Earthworm rearing
The culture stocks of earthworm, Eudrilus eugeniae was cultured at Centre for Molecular and Nanomedical Sciences, International Research Centre, Sathyabama Institute of Science and Technology, Chennai, Tamilnadu, India. Earthworms are maintained as per the standard protocol of Subbiahanadar Chellathurai et al.11. The bottom layer, containing coir, effectively maintained the necessary moisture condition. Every week, the earthworm compost was substituted with soil, dried cow dung, and leaf litter19.
Ethical statement
The experiments are conducted using lower invertebrate, earthworm so an ethical statement is not required. Great care is always taken in experimental procedures to prevent any unnecessary pain and suffering to the animals involved.
Tissue samples from the amputated and unamputated worms
Earthworms are divided into three groups, with each group containing 10 individuals. They are then placed in separate plastic boxes and allowed to acclimate for a period of up to 2 days. The initial set of worms remained intact and served as the control group. The clitellum of the control worm, specifically segments 13–18, was dissected to obtain a sample for preparing the Clitellum Factors (CF). In the second group, the worms were amputated at the 10th segment using a sterile scalpel blade (Lister Surgical Blade; Size 15, Kanpur, India). In this experiment, the amputated posterior segments are given the occurrence to regenerate their head for a period of 4 days. After the 4th day, the clitellum is dissected from the regenerating worm and used as a sample for further study on Regenerative Clitellum Factors (RCF). The third group of worms consists of unamputated controls, with tissue extract samples being prepared from specific body segments (30-37th segments).
Tissue extract preparation from clitellum factors (CF), regenerative clitellum factors (RCF) and body segments
The three groups of worms are carefully rinsed in running tap water, ensuring they are clean. The samples from clitellum, regenerating clitellum and body segments (30-37th segments) from the respective groups were dissected from the worms. The dissected samples were washed with distilled water for thrice, then those individual samples were transferred into 1X Phosphate Buffer Saline (PBS) for surface sterilization. Following that, clitellum, regenerating clitellum and body segments were kept in 10% DMEM (Dulbecco’s Modified Eagle Medium) (45 ml of 0% DMEM media + 5 ml Fetal Bovine Serum (FBS)) (DMEM – Sigma-Aldrich; United States. Catalogue: D1152-10 L) (FBS – Gibco; United States. Catalogue: 10270106) and crushed with tissue homogenizer (Bel-Art Disposable Plastic Pestle; Spectra Services, Wayne County, Michigan. Catalogue: SKU: 19923-0000) at 4˚C. The crushed tissue samples were subjected to short spin in centrifuge and their supernatant were collected and stored in -20˚C freezer. The prepared mixture of CF, RCF and body segments were used for cell culture studies and animal studies.
Cell culture
Mouse myoblast C2C12 cells were purchased from (NCCB) National Center for Cell Science, Pune, India. The C2C12 cells were maintained in T-flasks containing DMEM supplement with 10% FBS and 1% antibiotics (such as penicillin [100 µg/mL] (HiMedia; Thane, India. Catalogue: SD028), streptomycin [100 µg/ml] (IBI Scientifc; Dubuque, Lowa. Catalogue: IB02180) and amphotericin B [100 µg/mL] (Thermo Fisher Scientifc; Waltham, Massachusetts. Catalogue: 15290018). The cells were kept in a CO2 incubator at 37˚C with a 5% CO2 supply. Cells were sub-cultured once they reached 90% confluence. The culture maintenance was carried out in a biosafety cabinet level II to ensure a sterile and aseptic condition. (Haier; Qingdao, China. Model No: HR40-IIA2).
MTT assay
To determine the cell viability, C2C12 cells (stock 1 × 105 cells/ml) were seeded in the 96-well plate (10,000 cells/well) and incubated at 37˚C in CO2 incubator. After 24 h of incubation, the plates were washed thrice with 0% DMEM media (without serum). Along with 10% DMEM medium the tissue extracts of CF, RCF and body segments (30th -37th ) were treated with following percentage 5, 25, 50, 75, 100% and incubated at 37ºC in CO2 incubator. Following incubation, MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) (stock 5 mg/10 ml in 1X PBS) (Sigma; Chennai, Tamil Nadu, India. Catalogue: 11465007001)) were added in 96 well plate (10 µl/well) and incubated for 2 h in dark condition. After incubation, media was carefully removed and DMSO (Dimethyl sulfoxide) (Himedia; Mumbai, India. Catalogue: MB058-100 ml; 100 µl/well) were added in plate for the purple colour formation, which is directly proportional to the number of viable cells. The 96-well plate was subjected to OD value at 570 nm using a microplate reader (BioTek Epoch Microplate Spectrophotometer; United States). The percentage of cell viability was calculated using the formulae namely (Treatment average OD/Control Average OD*100).
Wound healing assay
To determine the wound healing ability of CF and RCF, C2C12 cells were initially seeded in the 6-well plate and incubated at 37˚C in CO2 incubator. After 24 h of incubation, wound was created by manual method (scratch done by 200 µl tips with the help of 15 cm scale) the plates were washed thrice with 0% DMEM media (without serum). The scratched dead cells along with DMEM medium are removed and replaced with fresh 10% DMEM medium. Following tissue extract from CF, RCF and body segments were added in the following percentage 5, 25, 50, 75, 100% and incubated for 24 h at 37˚C in CO2 incubator. Wound healing activity were carefully observed and documented following 0th, 24th, 30th and 48th hours by using EVOS microscope (EVOS-FL).
Anti-bacterial assay
Bacterial strain of Bacillus subtillus (Gram-positive, MTCC (Microbial Type Culture Collection and Gene Bank) No. 736) and Salinivibrio sp (Gram-negative, MTCC No. 12905) were obtained from Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. The culture medium was prepared using nutrient agar (Himedia, M001). The bacterial suspension of B. subtillus and Salinivibrio sp were individually swapped over the medium to obtain the uniform growth. Discs with different concentrations of body extract (50%), CF (5%), RCF (25%), and a control (10% serum DMEM medium) were carefully placed on the agar surface. Combination of penicillin and amphotericin B (10 µg/ml) was used as a positive control and distilled water was used as a negative control. The plates were then incubated at 37 °C for 24 h. The area without visually apparent bacterial growth (clear zone) around each disc was observed. The diameter of the clear zone was measured using a scale.
Immunofluorescence assay
C2C12 cells were cultured in the six well plate with the supplement of CF (5%) and RCF (50%) along with control (10% FBS) and after attain the confluence, cells were washed with ice-cold 1X PBS buffer for thrice. The cells were fixed with 4% paraformaldehyde (Himedia; Maharashtra, India. Catalogue: GRM3660-500 mg) in 1% PBS for 10 min at room temperature, and then permeabilized with 0.5% Triton-X-100 (Triton™ X-100 Solution, 20% Sterile filtered; Himedia Labs, Maharashtra, India. Catalogue: TCL136) for 10 min on ice. The cells were blocked with 1% Bovine Serum Albumin (BSA) (SRL; Maharashtra, India. Catalogue: 9048-46-8) for 30 min and further incubate with primary antibody, Anti-VEGF antibody (Abcam, ab194806), Anti-p53 antibody (Abcam, ab26), Anti-HoxD3 antibody (Abcam, ab221101), Anti-Wnt3 antibody (Abcam, ab19925), Anti-TCTP antibody (Abcam, ab37506), Anti-H2AX antibody (Abcam, ab20669), Anti-Caspase-3 (Abcam, ab208161), Anti-H3 (Abcam, ab1791) and Anti-β-actin (Abcam, ab8227) for overnight at 4ºC. The cells were washed with 1X TBST and then treated with the appropriate secondary antibodies for 2 h at room temperature under dark condition. After that, cells were washed with 1X TBST buffer for thrice and counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Sigma Aldrich, United States Catalogue: 10236276001), mounted with DPX mount (SRL; Maharashtra, India. Catalogue: 88147) and observed under EVOS fluorescence microscope.
Western blotting
Protein samples were prepared from the control C2C12, CF and RCF treated C2C12 cells using RIPA (Radio-Immunoprecipitation Assay) buffer. Following that, samples were quantified by Lowry’s method. Subsequently, 80 µg of each protein samples were resolved in 12% SDS-PAGE. The resolved proteins in the SDS-PAGE gel are transferred to the PVDF (Polyvinylidene fluoride) membrane. Then protein membrane was treated with a 5% BSA in TBST buffer for blocking. Following that, membrane was treated with primary antibodies namely, Anti-VEGF antibody (Abcam, ab194806), Anti-Cox2 (ab179800), Anti-p53 antibody (Abcam, ab26), Anti-HoxD3 antibody (Abcam, ab221101), Anti-Wnt3 antibody (Abcam, ab19925), Anti-TCTP antibody (Abcam, ab37506), Anti-H2AX antibody (Abcam, ab20669), Anti-Caspase-3 (Abcam, ab208161), Anti-β-actin (Abcam, ab8227), Anti-H3 (Abcam, ab1791) at the dilution of 1:5000 at 4 °C for overnight. Following incubation, the membrane is washed with 1X PBST buffer thrice and incubated with secondary antibody, anti-rabbit IgG, HRP (Sigma, A0545), and Anti-Mouse IgG, HRP (Abcam, ab6789) at a dilution of 1:10,000 for 2 h at room temperature. The washing step was repeated for thrice then ECL substrate was added on the membrane and luminescence signals from the membrane are captured through ChemDoc (Biorad ChemiDoc XRS+; United States. Catalogue: 170–8265).
Survivability augmentation using CF and RCF
Worms were divided into three groups: (1) Control, (2) CF treated, (3) RCF treated. Across all the groups, the worms underwent amputation at the 10th segments, followed by daily treatment of the amputated 1-10th segments with CF and RCF. The first to tenth segments without any treatment serve as a control group. The survival ability of the worms treated with Control, CF, and RCF was monitored and documented using a Canon Digital camera from Tokyo, Japan. Model No: IXUS 285 HS).
UV-C treatment
The earthworm, Eudrilus eugeniae, were divided into three groups, with each group consisting of 6 worms. The entire group of worms undergoes UV-C (Ultraviolet-C rays) treatment for 2 min, following the established protocol by Subbiahanadar Chelladurai et al., 202011 . After 24 h of UV-C treatment, the worms receive 10% DMEM medium (control), CF and RCF tissue extract, which are applied to their skin wounds every 24 h. After treatments, the worms were closely observed to track their wound healing progress every 24 h.
Statistical analysis
The dead and live worms following CF and RCF treatment are plotted using survival graph (Kaplan–Meier), GraphPad Prism, Version 5.01. All other experiments are repeated for at least three times to obtain the statistical significance. The data obtained were analysed using the statistical software SPSS (version 22.0; IBM Corp., USA) and expressed as mean ± SD. The results were considered as statistical significance when the p-value < 0.05.
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- Source: https://www.nature.com/articles/s41598-024-79304-3