Dexmedetomidine Attenuates Lung Ischemia Reperfusion Injury Through The PI3K/Akt/HIF1-α Signal Pathway

Bankground and objectiveLung ischemia-reperfusion injury was widely existing in clinic, such as CPR、Lung transplantation、one lung ventilation、CABG、pulmonary artery embolization surgery and so on. This damage is a form of acute aseptic lung injury with high morbidity and mortality. To discuss the pathogenesis and effective clinical intervention pathways is helpful to reduce LIRI and to improve the prognosis of patients, to reduce the burden of patients and improve in the near future and long-term survival rate. Pathogennesis of LIRI is complex, oxidative stress damage、oxidation and antioxidant imbalance、 calcium overload、endoplasmic reticulum stress injury、 inflammatory damage、apoptosis and so on many other pathways and pathophysiological processes were involved. New alpha 2 agonists dexmedetomidine(DEX)could reduce the presynaptic membrane,has sedative and analgesic effect, it could inhibit the stress reaction and play a wide range of clinical efficacy. In recent years, more and more studies show that DEX plays a protective role in a variety of important organs such as heart 、 brain 、 kidney. Mechanisms were involved in these studies, such as inflammatory mediator、oxidative stress damage、apoptosis and so on. However, so little studies were focused on the LIRI protection, especially the signaling pathways is uncertain, our study was designed to discuss the further mechanism of DEX on LIRI and maybe helpful to guide clinical drug use. Phosphoric acid acyl inositol 3- kinase(phosphoinositide 3 kinese, PI3K) is a kind of lipid kinase, existed in all kinds of cells, participated in emembrane related phosphatidyl inositol phosphorylation process, and the activation of the second messenger.It was involved in cell differentiation 、 proliferation 、 apoptosis and migration, etc; Akt is a serine/threonine protein kinase, main point of PI3 K signaling pathways downstream molecules.PI3K/Akt signaling pathway was closely associated with lung injury. In recent years, research has shown that the DEX could raise HIF- 1 alpha expression through PI3K/Akt signaling pathway to reduce rat C6 cells from hypoxic damage; Another study showed that the right supporting the DEX can reduce cerebral ischemia reperfusion injury in rats, PI3K/Akt signaling pathway was involved in the effect.Hypoxia inducing factor 1(HIF- 1)was found firstly by Semenza, etc.(1992).It is a nuclear transcription factor founded in the nucleus extract induced by hypoxia.HIF-1 can regulate a variety of target gene transcription, in which many biological effects were included. HIF- 1 alpha(HIF-1α) is one of the two subunits, studies have shown that it was HIF-1 which could participate in the pathophysiological process of many organs. Studies have shown that PI3K/Akt signaling pathway is closely related to HIF-1α. m TOR pathways were involved in the inflammatory cytokines and other signal molecules which could stimulate the synthesis of HIF-1α. Akt is a central part of the PI3K/Akt pathway, PI3 K activates Akt through PDK phosphorylated and then play a biological effect. PI3K/Akt is a classic of m TOR upstream pathways, thus it was speculated that PI3K/Akt pathway is closed to the HIF- 1α.The first part of this study was designed to establish the lung ischemia-reperfusion model by which to discuss the lung protection of DEX. The further mechanism was revealed by the changes of the PI3K/Akt signal pathway; the second part was designed to dicuss the changes of HIF-1α downstream target genes protein expression, tring to understand the further effect of DEX on PI3K/Akt pathway; the third part was designed to discuss the effect of DEX on one-lung ventilation in humans and to provide the theoretical basis for clinical medication. Materals and methods Part Ⅰ Establishment of Ischemia-reperfusion model in rats and detection of PI3K/Akt / HIF- 1α signal pathway.Forty-eight clean level male rats(provided by Henan Province Experimental Animal Center), aged 8 to 10 weeks, weighing 250-350 g were enrolled. HX- 100- e animal breathing machine were provided by China chengdu Thai union technology co., LTD.Rats were randomly divided into six groups: Sham group : no lung ischemia-reperfusion operation; IR group: lung ischemia-reperfusion operation; LD group : 1μg/kg DEX; LDL group : 1μg/kg DEX + 0.3 mg/kg LY294002; HD group:10μg/kg DEX; HDL group: 10μg/kg DEX + 0.3 mg/kg. 10% chloral hydrate at dose of 4 ml/kg were injected intraperitoneally followed by establishment of caudal vein punctureing for fluid circuit open,rats were placed in the supine position and limbs were fixed. Endotracheal intubation by homemade endotracheal tube were established after tracheotomy.Rats were then connected to breathing machine, left femoral artery puncture catheter were used for connection with a pressure sensor and detection of pressure and blood gas analysis. IR model: rats were put in a left side after the success of the anesthesia,the hilus of the left lung were exposed, A 60-min lung ischemia was induced by occluding the hilum of the left lung with a non-invasive microvascular clip, followed by a 120-min reperfusion by removing occlusion of the hilum. After the rats were sacrificed and the left lung was removed, the wet lung weight to dry lung weight(W/D) was determined. Lung tissues were fixed by paraffin and lung injury were evaluated after HE staining.Pathological changes of lung tissues were evaluated by light and transmission electron microscope. SOD activity and MDA content were determined by Colorimetric method; HIF- 1αm RNA and Aktm RNA were dectected by RT-PCR method; HIF- 1α and p-Akt were determined by Western-blotting method. PartⅡ The effects of DEX on HIF-1α downstream proteins:HO-1 and apoptosis related proteins Materials and Methods were same with part Ⅰ. After the rats were sacrificed and the left lung was removed, apoptosis of lung tissue were detected by TUNNEL method; BNIP3 and HO-1 were detected by western-blotting methods. Part Ⅲ The DEX upregulate the expressions of HIF1α and its downstream target genes protein in lung injury induced by one-lung ventilation. Forty patiens scheduled for elective general anesthesia undergoing thoracoscope lung cancer, no limit on the gender, aged 18 to 64 years old, body mass index 18-25 kg/m2, ASA class Ⅱ or Ⅲ. There is no history of diabetes, blood disease and other metabolic disorder, hypertension, chronic obstructive or restrictive lung disease, forced vital capacity > 80% expected, the first second rate > 70% expected, no smoking history in last 2 weeks, expected operating time between 1h and 4 h. The patients were randomly divided into two groups by use of the random number table methd(n = 20) : the control group(group C) and DEX group(group D). Preoperative routine banned from food, drink 8 h, without preoperative medication. Routine monitoring including ECG、BP and Sp O2.The right radial artery was opened for blood sampling and ABP monitoring. Anesthesia induction: 0.08- 0.12 mg/kg, sufentanil 0.1 ~ 1.0μ g/kg, etomidate 0.2- 0.6 mg/kg, Cis atracurium 0.15 mg/kg, double cavity endotracheal tube were intubated and fiber bronchoscope for positioning, Two lung ventilation:inhaled oxygen concentration 70%, oxygen flow rate is 1.0- 1.5 L/min, VT 6- 8 ml/kg, RR 10- 14 times/min,I:E 1:2;One lung ventilation: RR 12-16 times/min, other ventilation parameters were constant with one lung ventilation, maintaining PETCO2 35- 40 mm Hg(1 mm Hg = 0.133 k Pa). DEX with loading dose of 0.5 μg/kg infused intravenously within 20 min, and then continued at rate of 0.5μg/kg/h until the time immediately after lung resection.Normal saline were intravenously infused in group C with equivalent capacity. Maintaince of anesthesia: propofol in the 2 groups, 4- 8 mg/ kg/ min, intermittent intravenous of sufentanil 5-10μg and Cis atracurium 5 mg, the propofol infusion rate was adjusted to maintain the BIS value of 40-50, HR and MAP volatility not exceed 20% of the basic values. Intraoperative intravenous sodium lactate ringer’s solution and hydroxyethyl starch 130/0.4, capacity ratio of 3:1. Samples were obtained at time belows:immediately the begin of One lung ventilation(T1), 60 min after one lung ventilation started(T2) and the time of lung tumor resection(T3). Normal lung tissue about 1.0 cm x 1.0 cm x 1.0 cm size surrounding the tumor were cut off. The samples were washed by PBS and divided into two parts. One of the sample was preserved by liquid nitrogen for determination of HIF-1αand HO-1; another sample was fixed by formaldehyde, embedding by paraffin for HE staining. Results Part Ⅰ Establishment of Ischemia-reperfusion model in rats and detection of PI3K/Akt/ HIF- 1α signal pathway. 1. W/D ratio Compared with the Sham group, W/D in IR group were increased;W/D wered decreased in HD and HDL group when compared with IR group;W/D were lower in HD and HDL group than LD and LDL groups. 2. Score of lung injury Compared with the Sham group, the lung injury scorewere higher in the IR group; Lung injury score were lower in the HD group andHDL than IR group; When compared with LD group, lung injury score weredecreased in HD group and HDL group. 3. Compared with the Sham group, SOD activity were decreased and MDA werehigher in the IR group; Compared with IR group and LD group, SOD activitywere increased and MDA content were higher in HD and HDL group. 4. Compared with the Sham group, the Aktm RNA、HIF-1αm RNA 、Akt andHIF-1αwere downregulated IR group; Aktm RNA、HIF-1αm RNA、Akt andHIF-1αin the HD group were higher than IR group; Compared with LD group,Aktm RNA、HIF-1αm RNA、Akt and HIF-1α were higher in HD group. PartⅡ The effects of DEX on HIF-1α downstream proteins:HO-1 and apoptosis related proteins 1. Compared with the Sham group, the AI index were higher in IR group; AI indexwere lower in LD group、HD group and HDL group; No difference between LDand HD group were observed. 2. Compared with the Sham group, HO-1 in the treatment groups weredecreased;HO-1 were upregulated in LD group 、 LDL group and the HDgroup;HO- 1 in the HD group were higher than LD group. 3. Compared with the Sham group, BNIP3 expression were upregulated in the IRgroup、the LD and the HD group; Compared with IR group, BNIP3 expressionwere downregulated in LDL and HDL group. Part Ⅲ The DEX increase HIF1α and its downstream target genes protein in lung injury induced by one-lung ventilation 1. General condition、operation time、duration of one lung ventilation、the amountintraoperative transfusion、the amount of bleeding and the dosage of DEX has nodifferences between the two groups. 2. Compared with the T1 and T2, lung injury score at T3 was higher both in the twogroups during one lung ventilation(p < 0.05); When compared with group C,lung injury score at T3 were lower in group D(p < 0.05). 3. Compared with the T1 and T2, HIF-1αand HO-1 at T3 was higher both in the twogroups during one lung ventilation(p < 0.05); When compared with group C,HIF-1αand HO-1 at T3 were higher in group D(p < 0.05). Conclusion 1. Severe lung damage can be caused by lung ischemia-reperfusion accompanied byoxidative stress injury. 2. The DEX could attenuate lung ischemia-reperfusion injury in rats, with decreaseof oxidative stress injury 3. The DEX can attenuate lung ischemia reperfusion lung injury throughPI3K/Akt/HIF- 1αsignaling pathway. 4. Apoptosis and HO-1 which was regarded as downstream protein of HIF- 1αweredownregulated by DEX. 5. Lung injury induced by one lung ventilation could be attenuated byDEX,upregulation of HIF- 1αand its downstream protein HO-1 maybe one of itsmechanism.