[Modulatory effect of insulin on scalded rat serum-induced apoptosis of skeletal myoblast].
OBJECTIVE
To study the modulatory effect of insulin on apoptosis of skeletal myoblast (L6 cells) by serum of scalded rat and its mechanism.


METHODS
L6 cells cultured with DMEM medium containing 10% FBS were divided into control (C, added with 20% normal rat serum), serum from rat with scald injury (S, added with 20% serum from scalded rat), insulin (I, added with 20% normal rat serum and 100 nmol/L insulin), and serum of scalded rat + insulin (SI, added with 20% serum of scalded rat + 100 nmol/L insulin) groups according to the random number table. After being cultured for 48 hours, apoptosis was observed with Hoechst 33258 staining and its number counted, annexin V -FITC/PI double-labeling method was used to assess apoptosis rate, the protein levels of phosphorylated (p-) Akt, p-PI3K, Bax, Bcl-2, and active caspase-3 were determined by Western blotting. Data were processed with grouped or paired t test.


RESULTS
(1) The amount of apoptosis with typical morphological change in S group [(59.6 +/- 3.9) per visual field] was more than that in C, I, and SI groups [(4.9 +/- 2.6), (5.5 +/- 2.1), (19.7 +/- 2.3) per visual field, with t value respectively 28.53, 29.86, 21.53, P values all below 0.01]. (2) Apoptotic rate in S group was (18.5 +/- 1.8)%, which was markedly higher than that in C, I, and SI groups [(1.1 +/- 0.6)%, (1.5 +/- 0.3)%, (7.8 +/- 0.6)%, with t value respectively 22.41, 22.83, 13.92, P values all below 0.01]. (3) Compared with those in C group, the protein levels of Bax and active caspase-3 in S group were up-regulated (1.12 +/- 0.63 vs. 0.16 +/- 0.03, 2.15 +/- 0.51 vs. 0.21 +/- 0.03, with t value respectively 3.80, 10.69, P values all below 0.01), the protein level of p-Akt was lowered (0.20 +/- 0.03 vs. 0.42 +/- 0.07, t = -8.46, P < 0.01), and the protein levels of p-PI3K and Bcl-2 showed no statistical difference (0.19 +/- 0.03 vs. 0.26 +/- 0.09, 0.17 +/- 0.03 vs. 0.28 +/- 0.07, with t value respectively -2.73, - 1.14, P values all above 0.05). The protein levels of Bax (0.40 +/- 0.14) and active caspase-3 (0.83 +/- 0.18) in SI group were lowered (t = -3.23, P < 0.05; t = 6.66, P < 0.01) and the protein levels of p-Akt, Bcl-2, and p-PI3K in SI group were elevated (0.39 +/- 0.10, 0.78 +/- 0.03, 0.47 +/- 0.12, with t value respectively 4.07, 18.71, 5.05, P < 0.05 or P < 0.01) as compared with those in S group.


CONCLUSIONS
Serum from scalded rat can induce apoptosis in skeletal myoblast, and the effect can be inhibited by insulin through PI3K/Akt signal pathway.
Burns Institute, First Hospital Affiliated to the PLA General Hospital, Beijing 100048, China. cjk304@126.com
Jia-Ke
JK
Chai
Chuan-An
CA
Shen
Yun-Fei
YF
Chi
Rui
R
Feng
Hu-Ping
HP
Deng
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002056:Burns / Q000097:blood / Q000378:metabolism* / Q000473:pathology; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D007328:Insulin / Q000494:pharmacology*; D008297:Male; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000187:drug effects* / Q000473:pathology; D051381:Rats; D017208:Rats, Wistar; D044967:Serum / Q000276:immunology; D015398:Signal Transduction; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494828:Bax protein, rat; D007328:Insulin; D051028:bcl-2-Associated X Protein; C505410:Casp3 protein, rat; D053148:Caspase 3