The Role of Acod1 in Prognostic Evaluation of Sepsis: A Prospective, Single-Center, Observational Study
Conditions: Sepsis
Sponsor: Union Hospital, Tongji Medical College, Huazhong University of Science and Technology

1
Union Hospital Affiliated to Tongji
Medical College, Huazhong University
of Science and Technology
Clinical Research Project Protocol
Project Name
The Role of Mitochondria and
Related Genes in the Diagnosis and
Prognostic Assessment of Sepsis: A
Multicenter, Prospective Cohort
Study
Protocol Number
Mit01
Version Number
V2.0
Version Date
2024-09-18
Principal
Investigator
Zhang Jiancheng
Specialty
Department
Critical Care Medicine
Planned Start and
End Date
2024-10～2026-10

2
Protocol Signature Page
Project Name: The Role of Mitochondria and Related Genes in the
Diagnosis and Prognostic Assessment of Sepsis: A Multicenter,
Prospective Cohort Study
Version Number/Version Date: V2.0/2024-09-18
I have read and understood this research protocol, confirmed that it
contains all necessary content for the implementation of the study, and
clarified the investigators' responsibilities associated with this protocol.
As the Principal Investigator, I will provide a copy of this protocol and all
relevant materials to all researchers participating in the study. I will
discuss these materials with them to ensure their full understanding of the
protocol implementation procedures. I agree to and will strictly fulfill the
relevant responsibilities in accordance with the currently applicable laws
and regulations, the Declaration of Helsinki, Good Clinical Practice (GCP)
for drug clinical trials, and this research protocol.
Principal Investigator's Signature (in block letters): ____________
Principal Investigator's Signature/Date: ____________ / ____________
Clinical Research Institution: Wuhan Union Hospital Affiliated to
Huazhong University of Science and Technology

3
Protocol Abstract
Study Name
The Role of Mitochondria and Related Genes in
the Diagnosis and Prognostic Assessment of
Sepsis: A Multicenter, Prospective Cohort Study
Study Introduction
Sepsis is a severe multiple organ dysfunction
syndrome induced by infection, which has become
one of the major challenges to global public health.
Studies have shown that mitochondrial DNA
(mtDNA)
may
play
a
crucial
role
in
the
pathogenesis of sepsis as a damage-associated
molecular pattern (DAMP). mtDNA can promote
the spread of inflammatory responses through its
interaction with the immune system, and may serve
as a potential biomarker for assessing disease
severity and prognosis. A number of clinical
studies have found that plasma mtDNA levels are
significantly elevated in patients with sepsis, which
is associated with high mortality. For example,
some studies have demonstrated that high mtDNA
levels are closely correlated with an increased risk
of death in patients admitted to the intensive care
unit (ICU). In addition, incorporating mtDNA into
clinical
prediction
models
can
significantly
improve their ability to predict patient prognosis.
These studies provide a scientific basis for further
exploring the potential of mtDNA as a biomarker
for sepsis, and contribute to the improvement of

4
early diagnosis and treatment strategies for sepsis.
Research Objective
To clarify and verify the diagnostic value of
mitochondrial-related genes in sepsis and their
value in judging poor prognosis of sepsis patients.
Study Design
This study plans to collect data from patients
diagnosed with sepsis in Hubei Province from
September
2024
to
September
2025,
comprehensively evaluate the role of mitochondrial
genes in the diagnosis and prognosis of sepsis, and
hope to provide insights for clinical diagnosis and
treatment as well as future clinical research.
Total Number of
Enrolled Patients
200 cases
Number of Study
Groups / Control
Groups
100 subjects in the study group, 100 subjects in the
control group
Diagnosis
1. Confirmed or suspected infection;2. Change in
SOFA score ≥2 points
Inclusion Criteria
• Patients with sepsis diagnosed according to
Sepsis 3.0, regardless of ethnicity and gender
• Aged 16 to 90 years old
• Signed written informed consent form
Exclusion Criteria
• Patients over 90 years old or under 16 years old•
Pregnant and lactating women
•
Patients
with
hematological
tumors,
post-
radiotherapy/chemotherapy tumors, and immune
system diseases
•
Patients
with
blood-borne
diseases
(febrile

5
diseases, syphilis, HIV, hepatitis B, hepatitis C)
• Patients who have received systematic in-hospital
treatment for more than 1 week after the diagnosis
of sepsis
• Hemoglobin < 40g/L
• Subjects who participated in other clinical studies
at the time of enrollment or within 3 months before
enrollment
• Refused to sign the written informed consent
form
Study Intervention
No intervention
Evaluation Criteria
Primary Endpoints
Secondary
Endpoints Safety
Evaluation
Primary Endpoints:(1) To explore the guiding
value of mitochondrial-related genes combined
with SOFA score and APACHE Ⅱscore for the
prognosis and survival rate of sepsis patients.(2) To
compare the differences in the area under the
receiver
operating
characteristic
(ROC)
curve
(AUC)
of
mitochondrial-related
genes,
SOFA
score, APACHE Ⅱscore, IL-6, IL-1β, and HMGB1
in predicting death in sepsis patients.Secondary
Endpoints:To compare the correlations between
mitochondrial-related
genes
and
SOFA
score,
APACHE
Ⅱ
score,
IL-6,
IL-1β,
and
HMGB1.Safety Evaluation:1. Safety of sample
collection: Record and evaluate adverse events or
complications occurring during peripheral blood
sample
collection,
including
blood
collection-
related reactions (e.g., local bleeding, infection,

6
etc.).2.
Stability
and
reliability
of
biomarker
detection: Ensure the consistency and reliability of
the
detection
process
of
mitochondrial
gene
expression, and monitor potential problems in the
laboratory
detection
process
(e.g.,
equipment
failure, operational errors, etc.).3. Patient safety
monitoring: Regularly monitor and record the
health status of patients and any study-related
adverse reactions during the study to ensure the
overall safety of the study for patients.
Statistical
Methods：
Sample Size
Analysis Set
Primary
Efficacy Endpoint
Safety
Endpoint
1. Sample Size: Based on the expected effect size
(moderate correlation between mitochondrial gene
expression and patient prognosis, Cohen’s d = 0.5),
we set the significance level at 0.05 and the
statistical power
at 90%.
Using sample
size
calculation formulas and statistical software, it is
determined that 200 samples are required to ensure
sufficient statistical power to detect the effect and
reduce the probability of Type Ⅱerror, thus
providing reliable research results.2. Statistical
Analysis Set: Full Analysis Set (FAS)3. Primary
Efficacy Endpoint: Evaluate the predictive ability
of mitochondrial gene expression on primary
efficacy outcomes (e.g., overall survival [OS]),
especially to analyze whether the expression level
of mitochondrial genes in peripheral blood can
independently predict these clinical outcomes.4.
Safety Endpoint: Refer to the safety evaluation

7
content in the above evaluation criteria
Study Duration
2 years
Subject
Participation Time
2 years
Study
Institution/Location
Multicenter study (Department of Critical Care
Medicine, Wuhan Union Hospital)
Principal Investigator's Information
1. Name and Contact Information: Zhang Jiancheng, Tel: 13554105815
2. Qualifications:(1) Obtained the Physician Qualification Certificate on
December 18, 2013(2) Obtained the Medical Practitioner Certificate on
December 11, 2015, with the practice category of clinical medicine,
practice scope of Critical Care Medicine, and practice location at Union
Hospital Affiliated to Tongji Medical College, Huazhong University of
Science and Technology(3) Obtained the GCP Certificate: Training Time
(August 2021), Class Hours (32), Training Institution (Senior Executive
Training Institute of the National Medical Products Administration),
Training Level (National Level), Training Method (Lectures), Certificate
Issuance Date (August 30, 2021)
Study Flow Chart
1. Enroll patients with sepsis in the General ICU of Union Hospital
Affiliated to Tongji Medical College, Huazhong University of Science
and Technology in accordance with the inclusion and exclusion criteria,
regardless of gender and ethnicity.
2. Collect 5ml of venous blood samples from eligible patients who have
signed the informed consent form and store the samples at low
temperature.
3. Baseline data collection: Collect clinical data including ventilator use,

8
vasoactive drug use, routine blood test, biochemistry, coagulation
function, myocardial enzymes, CRP, PCT, vital signs, length of hospital
stay and clinical outcomes.
4. Laboratory tests: Detect the expression of mitochondrial genes and
inflammatory genes in PBMC and bronchoalveolar lavage fluid by PCR,
ELISA and WB.
5. Follow-up: Record ICU length of stay, ICU mortality, in-hospital
mortality and 28-day mortality.
6. Conduct clinical data collation, laboratory test result analysis and
statistical data analysis.
Abbreviation Table
Abbreviation
Full Name
mtDNA
Mitochondrial
deoxyribonucleic acid
APACHE Ⅱ
Acute Physiology Chronic
Health EvaluationⅡ
SOFA
Sequential
organ
failure
assessment
DAMP
Damage
associated
molecular pattern

9
1. Introduction
1.1 Research Background
1.1.1 General Overview
Sepsis is a life-threatening multiple organ dysfunction caused by the
host's abnormal response to infection, which can lead to various
pathological and biochemical abnormalities in the body. With the
increasing incidence of sepsis, it has become one of the major global
public health problems. According to a 2020 statistical study on sepsis
biomarkers, there are currently 258 sepsis biomarkers, among which 69
are mainly used to evaluate diagnostic value, 100 for prognostic value,
and 89 for both diagnostic and prognostic values. Exploring effective
diagnostic and prognostic biomarkers for sepsis will undoubtedly help us
timely and accurately understand the diagnosis, progression and
prognosis of the disease.
To date, most studies on mitochondria have focused on their role as
organelles responsible for energy production, protein synthesis,
catabolism and cell death. However, there is a growing interest in
mtDNA as a potential damage-associated molecular pattern. Among the
known mitochondrial damage-associated molecular patterns, mtDNA has
received the most attention. A growing body of literature has shown that
mtDNA is not only released in critical illnesses but also can spread
inflammatory responses through its interaction with the immune system.

10
This raises the possibility that mtDNA may serve as a surrogate for
disease severity and even a predictor of mortality in critically ill patients.
Given the function of mtDNA as a DAMP, there is an increasing interest
in using mtDNA as a predictor of disease severity and/or mortality in
critically ill patients. In 2013, Nakahira et al. published one of the first
clinical trials on this topic. In their study, 200 patients in the medical ICU
were enrolled in the main cohort. They found that mtDNA levels were
significantly higher in patients who died within 28 days of ICU admission
than in survivors. In addition, the odds of death within 28 days of ICU
admission increased with the elevation of mtDNA levels. These findings
were replicated in a validation cohort of 243 critically ill patients with
ARDS. Their data also indicated that mtDNA levels may allow clinicians
to distinguish patients with high and low mortality rates, as adding this
value to the clinical model increased the AUC of mortality prediction
from 0.76 to 0.83.
Since 2013, several studies have attempted to replicate or extend the
findings of Nakahira et al. In the past two years alone, five studies have
examined mtDNA in critically ill adults admitted to medical or cardiac
ICUs. In 2015, Bhagirath et al. published a translational study aiming to
clarify the roles of nuclear DNA (nucDNA), mtDNA and bacterial DNA
(bacDNA) in sepsis. The clinical part of their study included measuring
plasma nucDNA and mtDNA levels in 12 consecutive patients admitted

11
to the ICU with severe sepsis. In this cohort, plasma nucDNA and
mtDNA levels were 200-fold and 50-fold higher than those in the healthy
control group, respectively. Recognizing that the quantity and quality of
stimulation are not necessarily correlated, Bhagirath et al. conducted
subsequent in vivo experiments to measure the effects of different levels
of nucDNA, mtDNA and bacDNA on neutrophil viability.
A few months later, Krychtiuk et al. published a prospective
observational study investigating mtDNA in 228 critically ill patients; the
cohort was highly heterogeneous, including critically ill surgical patients
(cardiothoracic) and medical ICU patients (68% of the cohort). They
found that plasma mtDNA levels in critically ill patients were
significantly higher than those in healthy counterparts. In addition, in the
medical population, the highest mtDNA levels were found in patients
with sepsis or cardiogenic shock. In contrast, mtDNA levels in surgical
patients were almost the same as those in the healthy control group.
Although a statistically significant elevation of mtDNA was found, the
researchers did not observe an association between mtDNA and
vasopressor use, mechanical ventilation requirement, renal insufficiency,
or disease severity scores (APACHE Ⅱ, SAPS Ⅱ, SOFA). However, they
did note that the median mtDNA level was significantly higher in patients
who died within 30 days of ICU admission, independent of demographics,
initial diagnosis and APACHE Ⅱscore. They also observed a significant

12
correlation between mtDNA and mortality in medical patients, but not in
surgical patients. Finally, they calculated that the AUC of the ROC curve
for mtDNA and the clinical model (involving APACHE Ⅱand gender)
were 0.6 and 0.79, respectively. When mtDNA was added to the clinical
model, the AUC was further increased to 0.81, which was statistically
significant.
To explore the relationship between plasma nucDNA, plasma mtDNA
and various inflammatory markers (TNF-α, IL-6, IL-8, IL-10 and IL-
1RA), Timmermans et al. conducted a prospective observational study by
collecting samples on days 1, 3, 5, 6, 9, 14, 21 and 28 of ICU admission
in 121 patients with septic shock. Their data showed that nucDNA,
mtDNA and inflammatory cytokine levels in sepsis patients were
significantly elevated compared with the healthy control group, and
remained elevated at all time points.
In summary, sepsis is a severe infection-induced multiple organ
dysfunction syndrome and a major challenge to global public health.
Studies have shown that mtDNA may play an important role in the
pathogenesis of sepsis as a DAMP. mtDNA can promote the spread of
inflammatory responses through its interaction with the immune system
and may serve as a potential biomarker for assessing disease severity and
prognosis. Multiple clinical studies have found that plasma mtDNA levels
are significantly elevated in sepsis patients and associated with high

13
mortality. Therefore, understanding and studying the role of mtDNA in
sepsis not only helps to reveal its pathogenesis, but also provides
potential possibilities for the development of new diagnostic biomarkers
and therapeutic targets.
In conclusion, the study of mitochondrial genes, especially mtDNA, in
the pathogenesis of sepsis has important biological and clinical
significance. In-depth exploration of their role in the regulation of
inflammatory responses is expected to contribute to the development of
early diagnosis, disease assessment and individualized treatment
strategies for sepsis.
1.2 Study Type
Multicenter, prospective cohort study
1.3 Risk/Benefit Assessment
This study involves no clinical intervention. Only 5ml of venous blood
will be collected from patients, with minimal risk. The collected venous
blood will be used for PCR and WB detection, and the results will be fed
back to clinicians in a timely manner to indicate the patient's infection
status, but there is no direct benefit for the enrolled patients from this
study.
1.3.1 Known Potential Risks
Only 5ml of venous blood will be collected from patients in this study,
with minimal risk.

14
1.3.2 Severity of Harm
Only 5ml of venous blood will be collected from patients in this study,
with minimal harm.
1.3.3 Known Potential Benefits
The collected venous blood will be used for PCR and WB detection, and
the results will be fed back to clinicians in a timely manner; there is no
direct benefit for the enrolled patients from this study.
1.3.4 Potential Risk/Benefit Assessment
No potential risks
1.3.5 Discussion
No potential benefits or risks
2. Research Objectives/Endpoints
The study endpoint is reached when the following conditions are met:
complete collection of basic information (e.g., demographics), medical
history, vital signs, laboratory tests, disease severity assessment and other
data at enrollment and 7 days after enrollment, and completion of follow-
up on the survival status of patients at ICU discharge after enrollment.
3. Study Design
3.1 Overall Design
We will strictly enroll sepsis patients in accordance with the inclusion
and exclusion criteria, and divide them into a death group and a survival
group according to their clinical outcomes. We will compare the

15
differences in mitochondrial-related genes, SOFA score and APACHE Ⅱ
score at admission between the two groups; analyze the correlations
between mitochondrial-related genes and SOFA score/APACHE Ⅱscore,
and compare the differences in the AUC of ROC curve of mitochondrial-
related genes in predicting death in sepsis patients. This is a multicenter,
prospective study.
3.2 Sample Size
Based on the expected effect size (moderate correlation between
mitochondrial gene expression and patient prognosis, Cohen’s d = 0.5),
we set the significance level at 0.05 and the statistical power at 90%.
Using sample size calculation formulas and statistical software, it is
determined that 200 samples are required to ensure sufficient statistical
power to detect the effect and reduce the probability of Type Ⅱerror, thus
providing reliable research results.
4. Study Population
4.1 Diagnostic Criteria
Sepsis is diagnosed in accordance with the Sepsis 3.0 diagnostic criteria.
4.2 Inclusion Criteria
• Patients with sepsis diagnosed according to Sepsis 3.0, regardless of
ethnicity and gender
• Aged 16 to 90 years old
• Signed written informed consent form

16
4.3 Exclusion Criteria
• Patients over 90 years old or under 16 years old
• Pregnant and lactating women
• Patients with hematological tumors, post-radiotherapy/chemotherapy
tumors, and immune system diseases
• Patients with blood-borne diseases (febrile diseases, syphilis, HIV,
hepatitis B, hepatitis C)
• Patients who have received systematic in-hospital treatment for more
than 1 week after the diagnosis of sepsis
• Hemoglobin < 40g/L
• Subjects who participated in other clinical studies at the time of
enrollment or within 3 months before enrollment
• Refused to sign the written informed consent form
5. Evaluation
5.1 Primary and Secondary Endpoint/Outcome Evaluation
Primary Objectives
(1) To explore the guiding value of mitochondrial-related genes
combined with SOFA score, APACHE Ⅱscore, IL-6, IL-1β and HMGB1
for the prognosis and survival rate of sepsis patients.
(2) To compare the differences in the AUC of ROC curve of
mitochondrial-related genes, SOFA score, APACHE Ⅱscore, IL-6, IL-1β
and HMGB1 in predicting death in sepsis patients.

17
Secondary Objectives
(1) To compare the correlations between mitochondrial-related genes and
SOFA score, APACHE Ⅱscore, IL-6, IL-1β and HMGB1.
(2) To explore the effect of mitochondrial-related genes combined with
APACHE Ⅱscore and SOFA score on the total hospital stay and
infection-related complications during hospitalization in ICU sepsis
patients.
5.2 Safety Evaluation
Safety of sample collection: Record and evaluate adverse events or
complications occurring during peripheral blood sample collection,
including blood collection-related reactions (e.g., local bleeding, infection,
etc.).
Stability and reliability of biomarker detection: Ensure the consistency
and reliability of the detection process of mitochondrial gene expression,
and monitor potential problems in the laboratory detection process (e.g.,
equipment failure, operational errors, etc.).
Patient safety monitoring: Regularly monitor and record the health status
of patients and any study-related adverse reactions during the study to
ensure the overall safety of the study for patients.
6. Adverse Events and Serious Adverse Events

18
Patients only need to undergo a single 5ml blood collection throughout
the study with minimal risk, and there is no possibility of adverse events
or serious adverse events occurring.
7. Statistical Analysis and Statistical Methods
SPSS 20 software will be used for statistical analysis. Descriptive
statistics will be adopted to summarize the baseline characteristics of
patients. Continuous variables will be described by mean, median,
standard deviation, minimum and maximum values; all categorical
variables will be described by frequency and percentage. All statistical
tests will be two-tailed unless otherwise specified, and a two-tailed P
value < 0.05 will be considered statistically significant. Binary logistic
regression analysis will be used to analyze the correlation between
mortality and various factors.
8. Medical Care and Protection of Subjects
8.1 Assessment of Risks to Subjects in the Study and Risk Disposal
Measures & Contingency Plans
Patients only need to undergo a single 5ml blood collection throughout
the study with minimal risk. Aseptic operation will be strictly performed
before blood collection, and blood infection indicators will be closely
monitored after blood collection.
9. Supporting Documents and Notes
9.1 Privacy Protection

19
To protect patients' privacy, all case report forms, research reports and
study-related communications will use patient code and enrollment
number as identification marks instead of real names. In accordance with
the relevant national and local laws and regulations, investigators shall
allow the sponsor or designated personnel to access relevant medical
records within the permitted scope to verify the data collected in the case
report forms and audit the data collection process. Regulatory authorities
may also request access to all study records. In accordance with the
relevant requirements of the informed consent form, clinical data shall
not be disclosed without the patient's written permission.
9.2 Collection and Use of Specimens and Data
The data used in this study are derived from the hospital's case
management system, which will be exclusively used for this study and
strictly kept confidential.
9.3 Quality Control and Quality Assurance
(1) There will be 1 principal investigator and 3 fixed research team
members, who will strictly implement the clinical trial protocol.
(2) All experimental detection items must adopt the national statutory
measurement units. Experimental test reports must include complete
items, including test date, detection items, test results and their normal
value ranges, and be signed by the relevant personnel.

20
(3) Retained test specimens shall be properly stored. The kits purchased
for laboratory detection must be products with international or domestic
quality assurance, and the detection shall be strictly carried out in
accordance with the kit instructions.
9.4 Data Processing and Record Keeping
9.4.1 Data Collection and Management
Paper case report forms will be used to record clinical data, laboratory
results, various scores, adverse events, follow-up information and other
data. After the completion of paper data collection, all data will be
entered into electronic case report forms, and other members of the
research team will recheck the collected data.
Data collection will be conducted by clinical researchers under the
supervision of the principal investigator, who is responsible for ensuring
the accuracy, completeness and timeliness of the reported data.
9.4.2 Retention of Research Data
All study materials and data, including ethical approval documents,
signed informed consent forms, research protocols and completed case
report forms, will be stored in a locked location in the General ICU of
Union Hospital, and retained for at least 5 years.
9.5 Conflict of Interest Statement
No conflict of interest.
10. References

21
(1) Wilson DF. Oxidative phosphorylation: regulation and role in cellular
and tissue metabolism. J Physiol. 2017;595(23):7023-7038.
(2) Davies KM, Daum B. Role of cryo-ET in membrane bioenergetics
research. Biochem Soc Trans. 2013;41:1227-1234.
(3) Baker N, Patel J, Khacho M. Linking mitochondrial dynamics, cristae
remodeling
and
supercomplex
formation:
how
mitochondrial
structure can regulate bioenergetics [published online ahead of print
June 15, 2019]. Mitochondrion. doi.org/10.1016/j.smito.2019.06.033.
(4) Haddad A, Mohiuddin SS. Biochemistry, citric acid cycle. In:
StatPearls [internet]. Treasure Island, FL: StatPearls Publishing,
StatPearls Publishing LLC; 2019.
(5) Kunji ER, Aleksandrova A, King MS, et al. The transport mechanism
of the mitochondrial ADP/ATP carrier. Biochim Biophys Acta.
2016;1863(10):2379-2393.
(6) Spiegelman BM. Transcriptional control of mitochondrial energy
metabolism through the PGC1 coactivators. Novartis Found Symp.
2007;287:60-63.
(7) Whitaker
RM,
Corum
D,
Beeson
CC,
Schnellmann
RG.
Mitochondrial biogenesis as a pharmacological target: a new
approach to acute and chronic diseases. Annu Rev Pharmacol Toxicol.
2016;56:229-249.
(8) Bravo-Sagua R, Parra V, Lopez-Crisosto C, Diaz P, Quest AF,

22
Lavandero S. Calcium transport and signaling in mitochondria.
Compr Physiol. 2017;7(2):623-634.
(9) Li X, Fang P, Mai J, Choi ET, Wang H, Yang XF. Targeting
mitochondrial
reactive
oxygen
species
as
novel
therapy
for
inflammatory diseases and cancers. J Hematol Oncol. 2013;6:19.
(10)
Shoag J, Arany Z. Regulation of hypoxia-inducible genes by
PGC-1 alpha. Arterioscler Thromb Vasc Biol. 2010;30(4):662-666.
(11)
McBride HM, Neuspiel M, Wasiak S. Mitochondria: more than
just a powerhouse. Curr Biol. 2006;16(14):R551-R560.
(12)
Oh-hama T. Evolutionary consideration on 5-aminolevulinate
synthase in nature. Orig Life Evol Biosph. 1997;27(4):405-412.
(13)
Rossier MF. T channels and steroid biosynthesis: in search of a
link with mitochondria. Cell Calcium. 2006;40(2):155-164.
(14)
Brand
MD.
Mitochondrial
generation
of
superoxide
and
hydrogen peroxide as the source of mitochondrial redox signaling.
Free Radic Biol Med. 2016;100:14-31.
(15)
Moncada S, Erusalimsky JD. Does nitric oxide modulate
mitochondrial energy generation and apoptosis? Nat Rev Mol Cell
Biol. 2002;3(3):214-220.
(16)
Gellerich FN, Trumbeckaite S, Opalka JR, et al. Mitochondrial
dysfunction in sepsis: evidence from bacteraemic baboons and
endotoxaemic rabbits. Biosci Rep. 2002;22(1):99-113.

23
(17)
Adrie C, Bachelet M, Vayssier-Taussat M, et al. Mitochondrial
membrane potential and apoptosis peripheral blood monocytes in
severe human sepsis. Am J Respir Crit Care Med. 2001;164(3):389-
395.
(18)
Callahan LA, Supinski GS. Sepsis induces diaphragm electron
transport chain dysfunction and protein depletion. Am J Respir Crit
Care Med. 2005;172(7):861-868.
(19)
Mantzarlis K, Tsolaki V, Zakynthinos E. Role of oxidative stress
and mitochondrial dysfunction in sepsis and potential therapies. Oxid
Med Cell Longev. 2017;2017:5985209.
(20)
Fakhruddin S, Alanazi W, Jackson KE. Diabetes-induced reactive
oxygen species: mechanism of their generation and role in renal
injury. J Diabetes Res. 2017;2017:8379327.
(21)
Stepien KM, Heaton R, Rankin S, et al. Evidence of oxidative
stress and secondary mitochondrial dysfunction in metabolic and
nonmetabolic disorders. J Clin Med. 2017;6(7):71.
(22)
Zapelini PH, Rezin GT, Cardoso MR, et al. Antioxidant treatment
reverses mitochondrial dysfunction in a sepsis animal model.
Mitochondrion. 2008;8(3):211-218.
(23)
Arulkumaran
N,
Deutschman
CS,
Pinsky
MR,
et
al.
Mitochondrial function in sepsis. Shock. 2016;45(3):271-281.
(24)
Cuzzocrea S, Mazzon E, Di Paola R, et al. A role for nitric

24
oxidemediated peroxynitrite formation in a model of endotoxin-
induced shock. J Pharmacol Exp Ther. 2006;319(1):73-81.
(25)
Boulos
M,
Astiz
ME,
Barua
RS,
Osman
M.
Impaired
mitochondrial function induced by serum from septic shock patients
is attenuated by inhibition of nitric oxide synthase and poly(ADP-
ribose) synthase. Crit Care Med. 2003;31(2):353-358.
(26)
Borutaite V, Budriunaite A, Brown GC. Reversal of nitric oxide-,
peroxynitrite- and S-nitrosothiol-induced inhibition of mitochondrial
respiration or complex I activity by light and thiols. Biochim Biophys
Acta. 2000;1459(2-3):405-412.
(27)
Orrenius
S,
Gogvadze
V,
Zhivotovsky
B.
Calcium
and
mitochondria in the regulation of cell death. Biochem Biophys Res
Commun. 2015;460(1):72-81.
(28)
Sharma P, Sampath H. Mitochondrial DNA integrity: role in
health and disease. Cells. 2019;8(2):100.
(29)
Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial reactive
oxygen species (ROS) and ROS-induced ROS release. Physiol Rev.
2014;94(3):909-950.
(30)
Shadel GS, Horvath TL. Mitochondrial ROS signaling in
organismal homeostasis. Cell. 2015;163(3):560-569.
(31)
Poderoso JJ. The formation of peroxynitrite in the applied
physiology of mitochondrial nitric oxide. Arch Biochem Biophys.

25
2009;484(2):214-220.

Informed Consent Form
Project Name
The Role of Mitochondria and
Related Genes in the Diagnosis
and Prognostic Assessment of
Sepsis:
A
Multicenter,
Prospective Cohort Study
Protocol Number
Mit01
Version Number
V2.0
Version Date
2024-09-18
Principal
Investigator
Zhang Jiancheng
Specialty
Department
Department
of
Critical
Care
Medicine

Dear Patient and Family Members,
You are invited to participate in a clinical study entitled The Role of
Mitochondria and Related Genes in the Diagnosis and Prognostic
Assessment of Sepsis: A Multicenter, Prospective Cohort Study, initiated
by Union Hospital. The following sections describe the research
background, objectives, methods, potential benefits, possible risks or
inconveniences to you during the study, and your rights and interests.
Please read this information carefully before deciding to participate. This
informed consent form is designed to help you make an informed
decision about participation. If you have any questions, please ask the
investigator in charge of the study to ensure you fully understand the
content. Your participation is entirely voluntary. If you agree to take part,
please sign the statement at the end of this form.
This study has been approved by the Ethics Committee of Union Hospital
Affiliated to Huazhong University of Science and Technology.
Research Background
Sepsis is a systemic inflammatory response syndrome caused by severe
infection, which can lead to multiple organ dysfunction and even death in
severe cases. In recent years, increasing research has focused on the role
and importance of mitochondrial genes in the pathogenesis of sepsis.
Mitochondria are vital organelles within cells, primarily responsible for
energy production, cellular signal regulation, and involvement in life

processes such as cell death. Beyond these traditional functions,
mitochondria contain their own DNA, known as mitochondrial DNA
(mtDNA). Under normal conditions, mtDNA stably exists within
mitochondria; however, when cells are damaged or undergo death,
mtDNA can be released outside the cell and act as a damage-associated
molecular pattern (DAMP) signaling molecule.
In the pathophysiological process of sepsis, a large number of
inflammatory mediators are activated and released, triggering abnormal
immune and inflammatory responses in the body. These responses may
cause mitochondrial damage and dysfunction within cells, subsequently
leading to the release of mtDNA. Once released into the bloodstream,
mtDNA acts as a potent immunologically active molecule, promoting the
spread and exacerbation of inflammatory responses by activating
Toll-like receptors and other inflammatory signaling pathways of the
immune system.
Studies have shown that plasma mtDNA levels are typically significantly
elevated in patients with sepsis, and this elevation is closely associated
with disease severity and prognosis. For example, high mtDNA levels in
sepsis patients are linked to higher mortality and more severe organ
damage. Therefore, mtDNA, as a potential biomarker, can assist
clinicians in assessing the severity of a patient's condition and predicting
the likelihood of disease progression. In addition, researchers have found

that mtDNA release is closely associated with the activation of
inflammatory responses and may be involved in regulating the role of the
immune system during sepsis.
Understanding and investigating the role of mtDNA in sepsis not only
helps to reveal its pathogenesis but also provides potential opportunities
for the development of new diagnostic biomarkers and therapeutic targets.
In summary, research on mitochondrial genes, especially mtDNA, in the
pathogenesis of sepsis holds significant biological and clinical importance.
In-depth exploration of their role in the regulation of inflammatory
responses is expected to contribute to the development of early diagnosis,
disease assessment, and individualized treatment strategies for sepsis.
Research Objective
To clarify and verify the diagnostic value of mitochondrial-related genes
in sepsis and their value in determining poor prognosis.
Eligibility for Participation
Inclusion Criteria
Adult patients (≥18 years old) diagnosed with sepsis or septic shock
according to the Sepsis 3.0 criteria.
Exclusion Criteria
1. Patients over 90 years old or under 16 years old;
2. Pregnant and lactating women;

3. Patients with hematological tumors, post-radiotherapy/chemotherapy
tumors, or immune system diseases;
4. Patients with blood-borne diseases (febrile diseases, syphilis, HIV,
hepatitis B, hepatitis C);
5. Patients who have received systematic in-hospital treatment for more than
1 week after a confirmed diagnosis of sepsis;
6. Hemoglobin < 40g/L.
Study Brief
This is a multicenter, prospective study with the objective of clarifying
and verifying the diagnostic value of mitochondrial-related genes in
sepsis and their value in determining poor prognosis. The study requires
only a single collection of 5ml venous blood upon admission to the ICU.
The collected blood will be used for PCR and WB testing, and the results
will be promptly fed back to clinicians to indicate the patient's infection
status and guide clinical treatment.
Biological Sample Collection
A single 5ml venous blood sample will be collected from the patient for
this study.
Detection, Storage and Disposal of Biological Samples
The collected venous blood will be used for PCR and WB testing and
stored for 48 hours. Testing will be conducted at the Experimental Center

of Tongji Medical College, Huazhong University of Science and
Technology, and the results will be promptly fed back to clinicians.
After the completion of the study, the remaining blood samples will not
be used for other related or future research. They will be treated by
high-temperature and high-pressure sterilization and then transferred to a
designated unit for centralized incineration disposal.
Subject's Obligations
The subject shall cooperate with a single venous blood collection for the
study.
Potential Risks of Participation
Only a 5ml venous blood sample will be collected for this study, with
minimal risk.
Potential Benefits of Participation
The collected venous blood will be used for PCR and WB testing, and the
results will be promptly fed back to clinicians to indicate the patient's
infection status and guide clinical treatment.
Management of Study-Related Injuries
Only a single venous blood collection is required upon admission to the
department, with an extremely low risk of injury.
Associated Costs
In addition to routine ICU tests, the costs of PCR, WB and ELISA testing
will be borne by the investigators.

Notification of New Clinical Study Information
The collected venous blood will be used for PCR and WB testing, and the
results will be promptly fed back to clinicians.
Circumstances for Study Termination
After the patient consents to enrollment, only a single venous blood
collection is required. The clinical study will be terminated if the patient
experiences vital sign fluctuations that make blood sample collection
impossible.
Duration of Participation
Patient enrollment and screening will be conducted from September 1,
2024 to September 1, 2025. Case files will be established, the study will
be completed in October 2025, and data statistics will be performed
thereafter.
Sample Size
A total of 200 patients are expected to participate in this study.
Privacy and Confidentiality
Your personal information and health status will be strictly kept
confidential in this study. No information about you will be disclosed to
any third party except authorized regulatory authorities.
Voluntary Participation and Right to Withdraw
You may choose not to participate in this study, or withdraw from the
study at any time by notifying the investigator, without facing

discrimination or retaliation. Your medical treatment and rights will not
be affected in any way.
The investigator may terminate your participation in the study if you
require additional diagnosis/treatment, fail to comply with the study
protocol, or for any other reasonable cause. You may access information
and progress related to the study at any time.
How to Obtain Assistance
If you have questions about trial information, study progress, or subject
rights and interests, or experience any trial-related discomfort or harm,
you may contact the investigator Zhang Jiancheng at 13554105815
(24-hour hotline), or contact the Ethics Committee of our center: Medical
Ethics Committee of Union Hospital Affiliated to Tongji Medical College,
Huazhong University of Science and Technology, Tel: 027-85726375.
Subject's Statement
I have carefully read this informed consent form, have had the
opportunity to ask questions, and all my questions have been answered. I
understand that my participation in this study is voluntary. I may choose
not to participate, or withdraw from the study at any time by notifying the
investigator, without facing discrimination or retaliation, and my medical
treatment and rights will not be affected in any way.

The investigator may terminate my participation in this clinical study if I
require additional diagnosis/treatment, fail to comply with the study
protocol, or for any other reasonable cause.
I voluntarily agree to participate in this clinical study and will receive a
signed copy of this informed consent form.
Subject's Full Name (in block letters): ____________Contact Phone
Number: ____________Subject's Signature: ____________Date: ______
Year ______ Month ______ Day
If the subject is incapable of signing the informed consent form (e.g., due
to lack of capacity) or is a minor, the guardian shall sign on their behalf.
Guardian's Full Name (in block letters): ____________Contact Phone
Number: ____________Guardian's Signature: ____________Date:
______ Year ______ Month ______ DayRelationship with Subject:
____________Reason the Subject Cannot Sign the Informed Consent
Form: ____________
If the subject or their guardian is illiterate, an impartial witness shall sign
the form.
Impartial Witness's Full Name (in block letters): ____________Contact
Phone Number: ____________Impartial Witness's Signature:
____________Date: ______ Year ______ Month ______ Day
Investigator's Statement

I have accurately informed the subject of the content of the informed
consent form, answered all the subject's questions, and the subject has
voluntarily agreed to participate in this clinical study.
Investigator's Full Name (in block letters): ____________Contact Phone
Number: ____________Investigator's Signature: ____________Date:
______ Year ______ Month ______ Day