In this study, we used male SD rats. Among them, young rats are eight weeks old and weigh about 250g; old rats are 16 months old and weigh about 550g. We performed all operations under pentobarbital sodium anesthesia. The procedures for care and use of animals were approved by the Ethics Committee of the Southern Medical University and all applicable institutional and governmental regulations concerning the ethical use of animals were followed.
Establishment of rat scald skin model
We anesthetized male rats by intraperitoneal injection of 3% pentobarbital sodium according to 1.7 mL/kg, exposed the skins, and used a constant temperature and pressure scald instrument at 94℃ for 8 seconds with the pressure of 500g to cause deep second-degree scald on the back of the rats. The back of each rat had the same wound surface(1cm x 3cm), and we gave the rats an intraperitoneal injection of 5mL saline to prevent shock. Subsequently, we divided the rats into young and old groups, divided into the control group and the 1st day, 4th day, and 7th day groups after the scalding.All animals were kept in a pathogen-free environment and fed with national standard rodent feed. To avoid other drugs from affecting wound healing, we only use sterile saline gauze wet compress. We raised three rats in each group. To observe and record the scab formation, scab removal, and healing time of the rat wound.
Enzyme-Linked Immunosorbent Assay (ELISA)
Before scald and 1st day, 4th day, and 7th day after scald, blood was taken from the eye socket, placed at room temperature for 20 minutes, centrifuged at 3000r/min for 15 minutes, and collected the liquid supernatant. Firstly, we diluted the antigen to an appropriate concentration with the coating diluent, labeled the reaction wells with a blocking enzyme, and removed the bubbles in each well. Then establish a suitable concentration gradient to add the sample to be tested and the enzyme-labeled antibody. Finally, add 100µl of TMB-hydrogen peroxide urea solution to each well, place it at 37°C in the dark for 3–5 minutes, and add stop solution to develop color. The expression levels of rat serum TNC, VEGF, and CCL2 were determined using the ELISA method.
Quantitative Real-time PCR(RT-qPCR)
We took the normal skin tissues and scald skin tissues of the control group, 1st day, 4th day, 7th day groups of young rats and old rats. First, we used TRIzol one-step method to extract RNA from rat scalded tissues and dried RNA at room temperature. Then add an appropriate amount of RNose-free solution to the sample to dissolve the precipitate. Follow the instructions of TaKaRa for reverse transcription. In the fourth step, after preparing the qPCR reaction system, we put the sample on the qPCR reaction machine for the reaction. Finally, we calculated the gene expression with a CT value. The mRNA expression levels of TNC, VEGF, and CCR2 in skin tissues were detected using RT-qPCR.
We took the normal skin tissues and scald skin tissues of the control group, 1st day, 4th day, 7th day groups of young rats and old rats. Then we used western blot to detect the protein expression level of TNC, VEGF, and CCR2. Firstly the tissue protein samples were transferred to a polyvinylidene fluoride membrane after SDS-PAGE, blocked with 5% skimmed milk powder at 37°C for one hour, washed with TBST buffer three times, 5 min each time, and then added I-antibody and incubated overnight at 4°C. The next day, anti-II was added and incubated at 37°C for one hour and then developed with enhanced chemiluminescence (ECL) reagent.
Immunohistochemistry (IHC) staining
The normal skin tissues and scald skin tissues of the control group, 1st day, 4th day, 7th day groups of young rats and old rats were taken and fix them with formalin. Then the rat tissues were dehydrated, embedded in wax blocks, sectioned, and processed on glass slides. After the slides rinsing with distilled water, we added EDTA and sodium citrate antigen retrieval solution. Then the slides were rinsed with PBS buffer, 3% hydrogen peroxide solution was added to the block for 10 minutes, and then stained. IHC detected the protein expression of CD68 to observe the infiltration of macrophages.
To take the normal skin tissues and scald skin tissues of the control group, 1st day, 4th day, 7th day groups of young rats and old rats, and fix them in 4% formaldehyde solution for pathological preparation. Then the rat tissues were dehydrated, embedded in wax blocks, sectioned, and processed on glass slides. After rinsing the slides with distilled water, they were stained with hematoxylin for 3 minutes and then differentiated with hydrochloric acid and alcohol. Then rinse the slide with distilled water again and add eosin staining for 20 seconds. Finally, the slides are dehydrated and mounted with gum. To observe the pathological would changes, including inflammation and its strength by HE.
All data were analyzed using SPSS 20 software. We performed the comparison of means between two groups by using a t-test. Furthermore, we used a single-factor analysis of variance to perform the comparison between multiple groups. (P ≤ 0.05 asset as statistically significant)
The ELISA test showed that compared with the control group, the serum TNC concentration after scalding of young rats increased firstly and then decreased, while the TNC concentration of old rats decreased firstly and then slightly increased (A). The serum VEGF concentration of young rats increased after scalding and then decreased, while the serum VEGF concentration of old rats did not change significantly (B). The serum CCL2 concentration of young rats increased significantly after scalding, while the serum CCL2 concentration of old rats increased and then decreased to no significant difference from the control group (C). (*compared with the control group, P < 0.05; #compared with the scalded 1st day group, P < 0.05). (Fig. 2)
RT-qPCR detection showed that compared with the control group, the expression of TNC mRNA in young rats' skin after scalding decreased first and then increased significantly, while the expression of TNC mRNA in the skin of old rats decreased (D). The expression of VEGF mRNA in the skin of young rats after scald significantly reduced immediately, while the expression of VEGF mRNA in old rats significantly reduced on the 4th day after scalding (E). The expression of CCR2 mRNA in young rats increased firstly and then decreased after scalding, while CCR2 mRNA in old rats increased and then decreased, and then increased again on the 7th day (F). (*compared with the control group, P < 0.05; #compared with the scalded 1st day, P < 0.05)(Fig. 3)