the Mechanism of the Downregulation of Dopamine Receptor in Mechanical Ventilation Induced Lung Injury
Conditions: Acute Lung Injury
Sponsor: Xinhua Hospital, Shanghai Jiao Tong University School of Medicine

  
Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine 
 
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Research  Protocol 
 
 
 
 
 
 
TITLE: the Mechanism of the Downregulation of Dopamine Receptor in Mechanical 
Ventilation Induced Lung Injury 
 
PROTOCOL ID: XH-17-011 
 
SPONSOR: Xinhua Hospital, Shanghai Jiao Tong University School of Medicine 
 
INVESTIGATOR: Lai Jiang 
 DOCUMENT DATE: August 1, 2017 

  
Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine 
 
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Materials and methods 
1.Patients recruitment 
Research protocol was approved by Xinhua Hospital Ethics Committee Affiliated to 
Shanghai Jiao Tong University School of Medicine. With written informed consents given by all  
patients, individual information and lung tissue samples were collected. 
Forty-six colorectal patients, 18 to 60 years of age, were recruited for this study, meeting all the 
inclusion criteria shown as followed: undergo elective lobectomy with general anesthesia and 
mechanical ventilation; classified as physical status I to III according to the American Society of 
Anesthesiologists Physical Status Classification System; Written informed consent is approved. 
Participating subjects with any one of criteria shown as followed were excluded: Distant 
metastases: recent anaesthetics or mechanical ventilation treatment; children; women during 
pregnancy or lactation; being involved in other clinical subjects. 
2.Anesthetic management and mechanical ventilation 
General anesthesia protocol: anesthesia induction (midazolam 0.1mg/kg, sufentanil 
0.5ug/kg, etomidate 0.3mg/kg, cisatracurium 0.2mg/kg); anesthesia maintenance 
[sevoflurane 1.5-3%, cisatracurium 0.1mg/kg/h, sufentanil is supplemented during the 
entire surgical procedure according to patients' anesthetic situation, dexmedetomidine 
(used conditonly) 0.4ug/kg/h]. 
Mechanical ventilation protocol: tidal volume 6-8 ml/kg, positive end-expiratory 
pressure 5 cmH2O, oxygen concentration 40%; respiratory rate 10-15/min, 
inspiratory/expiratory ratio 1:1.5. 
3.Lung tissues collection and preparation:  
The lung tissues were homogenized, and incubated in cold RIPA lysis buffer (Beyotime, 
China) containing proteinase Inhibitor Cocktail (Sigma-Aldrich) to isolate the total proteins. The 
tissue extracts (40-50μg protein) were separated by 10%SDS-PAGE and subsequently transferred 
onto a PVDF membrane (Millipore Corp, Bedford, MA). After blocking, immunoblots were 
incubated with primary antibody against DRD1(1:1000; Abcam), DRD2(1:1000; Proteintech), 
TH (1:1000; Proteintech), DDC (1:1000; Abcam), Ac-a-tubulin (1:1000; Santa Cruz 
Biotechnology), β-actin (1:1000; Santa Cruz) at 4℃ overnight. The secondary antibodies 
employed were HRP-conjugated IgG (1:3000; Proteintech) for 1h at room temperature. The 
antibody-reactive bands were visualized by using the enhanced chemiluminescence Western 
blotting detection system(Millipore). Total β-actin protein levels were detected for sample loading 
correction and normalization. In determination of the expression level of above protein, we had a 
bulk preparation of normal placental protein which was set as a control. 
4. Statistical analysis 
ImageJ software were used to get the Integrated Optical Density(IOD) of the 
chemiluminescent signal from the membranes of dopamine receptor 1(DRD1), dopamine receptor 
2(DRD2), Tyrosine hydroxylase(TH), Dopa decarboxylase(DDC), acetylated-a-tubulin(Ac-a-

  
Xin Hua Hospital, Shanghai Jiao Tong University School of Medicine 
 
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tubulin). The normalization was carried out by DRD1, DRD2, TH, DDC and Ac-a-tubulin 
IOD/total β-actin IOD for sample loading correction respectively. In determination of the 
expression level of DRD1, DRD2, TH, DDC and Ac-a-tubulin in different time points, the 
investigators had a bulk preparation of normal placental protein which was set as a control. 
Analyze the correlation between duration of mechanical ventilation with DRD1, DRD2, TH, 
DDC and Ac-a-tubulin expression respectively. If p value is less than 0.05, then its change is 
statically significant.