| ObjectiveSepsis-induced muscle weakness has become a severe, frequent, and persistent complication among critically ill patients, which reduced the patients’ quality of life long after hospital discharge. Sepsis-induced muscle proteolysis aggravation is a main reason of muscle weakness. Many aspects including ubiquitin-proteasome system, proinflammatory cytokines, and oxidative stress contribute to muscle proteolysis. Hemin, an inducer of Heme oxygenase-1(HO-1), can enhance the activation of HO-1. HO-1 possesses a variety of functions like anti-inflammatory, anti-oxidative and anti-apoptosis effects. However, whether HO-1 exerts a protective effect in sepsis-induced muscle weakness is still unknown. This study was aimed to investigate the protective function of Hemin in sepsis-induced muscle weakness and the underling mechanisms. Methods1. C57BL/6 mice were used and cecal ligation and puncture(CLP) model was chosen to elicit sepsis. Mice were randomized divided into four groups, i.e., Control, CLP, Hemin, and ZnPP group. Control group was conducted with sham operation and were injected with 2% DMSO 200μl intraperitoneally(i.p). Mice of CLP group were administrated with 2% DMSO 200μl(i.p). Mice of Hemin group were injected with 200μl hemin(50mg/kg, i.p). Mice of ZnPP group were given with the same volume of the mixed drugs of hemin(50mg/kg, i.p) and ZnPP(20mg/kg, i.p). All treatment was given 3 times at a 48 hours interval and the last dose was given 24 h before surgery. All mice were anesthetized with 2%~3% sevoflurane before tissue or blood collection in our study. All experiments were conducted under animal welfare and ethics.2. At 3d after CLP operation, the skin of hind limbs was stripped for gross comparisons of muscle mass. The soleus of mice were collected and weighed at baseline, 1d, 3d, 5d, and 7d post operation.3. At 24 hours post operation, we evaluated the protein breakdown rates by testing net release of free tyrosine during soleus muscle proteolysis.4. At 5d post operation, the tibialis anterior(TA) muscles of mice were fixed with buffered formalin, then embedded in paraffin, and cut into sections. The sections were stained with hematoxylin–eosin(H&E) for morphological evaluation under a light microscope. Muscle fiber size was measured by the cross-sectional area(CSA) of skeletal muscle fiber.5. At 24 h post operation, blood of mice in all groups was collected via cardiac puncture. Serums were assayed for mouse TNF-α and IL-6 by a murine enzyme-linked immunosorbent assay kit.6. The TA muscles of mice in all groups were harvested at 1d, 4d, and 7d after surgery. We examined the mRNA levels of HO-1, Atrogin-1, and MuRF1 via RT-PCR. We collected the TA muscles of mice at 1d after surgery, and the protein content of HO-1, Atrogin-1, and MuRF1 were examined via Western-blot. We measured the bilirubin generation in the TA muscles of mice to evaluate the HO-1 activity 24 h after surgery.7. At 24 h after surgery, we harvested the TA muscle of mice of all groups, and measured the MDA quantity and SOD activity to determine the oxidative stress level.Results1. The size of hind limbs was significantly atrophic at 3d after CLP operation via gross comparisons; administration of hemin partly improved the situation. However, ZnPP reversed the protective effect of hemin. Weight of soleus among groups was not significantly different at the baseline. With the extension of time after operation, only slightly loss of soleus weight was found in control group. However, there was a significant deterioration of soleus weight in septic mice. Administration of hemin ameliorated muscle mass loss(P<0.05 at day3, day5 and day7 versus CLP group), while ZnPP reversed its protective effect.2. There was nearly a two-fold increase of protein breakdown rates in mice of CLP group(P<0.01). There was a significant decline of protein breakdown rates in mice of hemin group(P<0.01, versus CLP group). However, the protein breakdown rates in mice of ZnPP group were similar to that of CLP group.3. Smaller myofibers were observed in TA tissues of mice in CLP group compared with that of control group. However, muscle fibers of hemin group show a better condition than that of CLP group. The CSA of CLP group was significantly smaller than that of control group(P<0.01). Administration of hemin significantly improved the myofiber size(P<0.01), whereas the mice in ZnPP group showed a similar myofiber size to that of CLP group.4. The plasma levels of proinflammatory cytokines, i.e., IL-6 and TNF-α, were significantly up-regulated after CLP compared with that of control group(P<0.01). Hemin inhibited the release of IL-6 and TNF-α(P<0.01), but no differences were observed between CLP and ZnPP group.5. Results of RT-PCR showed that HO-1 levels and activities were higher in CLP group than that of control group. Administraion of hemin significantly increased the HO-1 level and its activities(P<0.01 versus CLP group). Levels of HO-1 expression in ZnPP group were also up-regulated, but its activity was suppressed by ZnPP(P<0.01). The mRNA expression of Atrogin-1 and MuRF1 were significantly up-regulated in TA tissues of CLP mice. The enhanced expression of HO-1 induced by hemin inhibited the expression of Atrogin-1 and MuRF1. Expression of Atrogin-1 and MuRF1 in ZnPP group was higher than that of hemin group.6. The protein level of HO-1 was elevated in CLP group compared with that of control group. Pretreatment with hemin enhanced the HO-1 protein expression, and ZnPP administration could not affect the its protein level. The protein levels of Atrogin-1 and MuRF1 were elevated in CLP group compared to that of control group. Pretreatment with hemin blunted protein expression of Atrogin-1 and MuRF1. In ZnPP group, the protein levels of Atrogin-1 and MuRF1 were also up-regulated.7. Compared with control group, the plasma levels of MDA showed a significant increase in CLP group(P<0.05). MDA levels were lower in hemin group than that of CLP group(P<0.05). There is no difference of MDA levels between ZnPP group and CLP group. SOD activity in hemin group was obviously higher than that of CLP group(P<0.01), and its level can be reversed by ZnPP. ConclusionUpregulation of HO-1 with hemin could alleviate sepsis-induced muscle wasting in mice. High expression and activation of HO-1 might protect against muscle wasting via downregulating the expression of proinflammatory cytokines, inhibiting activation of ubiquitin-proteasome system, and attenuating the level of oxidative stress. |
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