*4.9. Biochemical Analysis*

### 4.9.1. Estimation of Cytokines by ELISA

Enzyme-Linked Immunosorbent Assay (ELISA) was performed to determine the cytokine levels of IL-1β, IL-2, IL-6, and IL-10 in liver tissue as per the manufacturer's instructions [44].

### 4.9.2. qRT-PCR Analysis

In total, 10 mg of tissue samples from all the groups was used to isolate total mRNA via the Triazole technique in order to examine the amount of mRNA that is expressed for the gene of interest. The RNeasy small kit was used to purify the mRNA as per the manufacturer's instructions. Finally, qRT-PCR (Real-Time Quantitative Reverse Transcription PCR) was performed via the standard instrument with the help of the PCR master mix. Denaturation of the cDNA was performed at 94 ◦C for 5 min, followed by annealing at 58 ◦C for 30 s and subsequent extension at 72 ◦C for 35 s. Forty times repetition of the cycle was set by using qRT-PCR, which helps in the detection of the amplified DNA in real-time. To normalise mRNA, GAPDH was used as a housekeeping reference. For all the treated groups, ∆Ct values was normalised with untreated control samples (∆Ct = Ct gene of interest − Ct housekeeping gene). The relative changes in the expression level of a particular gene were measured in terms of 2−∆∆Ct (∆∆Ct = ∆Ct test − ∆Ct control) [45,46].

### *4.10. Histopathological Investigations*

The repaired skin of the wounds was taken out and preserved in a 4% paraformaldehyde solution on the 7th and 14th day following surgery for histopathological examination and to determine where collagen was deposited. To check the biocompatibility of biological macromolecules, viz., keratin and genistein, major tissues such as the liver and kidney were resected and fixed in 10% formalin for histological observations with respect to major changes associated with the tissue toxicity study.

Via selective staining with the dye haematoxylin and eosin, the histological analyses of wound skin, liver, and kidney were performed. The 7 mm to 10 mm thick sections were cut and initially fixed in formal calcium and embedded in paraffin wax. By employing xylene, the cut sections were de-waxed, treated with decreasing amounts of alcohol for hydration, and stained with haematoxylin. Then, the sections were dehydrated with alcohol to 70% concentration and further stained with 1% alcoholic eosin solution. Subsequently, the sections were differentiated in 90% alcohol solution, cleaned with xylene, mounted cautiously, and observed under the microscope [47,48].

### *4.11. Digital Image Analysis*

From the histological images of tissues, luminescence was identified and transformed into the heights of the final 3D, interactive surface plot, which recognises the pure stained areas adjacent to the adjoining sampling in a square dimension and represents the background

stains, nuclear sites, and other characteristics. A computerised scoring was allocated by examining and determining the staining pattern by using the Image J® program.

### *4.12. Statistical Analysis*

All values were provided as means with standard deviations (±SD), with n = 6; \*\* represented significance at *p* < 0.01 versus the control group, and \* represented significance at *p* < 0.05 in comparison with the standard gel and keratin–genistein combination groups. By using Statistica® v.17.0 software, one-way ANOVA was used to analyse the data, and then Bonferroni's Multiple Comparison Test was performed.

**Author Contributions:** N.A. and N.M.M. analysed the data and prepared the article; K.W. carried out the experimental portion. The design, in vivo work processing, and tissue histopathological analysis interpretation were all performed with help from M.I., N.A. and M.K. The manuscript has been reviewed by N.M.M., M.K., D.K.M., N.A. and A.R.B. N.M.M. supervised all experimental work and provided valuable recommendations for troubleshooting throughout the project. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded through Researchers Supporting Project no. RSPD2023R734, King Saud University, Riyadh, Saudi Arabia.

**Institutional Review Board Statement:** The animal study protocol was recommended and approved by the Institutional Animal Ethical Committee of Dadasaheb Balpande College of Pharmacy, Besa, Nagpur, Maharashtra, India (Registration no. 1426/PO/Re/S/11/CPCSEA).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors extend their appreciation to "Researchers Supporting Project number (RSPD2023R734)" King Saud University, Riyadh, Saudi Arabia" for their financial support. The authors are thankful to Dadasaheb Balpande College of Pharmacy for their support and for providing facilities.

**Conflicts of Interest:** The authors declare no conflict to interest.

### **Abbreviations**


### **References**


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