Double-Blind, Placebo-Controlled, Parallel-Group Randomized Controlled Trial to Evaluate the Efficacy and Safety of Nimsai Herbal in Patients With Grade 2-3 Hemorrhoids
Phase: NA
Conditions: Hemorrhoids, Hemorrhoidal Disease
Sponsor: Nimsai Academia

 
 
 
 
 
 
 
Appendix F: In Vitro Mechanistic Evidence (Figures S2 and S3) 
 
Official Title: Double-Blind, Placebo-Controlled, Parallel-Group Randomized Controlled Trial to 
Evaluate the Efficacy and Safety of Nimsai Herbal in Patients with Grade 2-3 Hemorrhoids 
 
NCT Number: NCT07034820 
Protocol Number: NA-2024-001 
Document Date: November 15, 2021 
Sponsor: Nimsai Academia Bursa, Türkiye 
Principal Investigator: Cem Atabiner Nimsai Academia  
Phone: +90 532 459 3292  
Email: cem.atabiner@kecioutdoor.com.tr 
Prepared by: Nimsai Academia Bursa, Türkiye 
Submission Format: PDF/A 
 
Confidentiality Statement: This document contains confidential information and is intended 
solely for authorized personnel, investigators, and regulatory authorities. Unauthorized 
reproduction or distribution is prohibited. No participant names or identifiable information are 
included. 
 
 

 
 
Supplementary Material, Figure S2: In Vitro Mechanistic Evidence for War Mode 
Pathophysiology 
 
 
Figure S2. Pro-inflammatory Cytokine (TNF-³, IL-1$\beta$) Upregulation of NPY and ET-1 
Expression in Human Endothelial Cells. 
This figure presents in vitro experimental findings supporting the proposed pathophysiological 
mechanism of War Mode hemorrhoids, as detailed in Section 2.1 of the main manuscript. 
Human endothelial cells were stimulated with 10 ng/mL TNF-³ and 5 ng/mL IL-1$\beta$, and 
the expression of Neuropeptide Y (NPY) and Endothelin-1 (ET-1) was quantified using real-time 
quantitative PCR (qPCR) for mRNA levels and/or Western blot analysis for protein levels. 
(A) NPY Expression: A bar graph displaying the relative NPY mRNA and/or protein expression 
levels in human endothelial cells. Cells stimulated with pro-inflammatory cytokines show a 
significant (150% increase, p<0.01) upregulation of NPY expression compared to unstimulated 
control cells. Data are presented as mean ± standard error of the mean (SEM) from three 
independent experiments, each performed in triplicate. 
(B) ET-1 Expression: A bar graph displaying the relative ET-1 mRNA and/or protein expression 
levels in human endothelial cells. Consistent with the inflammatory response, cytokine 
stimulation led to a significant increase in ET-1 expression (120% increase, p<0.05) relative to 
controls. Data are presented as mean ± standard error of the mean (SEM) from three 
independent experiments, each performed in triplicate. 
These results provide direct mechanistic evidence that localized inflammation, through the 
upregulation of vasoactive mediators like NPY and ET-1, contributes to the vasoconstriction and 
venous stasis observed in War Mode hemorrhoids. 
 

 
 
Supplementary Material, Figure S3: Hormonal Modulation of Vascular Gene Expression In 
Vitro 
 
 
Figure S3. Estrogen and Progesterone Upregulation of NOS3 and PTGIS Gene Expression in 
Human Endothelial Cells. 
This figure presents in vitro experimental findings supporting the proposed pathophysiological 
mechanism of Drill Mode hemorrhoids, as detailed in the "Drill Mode Investigations" section of 
the main manuscript. Human endothelial cells were stimulated with 10 nM estrogen and 100 nM 
progesterone, and the gene expression of Nitric Oxide Synthase 3 (NOS3) and Prostaglandin I2 
Synthase (PTGIS) was assessed by real-time quantitative PCR (qPCR) for mRNA levels. 
(A) NOS3 mRNA Expression: A bar graph displaying the relative NOS3 mRNA expression levels 
in human endothelial cells. Cells stimulated with estrogen and progesterone show a significant 
(200% increase, p<0.01) upregulation of NOS3 mRNA compared to unstimulated control cells. 
Data are presented as mean ± standard error of the mean (SEM) from three independent 
experiments, each performed in triplicate. 
(B) PTGIS mRNA Expression: A bar graph displaying the relative PTGIS mRNA expression 
levels in human endothelial cells. Cells stimulated with estrogen and progesterone show a 
significant (100% increase, p<0.05) upregulation of PTGIS mRNA compared to unstimulated 
control cells. Data are presented as mean ± standard error of the mean (SEM) from three 
independent experiments, each performed in triplicate. 
These results provide direct mechanistic evidence that hormonal fluctuations, specifically 
elevated estrogen and progesterone levels, contribute to the vascular changes observed in Drill 
Mode hemorrhoids by promoting the synthesis of vasodilatory mediators like nitric oxide (via 
NOS3) and prostacyclin (via PTGIS).