Experimental Objective

To investigate the expression levels of inflammatory cytokine proteins in rat lung tissue, it is necessary to fully release and collect proteins from the tissue. Grinding and ultrasonic methods were employed to homogenize and lyse the lung tissue, ultimately isolating proteins in solution for Western blot (WB) analysis to determine protein expression levels.

Experimental Procedure

1. Sample Preparation:
   • A 0.2 g sample of rat right lung tissue was finely minced.
   • An appropriate amount of RIPA lysis buffer (pre-mixed with protease inhibitors) was added at a ratio of 1 mL per 100 mg of tissue.
2. Tissue Homogenization:
   • The minced tissue was thoroughly homogenized using the FeiTong-96 high-throughput tissue grinder.
   • The homogenate was incubated at 4°C for 30-60 minutes to allow complete lysis.
3. Cell Disruption:
   • The lysed tissue was further disrupted using the FYT-1800 ultrasonic disperser.
   • The sample was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected.
4. Protein Extraction and Quantification:
   • Total protein was extracted, and its concentration was measured using a BCA assay kit.
   • The protein samples were denatured by boiling at 100°C for 5 minutes, rapidly cooled on ice, and stored at −20°C for further use.
5. Western Blot (WB) Analysis:
   1. Glass plates for gel casting were cleaned with distilled water and air-dried.
   2. SDS-PAGE was performed using separating gel (12%) and 5% stacking gel.
   3. After electrophoresis, proteins were transferred onto a pre-wetted NC membrane using a wet transfer system.
   4. The membrane was blocked for 1-2 hours, followed by overnight incubation with primary antibodies at 4°C on a shaker.
   5. The membrane was washed three times with TBST (10 minutes each), incubated with secondary antibody, and washed again.
   6. ECL working solution was applied for 30-60 seconds, and chemiluminescence signals were captured using an imaging system.
   7. Protein band intensities were quantified using ImageJ software.

Experimental Results

Comparison of Tissue Before and After Grinding

The relative expression levels of IL-17, IL-22, IL-10, and IL-35 proteins in rat lung tissues were analyzed:

• Compared to the blank group, the model and sham groups showed significantly higher expression of IL-17 and IL-22 (P < 0.05) and significantly lower expression of IL-10 and IL-35 (P < 0.05).
• Compared to the model and sham groups, the dexamethasone, fermented, and non-fermented groups exhibited significantly reduced IL-17 and IL-22 expression (P < 0.05) and increased IL-10 and IL-35 expression (P < 0.05).
• No statistically significant differences were observed among the dexamethasone, fermented, and non-fermented groups (P > 0.05).

Table 1: Comparison of relative protein expression levels (mean±SD) of IL-17, IL-22, IL-10, and IL-35 in lung tissues of blank and bronchial asthma model groups.

Figure 1: WB results showing IL-17, IL-22, IL-10, and IL-35 protein expression in lung tissues of blank and bronchial asthma model groups.

Key Findings

• The FeiTong-96 tissue grinder and FYT-1800 ultrasonic disruptor efficiently homogenized and lysed lung tissue, enabling high-quality protein extraction.
• WB analysis revealed significant differences in inflammatory cytokine expression across experimental groups, validating the efficacy of the sample preparation workflow.

Conclusion

The combined use of Tissue Grinder (FeiTong-96) and ultrasonic disruption (FYT-1800) provides a robust method for tissue homogenization and protein extraction, facilitating accurate detection of protein expression levels in biomedical research.