully elucidated.

The overall goal of this study is to evaluate the therapeutic effects of Acorus calamus L. extract in animal models of radiation-induced skin injury. We assess wound healing, inflammatory responses, and cellular behavior to elucidate the extract's mechanisms of action from multiple perspectives. The findings aim to establish a theoretical basis for using Acorus calamus L. in clinical management of skin lesions arising from cancer radiotherapy. In addition, the study will present the extract's functional characteristics, suitable intervention scenarios, and core efficacy data to help readers judge whether this natural-product-based approach complements their research or clinical needs. Ultimately, this work seeks effective therapeutic options to improve the quality of life for patients receiving tumor radiotherapy.

The animal experiments described in this study were approved by the Animal Medical Research Ethics Committee of the Northern Theater Command General Hospital (Ethics Approval Number: 2018-03). SPF-grade male SD rats（6-8 weeks, weight 190-220 g）were used for the study. The details of the reagents and equipment used are listed in the Table of Materials.

1. Housing the experimental animals

1. Select 108 SPF male SD rats, aged between 6 to 8 weeks and weighing between 190 to 220 g.
2. Maintain the animal room temperature at 22 ± 2 °C, relative humidity at 50 ± 5%, and a 12 h light-dark cycle. Allow the rats to have unrestricted access to food and water. After a one-week acclimatization period, proceed to the follow-up experiments.

2. Grouping of animals

1. Randomly assign the rats into five distinct groups: Normal group (Normal, n = 18), Model group (Model, n = 18), positive drug groups (Positive, n = 18, Betamethasone Cream)¹⁶, Low-dose experimental group (Experimental-L, n = 18), Medium-dose experimental group (Experimental-M, n = 18), and High-dose experimental group (Experimental-H, n = 18). Except for the normal group, all groups were exposed to radiation.

3. Establish an animal model of radioactive skin injury

1. Administer anesthesia with precision and minimal discomfort through intraperitoneal (i.p.) injection of a 1% sodium pentobarbital solution (40 mg/kg). Confirm the depth of anesthesia by observing the absence of the corneal reflex and the lack of response to toe pinching. Apply ointment to the rats' eyes to prevent dryness.
2. Remove the rats' back hair with an electric shaver. Then disinfect the back area with iodine and 75% alcohol.
3. Put each rat in a prone position. Secure the ³²P-β irradiator (surface dose rate: 0.9 Gy/min) over the bilateral spinal region (2 cm × 2 cm area) centered 1 cm lateral to the midline, ensuring the source contacts the epidermis with no gap. Secure the applicator with sterile medical tape to prevent displacement during irradiation. Irradiate the target skin area for 50 min to achieve a total absorbed dose of 45 Gy¹⁷.
4. Once the rat regains consciousness after irradiation, return it to itsindividual cage, ensuring access to water and food ad libitum, maintaining temperature at 22 ± 2 °C, relative humidity at 50 ± 5%, and a 12 h light-dark cycle.
   NOTE: The experimental process must strictly adhere to the established principles of biological and radioactive occupational protection. Additionally, all operational procedures must follow guidelines for the proper and compliant disposal of medical and radioactive waste to ensure the safety and health of all personnel involved.

4. Preparation of Acorus calamus L. extract and drug administration

1. Weigh the rhizomes of Acorus calamus L., ensure thorough cleansing, allow to air-dry, and grind into a coarse powder (40 mesh). Incorporate 10-20 volumes of 50% ethanol and homogenize at room temperature for 24 h
2. After permitting the extract to settle for 30 min, decant the supernatant and conduct preliminary filtration through 4-8 layers of gauze to eliminate coarse impurities. Subsequently, filter using filter paper, and evaporate the filtrate under reduced pressure in a rotary evaporator at 50-60 °C to recover the solvent, resulting in a solvent-free solid residue (AE, moisture content < 5%).
3. AE ointment preparation¹⁸
   NOTE: Formulate 10% (Experimental-L, w/w), 20% (Experimental-M, w/w), and 40% (Experimental-H, w/w) Acorus calamus L. extract ointments in accordance with the Chinese Pharmacopoeia 2020 Edition (General Chapter 0109)¹⁹.
   1. Accurately weigh AE (e.g., 20 g for 20% ointment) and corresponding Pharmaceutical-grade petrolatum (e.g., 80 g for 20% ointment).
   2. Add 10% of the total petrolatum to AE, triturate vigorously for 5 min to form a homogeneous paste (geometric dilution method, ensuring AE disperses evenly in the lipophilic base).
   3. Melt the remaining petrolatum at 60 °C, cool to 55 °C, and slowly add to the AE-petrolatum paste.
   4. Stir magnetically at 250 rpm for 30 min at 55 °C, then stir continuously while cooling to room temperature to obtain a smooth, particle-free ointment.
   5. Blank control preparation: Melt petrolatum at 60 °C, stir at 250 rpm for 30 min, and cool to room temperature (identical to the AE ointment base).
   6. Store all ointments and blank petrolatum at 4 °C in sealed sterile containers.
4. All groups were topically administered agents twice daily at 09:00 and 17:00 for 45 consecutive days following irradiation (the normal group, non-irradiated, received the same administration schedule synchronously). The applied dose was 0.2 g/cm², evenly distributed over a 2 cm × 2 cm area of dorsal skin. The normal group received vehicle (petrolatum), and the model group received vehicle (petrolatum). The positive control group received betamethasone cream. The experimental groups L, M, and H received creams containing AE at 10%, 20%, and 40% (w/w), respectively. After each application, the treated area was gently massaged for 10 s to enhance absorption, and rats were restrained for 5 min to prevent licking.

5. Collection of rat skin tissue and blood

1. Anesthetize the rats using a 1% sodium pentobarbital solution(40 mg/kg, i.p.). Disinfect surgical instruments and excise skin along the wound site.
2. Fix a portion of the tissue in 4% paraformaldehyde(10 mL), another portion in glutaraldehyde/osmium tetroxide (5 mL, 2.5%), and preserve the remainder in liquid nitrogen for further analysis (refer to Table of Materials).
3. Collect blood samples from the abdominal aorta of the rat. Centrifuge at 3,000 × g at 4 °C for 15 min; aspirate the supernatant into EP tubes; aliquot and store at -80 °C.

6. Healing evaluation

1. Observe the wound on the 7th, 15th, 30th, and 45th days after irradiation and take images of the wound surface with a high-definition camera. Import the images into ImageJ and draw the wound area along the edge of the wound. Compute the area in the image. Ensure that each operation is consistent to avoid measurement errors. Calculate the wound healing time and healing rate.
   NOTE: Wound healing rate (%) = (Initial irradiated area - Unhealed wound area) / Initial irradiated area. Wound healing criteria: Complete healing is defined as either scabbed area < 5% of wound area or healed area > 95% of wound area. Initial irradiated area: the wound area measured at the time when irradiated animals exhibited a stable wound (a baseline).

7. Electron microscopy examination

1. Rapid tissue sampling and fixation
   1. Employ a sharp blade to excise a skin tissue block measuring 1 mm³, encompassing the epidermis-dermis layer.
   2. Immediately immerse the specimen in pre-chilled 2.5% glutaraldehyde phosphate buffer (0.1 M, pH 7.4) at 4 °C for 24 h for fixation.
2. Buffer washing
   1. Conduct three washes with 0.1 M phosphate buffer (pH 7.4), allowing 15 min for each wash.
3. Post fixation
   1. Transition the sample to a 1% osmium tetroxide solution at 4 °C for fixation lasting 1.5 h.
4. Gradient dehydration
   1. Progressively pass through acetone concentrations of 50%, 70%, 80%, 90%, and 100%, allocating 15 min to each step.
   2. Repeat the treatment with 100% acetone three times, with each immersion lasting 10 min.
5. Epoxy Resin embedding
   1. Soak the specimen in a 2:1 mixture of acetone and epoxy resin for 2 h, followed by overnight immersion in pure epoxy resin.
   2. Polymerize the sample at 60 °C for 48 h to form a solid block.
6. Sectioning and staining
   1. Produce 70 nm ultrathin sections using an ultramicrotome.
   2. Stain with uranium acetate for 15 min, then with lead citrate for 5 min at 60 °C.

8. Hematoxylin-Eosin (HE) staining

1. Perform paraffin embedding
   1. Excise skin specimens, dehydrate using an alcohol gradient (e.g., 70%, 80%, and 95% ethanol, each for 15 min, followed by two changes of 100% ethanol, each for 15 min), and subsequently transfer to xylene until the tissue reaches a translucent state.
   2. Submerge in paraffin to ensure embedding, then allow solidification.
2. Execute dewaxing and rehydration
   1. Immerse paraffin sections in xylene I/II for 10 min each.
   2. Rinse xylene with 100% ethanol, then immerse in 95% ethanol for 3 min, 80% ethanol for 3 min, 70% ethanol for 3 min, and finally rinse with distilled water.
3. Conduct hematoxylin nuclear staining
   1. Soak in Harris' hematoxylin solution for 7 min at room temperature.
   2. Rinse under running water for 10 min until the blue coloration appears.
4. Implement differentiation and counterstaining
   1. Differentiate using 1% hydrochloric acid in ethanol for 3 s (observe nuclear clarity under the microscope).
   2. Counterstain with a 0.5% ammonia solution for 30 s.
5. Perform eosin counterstaining
   1. Stain with a 0.5% eosin solution (containing 1% glacial acetic acid) for 90 s.
   2. Quickly rinse with distilled water for 5 s to remove excess stain.
6. Execute dehydration and clearing
   1. Sequentially pass through 70%, 80%, 95%, 100% ethanol I/II (1 min each).
   2. Clear with xylene I/II for 2 min each.
7. Apply sealing and observation
   1. Apply neutral resin dropwise, then cover with a coverslip.
   2. Dry in a 60 °C oven for 30 min prior to microscopic examination.

9. Masson's trichrome staining

1. Perform routine dewaxing similarly to pre-HE staining.
2. Nuclear staining
   1. Soak in hematoxylin solution for 10 min.
   2. Rinse under running water for 5 min until the blue tint is reinstated.
3. Collagen fiber staining
   1. Immerse in a mixture of Alizarin Red and Acid Fuchsin for 5 min.
4. Phosphomolybdic acid differentiation
   1. Transfer to a 1% phosphomolybdic acid aqueous solution for 1 min.
5. Counterstaining of myofibers
   1. Directly transfer to a 2.5% aniline blue solution for 5 min.
   2. Quickly rinse with a 0.5% glacial acetic acid aqueous solution for 5 s.
6. Dehydration and mounting
   1. Execute gradient ethanol dehydration: 95% ethanol I, 100% ethanol II (30 s each).
   2. Clear with xylene.
   3. Mount using neutral resin, dry at 60 °C, and subsequently examine under the microscope.

10. ELISA detection of IL-1β, IL-6, and TNF-α levels

NOTE: IL-1β, IL-6, and TNF-α are important pro-inflammatory cytokines commonly used to assess both the presence and intensity of inflammation. Monitoring their levels is crucial for gaining insight into inflammatory processes and for determining the effectiveness of anti-inflammatory treatments.

1. Prepare serum samples
   1. Follow the protocol detailed in Step 5.3 to collect serum samples.
2. Dilute standards and samples
   1. Create a gradient dilution of standards utilizing the sample diluent; dilute serum samples at a ratio of 1:4.
3. Add samples and incubate
   1. Introduce 100 µL of the standard/sample into each well; cover the wells with a membrane; incubate at 37 °C for 90 min; then discard the liquid and blot the wells dry.
4. Detect antibody-enzyme reaction
   1. Dispense 100 µL of enzyme-labeled reagent specific for IL-1β, IL-6, and TNF-α into each well; incubate at 37 °C for 60 min; remove the liquid and blot dry; wash the plate four times with 200 µL of wash buffer.
   2. Add 100 µL of streptavidin-HRP working solution (1:100 dilution) to each well; incubate at 37 °C in the dark for 30 min; wash the plate four times with 200 µL of wash buffer.
5. Develop color and terminate
   1. Introduce 100 µL of TMB substrate solution to each well; incubate at room temperature in the dark for 10 min; subsequently add 50 µL of stop solution to each well.
6. Read data
   1. Measure the absorbance at 450 nm (with a calibration wavelength of 630 nm) using an enzyme-linked immunosorbent assay reader within 30 min; calculate the concentration (pg/mL) utilizing the standard curve.

11. TUNEL assay

1. Section pretreatment
   1. Dewax and dehydrate paraffin sections until they reach distilled water.
   2. Introduce a working solution of proteinase K at a concentration of 20 µg/mL, and incubate at 37 °C for 20 min.
   3. Wash the sections three times with PBS, with each wash lasting 2 min.
2. Labeling reaction
   1. Apply the TUNEL reaction mixture containing TdT enzyme and fluorescein-labeled dUTP.
   2. Incubate in a humidified chamber at 37 °C in the dark for 60 min.
3. Signal conversion
   1. Wash three times with PBS, allowing 5 min for each wash.
   2. Introduce an HRP-labeled anti-fluorescein antibody at a dilution of 1:500.
   3. Incubate at 37 °C for 30 min.
4. DAB chromogenic development
   1. After PBS washing, apply DAB chromogenic solution and monitor development under a microscope for 1 to 3 min.
   2. Halt the reaction using distilled water.
5. Counterstaining and mounting
   1. Counterstain nuclei with hematoxylin for 1 min.
   2. Dehydrate using graded ethanol, clear with xylene, and mount with neutral resin.
6. Result interpretation
   1. Identify apoptotic nuclei as brownish-yellow, while normal nuclei appear blue.
   2. Randomly select five high-power fields (400×) per slide to calculate the apoptosis rate.

12. Immunohistochemical staining

1. Dewaxing and antigen retrieval
   1. Soak paraffin sections in Xylene I for 10 min, then transfer to Xylene II for another 10 min. Subsequently, dehydrate using a gradient ethanol series, beginning with 5 min in 100% Ethanol I, followed by 5 min in 100% Ethanol II, 3 min each in 95%, 80%, and 70% ethanol, and finally rinse with distilled water for 2 min.
   2. Conduct high-pressure heat repair with 0.01 M sodium citrate buffer at pH 6.0, maintaining a temperature of 121 °C for 3 min.
2. Block endogenous interference
   1. Introduce a 3% H₂O₂ solution at room temperature for 10 min to inactivate endogenous enzymes.
   2. Apply a 5% BSA blocking solution to the tissue and incubate in a humidified chamber at 37 °C for 45 min; aspirate any excess blocking solution without rinsing.
3. Antibody incubation
   1. Dispense a 5% BSA blocking solution and incubate at 37 °C for 30 min.
   2. Add the primary antibody working solution at the optimized concentration (e.g., P53, Bax, Bcl-2, VEGF, and bFGF) and incubate overnight in a humidified chamber at 4 °C; wash three times with PBS-T buffer containing 0.1% Tween-20 for 5 min each.
   3. Introduce an HRP-labeled secondary antibody diluted 1:200; incubate in a humidified chamber at 37 °C for 60 min; wash three times with PBS-T buffer for 5 min each. Then, add the ABC conjugate solution and incubate at 37 °C for 30 min, followed by three washes with PBS buffer for 5 min each.
4. DAB color development
   1. Add DAB developing solution containing 0.02% H₂O₂ and monitor the development under a microscope at room temperature for 1-4 min.
   2. Immediately terminate the reaction with distilled water.
5. Counterstaining and mounting
   1. Immerse the sample in Harris's hematoxylin solution for 1 min and rinse under running water for 10 min to achieve blue recovery.
   2. Sequentially immerse in the following solutions: 70% ethanol for 30 s, 80% ethanol for 30 s, 95% ethanol for 30 s, 100% ethanol I for 1 min, and 100% ethanol II for 1 min.
   3. For complete clearing and fixation, immerse in xylene I for 2 min, then transfer to xylene II for 2 min. Add neutral resin mounting medium dropwise, cover with a coverslip, and dry in a 60 °C oven for 30 min.
6. Result quantification
   1. A brown color indicates a positive reaction; positive signals (brownish-yellow) should be contrasted against the negative background.
   2. Utilize Image-Pro Plus software to calculate the percentage of positive cells across at least 5 fields of view.

13. Western blotting detection

1. Tissue lysis preparation
   1. Introduce liquid nitrogen-frozen skin tissue into a pre-chilled stainless steel mortar at -80 °C; gradually add liquid nitrogen in three increments to mitigate vaporization; grind in a vertical manner until all particles are no longer visible; transfer the resulting powder into an EP tube containing lysis buffer.
   2. Incorporate protease inhibitor (1:100) and phosphatase inhibitor (1:50); vortex on ice for 30 s; conduct ultrasonic disruption; incubate at 4 °C in a shaking incubator for 2 h; centrifuge at 4 °C, 14,000 × g for 20 min; carefully aspirate the supernatant.
2. Protein quantification and processing
   1. Dispense 5 µL of the supernatant into a 96-well plate; add 200 µL of BCA working solution; incubate at 37 °C in the dark for 30 min; measure absorbance at 562 nm using a microplate reader; determine concentration according to the standard curve.
   2. Combine 5× Reduced Protein Loading Buffer with the protein solution at a 4:1 ratio; denature by placing in a boiling water bath for 15 min; store at -20 °C for future use.
3. SDS-PAGE electrophoresis
   1. Prepare a 12% acrylamide resolving gel; pour the gel until it reaches 1 cm below the comb teeth; cover with isopropanol to prevent air exposure; after solidification, remove isopropanol; pour a 4% concentrating gel; insert a 15-well comb; allow solidification at room temperature for 30 min; add 5 µL of pre-stained protein marker to loading well 1; add 20 µL of blank lysate to well 2; add 10 µL of sample containing 40 µg protein to each well from 3 to 14.
   2. Initiate electrophoresis at 80 V; switch to 120 V when the bromophenol blue migrates into the separating gel; maintain a temperature of 10-15 °C with ice-water circulation; run until the bromophenol blue reaches 0.5 cm from the gel's bottom.
4. Wet blotting procedure
   1. Prepare a transfer buffer containing 20% methanol; pre-cool to 4 °C. Activate the PVDF membrane by immersing it in methanol for 1 min; equilibrate the NC membrane in buffer. Assemble the transfer clamp in this order: "Cathode plate - Sponge pad - Filter paper - Gel - Membrane - Filter paper - Sponge pad - Anode plate"; thoroughly remove any bubbles with a roller; set parameters according to the target protein molecular weight: for proteins < 50 kDa, use a constant voltage transfer at 100 V for 60 min; for 50-100 kDa proteins, apply a constant current transfer at 250 mA for 90 min; for proteins > 100 kDa, implement a constant current transfer at 300 mA for 120 min; connect to a 4 °C circulating water bath.
5. Immunodetection procedure
   1. After the transfer, immerse the PVDF membrane in methanol for 1 min; wash three times with TBST for 5 min each; cover the membrane surface with 3% H₂O₂/TBST; incubate at room temperature in the dark for 15 min; aspirate the liquid; add a 5% BSA/TBST blocking solution; block for 1 h in a 37 °C shaking incubator; wash the membrane three times with TBST for 5 min each.
   2. Add the primary antibody working solution (such as P53, Bax, Bcl-2, VEGF, and bFGF); incubate overnight at 4 °C in a shaking incubator; wash the membrane four times with TBST for 10 min each; add HRP-labeled secondary antibody; incubate on a shaking incubator at room temperature for 90 min; wash the membrane six times with TBST + 0.5% SDS for 8 min each.
6. Chemiluminescence imaging
   1. Mix equal volumes of ECL A and B solutions; uniformly coat the membrane surface; allow the reaction to occur in the dark for 60 s; aspirate any excess liquid; set a single exposure time between 1-5 s for strong signal proteins; perform cumulative exposure in five segments (10/30/60/120/300 s) for weak signal proteins; capture images using a chemiluminescence imager.
7. Data analysis workflow
   1. Quantify proteins utilizing ImageJ software; establish a uniform background subtraction threshold; calculate the grayscale ratio of the target protein to the reference protein (β-actin or GAPDH)
   2. Import the data into GraphPad Prism for analysis.

14. Statistical analysis

1. Present data by mean ± standard error of mean. Employ independent samples t-tests for comparisons between two groups, and utilize one-way ANOVA for comparisons across multiple groups.
2. Analyze data using SPSS 26.0 and GraphPad Prism 8.0.2.
3. Consider P < 0.05 as indicative of statistically significant differences.

Therapeutic effect of Acorus calamus L. on radiation-induced skin injury
15 days post-irradiation, all groups of rats exhibited dry peeling, localized redness, swelling, ulceration, and scabbing. At 30 days post-irradiation, the model group showed the most severe skin damage, with scabbing and ulceration at the irradiated site reaching their peak. At 45 days, the model group showed minimal scab shedding, and the wound exhibited some contraction. In contrast, the Acorus calamus L. and positive drug-treated groups displayed significantly reduced skin damage, lighter ulceration, enhanced wound healing, and faster epithelial coverage (see Figure 1A). At 45 days post-irradiation, the medium-dose Acorus calamus L. group exhibited the highest wound healing rate at 88.97%, followed by the high-dose Acorus calamus L. group at 72.88% and the positive control group at 64.75% (see Figure 1B). Among the experimental groups, the medium-dose Acorus calamus L. group exhibited the fastest wound healing (see Table 1).

Ultrastructural changes in skin tissue observed by transmission electron microscopy
In the normal group, the epidermal cells were intact, and the structures of the organelles, such as mitochondria, were well preserved. In the model group, hair follicles were absent, fibroblasts exhibited severe edema, and collagen fibers were disorganized with fewer organelles and more free mitochondria. Mitochondria were moderately swollen, and the rough endoplasmic reticulum was significantly dilated and the damage was most severe 30 days after irradiation. However, the damage improved after treatment with both calamus and positive drugs. Compared with the positive drugs group, the medium and high-dose Acorus calamus L. exhibited substantial collagen fiber proliferation and a rich junction structure. The nuclei were oval-shaped, with uniform chromatin distribution, and the mitochondria and rough endoplasmic reticulum were more abundant, with moderate swelling. These findings suggest that Acorus calamus L. helps improve mitochondrial (see Figure 2).

Histopathological and Masson staining observations of skin tissue
Hematoxylin and eosin (H&E) staining and Masson staining revealed that in the normal group, skin cell arrangements were clear, and the structures of the skin appendages were intact. At 15 days post-irradiation, the model group exhibited partial epidermal necrosis, destruction of appendage structures, swelling of the dermis, collagen fiber rupture, dissolution, disorganization, and significant inflammatory cell infiltration. At 30 days, the model group showed extensive necrosis in both the epidermis and dermis, with severe collagen fiber breakdown and heavy inflammatory infiltration. After Acorus calamus L. and positive drug treatment, inflammatory cell infiltration was reduced, collagen fiber proliferation was observed, and the collagen fibers were more organized. In the medium-dose group, new blood vessels were formed, and epithelial tissue covered the wound. These results suggest that Acorus calamus L. accelerates the repair of radiation-induced skin wounds, with the medium dose showing the most significant effect (see Figure 3).

Effect of Acorus calamus L. on serum IL-1β, IL-6, and TNF-α levels
To assess the impact of Acorus calamus L. on serum inflammatory factors, ELISA kits were used to measure IL-1β, IL-6, and TNF-α levels in the rats' serum on day 30 post-irradiation. Results showed that the serum levels of IL-1β, IL-6, and TNF-α were significantly increased in the model group compared to the normal group (p < 0.05). After treatment with Acorus calamus L. and positive drugs, there was a significant reduction in these inflammatory indicators (p < 0.05) (see Figure 4). These data indicate that Acorus calamus L. effectively regulates the elevated serum inflammatory factors following radiation exposure, reducing the inflammatory response.

Effect of Acorus calamus L. on skin cell apoptosis in rats
TUNEL staining revealed significantly higher apoptosis in the model group compared to the normal group. In contrast, Acorus calamus L. and positive drugs treatment significantly reduced cell apoptosis, The medium-dose Acorus calamus L. demonstrated superior efficacy compared with both the high-dose Acorus calamus L. and the positive drugs group (p < 0.05) (see Figure 5).

Impact of Acorus calamus L. on P53, Bax, and Bcl-2 expression
Immunohistochemical analysis showed that the expression of P53 and Bax proteins was significantly increased in the model group compared to the normal group , while Bcl-2 expression was also elevated. In the Acorus calamus L. and positive drugs-treated groups, P53 and Bax expression was significantly reduced, while Bcl-2 expression was significantly increased. The treatment effect of the Acorus calamus L. was significantly higher than that of the positive control group (p < 0.05) (see Figure 6). Western blotting further confirmed these findings, showing a consistent trend in protein expression levels (p < 0.05) (see Figure 7). These results suggest that Acorus calamus L. can regulate apoptosis-related proteins in radiation-induced skin injury.

Effect of Acorus calamus L. on VEGF and bFGF expression in skin tissue
Immunohistochemistry showed that the expression of VEGF and bFGF was significantly higher in the model group compared to the normal group. In contrast, both the Acorus calamus L. and the positive drugs group exhibited significantly increased VEGF and bFGF expression with the Acorus treatment group showing a greater effect than the positive drugs group (p < 0.05) (see Figure 8). Western blot results confirmed these trends (p < 0.05) (see Figure 9). These findings suggest that Acorus calamus L. enhances the expression of VEGF and bFGF, which are important for angiogenesis and wound healing in radiation-induced skin injury.

Figure 1: Therapeutic effect of Acorus calamus L. on radiation skin injury in SD rats. (A) Diagram of radiation dermatitis wounds. The graph illustrates that the Acorus calamus L. treatment group exhibited superior skin wound healing effects compared to the model group and positive drugs group on the 30th and 45th days. Among the treatment groups, the medium-dose group demonstrated the most significant healing effect. (B) Wound healing rate. *p < 0.05 and **p < 0.01 vs model group; #p < 0.05 and ##p < 0.01 vs positive group. Please click here to view a larger version of this figure.

Figure 2: Electron microscope observation of ultrastructure changes in skin tissue. Following treatment with Acorus calamus L., there was a significant increase in dermal collagen fibers, even distribution of chromatin, and relatively abundant mitochondria and rough endoplasmic reticulum compared to the model group and positive drugs group. Scale bar =5 µm. Please click here to view a larger version of this figure.

Figure 3: Pathological damage of skin in rats. (A) H&E staining findings revealed that the model group exhibited epidermal coagulative necrosis, dermal swelling, and extensive infiltration of inflammatory cells after 30 days. In contrast, the treatment group demonstrated neovascularization, reduced inflammation, and complete re-epithelialization of the wound surface. (B) Masson staining illustrated that collagen fibers in the model group show a disorganized structure and fragmentation. By comparison, the treatment group exhibits an increased density of collagen fibers, which are orderly aligned. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 4: ELISA detection of inflammatory factors in SD rats. Perform ELISA to detect inflammatory factor concentrations in skin tissue homogenates of SD rats (30 days after irradiation). Bar charts showing the concentrations of (A) IL-6, (B) TNF-α, (C) IL-1β. *p < 0.05, ns =not significant (30 days after irradiation). Please click here to view a larger version of this figure.

Figure 5: TUNEL assay showing apoptosis in irradiated rat skin cells. (A) Detection of apoptotic cell distribution in skin tissues by TUNEL assay (DAB staining) (30 days after irradiation); (B) Statistical analysis of apoptotic cell positive rate. 30 days after irradiation, *p < 0.05, ns =not significant. Please click here to view a larger version of this figure.

Figure 6: Immunohistochemical detection of Acorus calamus L. effects on P53, Bax, and Bcl-2 protein expression in rat skin. (A) Graph shows the immunohistochemical staining of P53, Bax, and Bcl-2 proteins in each group (30 days after irradiation). (B,C,D) Graphs display the immunohistochemical expression levels of P53, Bax, and Bcl-2 in each group, respectively. *p < 0.05, ns =not significant. Please click here to view a larger version of this figure.

Figure 7. Western blot detection of P53, Bax, and Bcl-2 expression in rat skin. (A) P53, Bax, and Bcl-2 in rat skin tissue through western blotting (30 days after irradiation). (B,C,D) Statistical analysis of P53, Bax, and Bcl-2 expression. *p < 0.05, ns =not significant. Please click here to view a larger version of this figure.

Figure 8. Immunohistochemical detection of Acorus calamus L. effects on VEGF and bFGF protein expression in rat skin. (A) Graph shows the immunohistochemical staining of VEGF and bFGF in skin tissues from each group (30 days after irradiation). (B,C) Graphs display the analysis of the immunohistochemical expression levels of VEGF and bFGF in each group, respectively. *p < 0.05, ns =not significant. Please click here to view a larger version of this figure.

Figure 9. Western blot detection of VEGF and bFGF expression in rat skin. (A) VEGF and bFGF in rat skin tissue through western blotting (30 days after irradiation). (B,C,) Statistical analysis of VEGF and bFGF expression. *p < 0.05, ns =not significant. Please click here to view a larger version of this figure.

Table 1: Comparison of wound healing time of rats. Compared with the model group, the treatment of Acorus calamus L. and positive drugs could significantly shorten the wound healing time, with the best effect in the middle-dose group. Results are expressed as mean ± SEM, and statistical significance was assessed by the Student's t-test. *p < 0.05, compared with the model group, #p < 0.05, compared with the positive drugs group.

Radiation-induced skin injury remains one of the most common and challenging complications in clinical cancer radiotherapy. As the largest organ and the first target of radiation, the skin is susceptible to damage from free radicals and reactive oxygen species produced by radiation, which can impair cellular functions, including cell division, migration, and differentiation, ultimately leading to delayed wound healing and tissue damage^20,21. These injuries often manifest as erythema, edema, recurrent ulcers, and necrosis, and in severe cases, they can threaten the patient's life^20,21 . Current treatments for radiation-induced skin injury lack specificity, and as such, finding effective therapeutic agents is a major focus of medical research. The traditional Chinese medicinal Acorus calamus L. has demonstrated potential in promoting wound healing owing to its multifaceted pharmacological properties, including anti-inflammatory, antioxidant, antimicrobial activities, and free radical scavenging²². However, its therapeutic effects and underlying mechanisms in radiation-induced skin injury remain inadequately explored. This protocol is based on an animal model of radioactive skin injury to study the mechanism of Acorus calamus L. in treating radioactive skin injury in terms of inflammatory response, apoptosis, and angiogenesis.

The critical steps of this study are to establish a rat model of radiation-induced skin injury, to administer Acorus calamus L. as treatment, and to comprehensively elucidate the intervention mechanisms of Acorus calamus by quantitatively assessing therapeutic efficacy across multiple dimensions, including histopathology, serum cytokine analysis, and protein expression. In this study, we made several modifications. We optimized the establishment method of the rat radiation-induced skin injury model and used ³²P as the radiation source for back irradiation. During the modeling process, by precisely controlling the distance between the radioactive source and the skin surface as well as the radiation dose, the uniform distribution of the radiation field is ensured, thereby achieving repeatable damage induction within a small range. The back skin model is particularly suitable for systematic monitoring of wound healing, tissue fibrosis, and regeneration processes due to its large area, ease of operation, and observation¹⁶. This model provides a reliable in vivo platform for evaluating the therapeutic effects of topical preparations (such as the extract of the Acorus calamus L.). Troubleshooting mainly lies in optimizing the dosage and duration of drug treatment to ensure the reproducibility of the model, the consistency of treatment, and its correlation with the pathological process of human radiation-induced skin injury.

Previous studies have demonstrated that ethanol extracts of Acorus calamus L. promote wound contraction, shorten epithelialization time, and accelerate healing^23,24. Our study further supports these findings by showing that Acorus calamus L. treatment significantly improved the healing rate and reduced healing time in a rat model of radiation-induced skin injury. Histopathological analysis indicated that Acorus calamus L. reduced inflammatory cell infiltration, enhanced collagen fiber proliferation, and stimulated new blood vessel formation, leading to improved epithelial coverage of the wound. These findings confirm that Acorus calamus L. accelerates the repair of radiation-induced skin wounds and promotes tissue regeneration. The healing of radiation-induced skin injuries occurs in multiple stages, with the inflammatory phase being crucial²⁵. An overproduction of inflammatory cytokines during this phase can exacerbate tissue damage and impair cell proliferation, thereby delaying wound healing. Interleukins (IL-1β, IL-6) and tumor necrosis factor-alpha (TNF-α) are key indicators of inflammation, and their elevated levels are often associated with chronic inflammation and poor wound healing. Previous studies have demonstrated that Acorus calamus L. extracts can modulate the levels of inflammatory cytokines, suppressing the production of TNF-α, IL-1β, and IL-6^26,27. In our study, Acorus calamus L. treatment led to a significant reduction in serum levels of IL-1β, IL-6, and TNF-α, further indicating its ability to mitigate the inflammatory response and promote wound healing.

In addition to inflammation, cell apoptosis plays a key role in radiation-induced skin injury²⁸. Radiation exposure activates apoptotic pathways, increasing the number of apoptotic cells, slowing down cell proliferation, and impeding the wound healing process²⁹. P53, Bax, and Bcl-2 are pivotal regulators of apoptosis, with P53 and Bax promoting cell death, while Bcl-2 serves as an anti-apoptotic protein. The balance between Bcl-2 and Bax determines whether apoptosis is triggered³⁰. In this study, we observed that radiation exposure resulted in increased expression of P53 and Bax proteins, along with mitochondrial damage and elevated apoptotic cell numbers. Treatment with Acorus calamus L. significantly reduced P53 and Bax expression and increased Bcl-2 expression, suggesting that Acorus calamus L. can inhibit radiation-induced apoptosis, thereby promoting wound healing. Angiogenesis is another critical aspect of wound healing, as it ensures the supply of oxygen and nutrients to the injured tissue³¹. VEGF and bFGF are essential factors that promote angiogenesis and tissue repair. VEGF stimulates endothelial cell proliferation and microvascular permeability, while bFGF aids in the growth of fibroblasts and endothelial cells³². Our study showed that Acorus calamus L. treatment upregulated the expression of both VEGF and bFGF in skin tissue, which likely contributed to enhanced angiogenesis and accelerated wound healing.

Based on the above experimental results, this study was compared with existing treatment methods, and it was found that the standard treatments for traditional radiation-induced skin injuries include topical steroids, moisturizers, and non-steroidal anti-inflammatory drugs (NSAIDs). These treatments mainly target symptoms rather than the underlying pathophysiological process of the disease itself, and the therapeutic effects are often unsatisfactory for different patients^16,33. In contrast, Acorus calamus has multi-component and multi-target effects. It plays a synergistic role in inflammation, apoptosis, and angiogenesis to promote radiation-induced skin healing, embodying the overall treatment concept of traditional Chinese medicine. In addition, it has relatively few side effects and good safety, but its long-term side effects still require further study.

However, this technology has certain limitations. First, although animal models can simulate human radiation damage, species differences may limit the extrapolation of results²⁴. So far, the most severe pathological and physiological mechanisms of the animal model after irradiation on the 30th day have only been studied. Secondly, the complexity of components in Acorus calamus L. extracts may introduce variability. Medicinal plant extracts often contain multiple compounds whose synergistic or antagonistic effects have not been fully elucidated, which may affect the precise resolution of their mechanisms of action³⁴. In addition, further research is needed to elucidate the specific molecular pathways through which Acorus exerts its radioprotective effect, as well as to gain a deeper understanding of its potential role as a treatment for radiation injury.

The method used in this study can be used to screen other active ingredients of traditional Chinese medicine, discover new active ingredients and mechanisms of action for the treatment of radiation-induced skin damage, and facilitate the development of new therapeutic drugs. In summary, this study accelerates the healing of radiation skin damage through anti-inflammatory, anti-apoptotic, and pro-angiogenic mechanisms, laying a theoretical and experimental foundation for the development of new radioprotective agents.