Endoplasmic Reticulum Stress During Skeletal Muscle Wasting In Rats With Severe Scald Injury And Effects Of Insulin Therapy

Objetive: The metabolism post severe burn injury is characterized byhypercatabolism, with significant skeletal muscle wasting, which has profound influenceon prognosis of burned patients. Our previous studies have found thatubiquitin-proteasome mediated protein degradation and apoptosis are main processesinvolved in skeletal muscle wasting post-burn, and these can be prevented by insulintherapy. Endoplasmic reticulum is a critical site for the synthesis and modification ofproteins and also an organelle responsible for Ca2+storage, so its homeostasis is crucial forcellular function and fate. If endoplasmic reticulum homeostasis were broken, endoplasmicreticulum stress (ERS) would be activated, which plays important roles in metabolism,inflammation and tumorigenesis. ERS mediated apoptosis is also an important pathway foreukaryotic cellular death. To further clarify the mechanisms of skeletal muscle wastingfollowing severe burn injury and improve clinical management, we intend to investigatethe role of ERS in skeletal muscle wasting post-burn and effects of insulin therapy withthree divided studies:(1) Observation of ERS activation, insulin signaling changes, apoptosis and wastingof skeletal muscle in rats with severe scald injury;(2) Observation of ERS activation, insulin signaling changes, apoptosis and wastingof skeletal muscle in severely scalded rats after treatment with4-phenyl butyricacid(4-PBA), which is a molecular chaperone for ERS intervention;(3) Observation of ERS activation, insulin signaling changes, apoptosis and wastingof skeletal muscle in severely scalded rats after insulin therapy.Methods:(1) Sixty-four Wistar rats were randomly divided into the control (shamburn injury) and burn group [30%total body surface area (TBSA) full-thickness thermalinjury](n=32respectively). Body weights were monitored during research, tibialis anteriorand soleus were harvested at Day1,4,7,14post-burn. Transmission electron microscope (TEM) was used to observe the changes of tibialis anterior ultrastructure. In both types ofmuscles mentioned above, Caspase-3expressions were detected with immunochemistrymethods, apoptosis was assessed by the assay of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), and Western Blotting wasused to detect the expressions of endoplasmic reticulum stress associated proteins glucoseregulated protein78(GRP78), C/EBP-homologous protein(CHOP), and Caspase-12.Expression of phosphorylated phosphatidylinositol3kinase (p-PI3K) in tibialis anteriorwas also detected with Western Blotting.(2) Sixteen Wistar rats were randomly divided into the burn and treatment group (n=8respectively),30%TBSA full-thickness thermal injury model was produced in all rats.Rats in the treatment group were treated with4-PBA [diluted in0.6%Carboxymethylcellulose-Na (CMC-Na)](700mg/kg·d, i.g.) from Day1after injury, whilerats in the control group treated with equal quantity CMC-Na as placebo. Body weightswere monitored and rats were sacrificed at Day14post-burn to harvest tibialis anterior andsoleus, the weights of skeletal muscle were also recorded. Other experimental parameterswere detected as described in the first part.(3) Forty-eight Wistar rats were randomly divided into the burn and treatment group(n=24respectively),30%TBSA full-thickness thermal injury model was produced in allrats. Rats in the treatment group were treated with insulin glargin [diluted in0.9%normalsaline](1IU/kg·d, s.c.) from Day1after injury until Day7post-burn, while rats in thecontrol group treated with equal quantity normal saline as placebo at the same time. Bodyweights and blood glucose levels were monitored and rats were sacrificed at Day4,7,14post-burn to harvest tibialis anterior and soleus, the weights of both skeletal muscle wererecorded. Other experimental parameters were detected as described in the first part.Results:(1) Compared with rats in the control group at the same time, rats in the burngroup showed significant body weight loss at Day4,7,14post-burn (P<0.01),accompanied by tibialis anterior weight loss and lower tibialis anterior weight/bodyweight ratio at every observational time point, while soleus weight loss was onlysignificant at Day7,14post-burn (P<0.01). TEM observation indicated that after burn injury tibialis anterior showed obvious apoptotic alterations and ER expansion. At all timepoints, the apoptotic index (%) of tibialis anterior and soleus were significantly higher(P<0.05) accompanied by increased Caspase-3expressions (P<0.01). Compared with thecontrol group, in tibialis anterior, expressions of GRP78became evaluated at Day4post-burn (P<0.01), active Caspase-12expressions were higher at Day4,14post-burn(P<0.05), while CHOP increased at all time points post-burn (P<0.01); In soleus,expressions of GRP78became evaluate at Day4,7post-burn (P<0.05), activeCaspase-12expressions were higher at Day1,4post-burn (P<0.05), while CHOPincreased at Day1,4,7post-burn (P<0.05). In addition, expressions of p-PI3K in tibialisanterior were lower at Day7,14post-burn compared with the control group (P<0.05).(2)Compared with the burn group, rats in the4-PBA treatment group exhibitedtendency of less body weight loss and body weight was higher at Day7post-burn (P<0.01),accompanied by increased tibialis anterior weight at Day14post-burn (P<0.05), withoutno alteration in tibialis anterior weight/body weight ratio (P>0.05). There were no changesin soleus weight and soleus weight/body weight ratio either (P>0.05). TEM observationindicated that after4-PBA treatment tibialis anterior displayed slighter apoptotic alterationsand ER expansion. At Day14post-burn, the apoptotic index (%) of tibialis anterior andsoleus was significantly lower (P<0.01) accompanied by depressed Caspase-3expressions(P<0.01) after4-PBA treatment. At Day14, compared with the burn group, expressions ofGRP78, active Caspase-12and CHOP were all significantly lower both in tibialis anteriorand soleus in the treatment group (P<0.05). In addition, expressions of p-PI3K in tibialisanterior increased at Day14after4-PBA treatment (P<0.01).(3) Compared with the burn group, rats in the insulin treatment group displyedincreased body weight (P<0.05) and tibialis anterior weight at Day14(P<0.01). The bloodglucose levels were lower in the treatment group during insulin therapy period (P<0.01).TEM observation indicated that insulin therapy also inhibits tibialis anterior apoptoticalterations and ER expansion. At Day4,7,14post-burn, the apoptotic index (%) of tibialisanterior was lower in the treatment group, while the apoptotic index (%) of soleus waslower at Day7,14(P<0.01), both of them were accompanied by depressed Caspase-3 expressions at the same time (P<0.05). Compared with the burn group, expressions ofGRP78became evaluate at Day4,7,14in tibialis anterior and soleus with insulin therapy(P<0.05),and active Caspase-12expressions were lower at Day7,14post-burn (P<0.01).CHOP expressions were depressed at all time points in tibialis anterior (P<0.01) withinsulin therapy while they only became lower in soleus at Day7,14(P<0.01). Expressionsof p-PI3K in tibialis anterior increased at Day7,14with insulin therapy (P<0.01).Conclusions:(1) ERS is activated in skeletal muscle of rats with severe scald injury,and this may be a mechanism of skeletal muscle apoptosis, insulin signaling alterations andskeletal muscle wasting post-burn.(2) Insulin therapy can ameliorate ERS, inhibit skeletal muscle apoptosis, insulinsignaling alterations and skeletal muscle wasting after burn injury. The intervention effectsof insulin on ERS may be a mechanism involved in insulin therapy.