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ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 5 

 

CTMJ | traditionalmedicinejournals.com  

 

 

A RARE INSULMAME-MEDIATED PYROPTOSIS CURE THAT 

MAY BE USED TO ADDRESS ACUTE PANCREATITIS 

Dr.Aneela 
1
, Dr.Y.Sirisha 

2
,Adithyamathur 

3
,Ashraful alom 

4,
 

Assistant professor 1,2,3,4, 
Department of Pharmacy, 

Samskruti College of Pharmacy, 
Kondapur (V), Ghatkesar (M) Medchal Dist, Telangana, India. 

 

ABSTRACT 

This study aims to examine the role of membrane-associated ring-CH-type finger 9 (MARCH9) in the regulation of 

acute pancreatitis (AP). Methods: Ten healthy persons and fifteen AP patients hospitalized to Changzhou Second 

People's Hospital Nanjing, China, took part in the research. We tested the individuals' serum samples for MARCH9 

expression. Inducing rat pancreatic acinar (PA) cells with ceruletide and then transfecting them with a MARCH9 

overexpression vector allowed for the establishment of an AP cell model. We found the molecular structure and 

level of MARCH9, which are associated with pyroptosis and inflammatory cytokines. Caspases 1 activity and cell 

survival rate were measured. The results showed that both the serum of AP patients and ceruletide-induced PA 

cells have low levels of MARCH9. The survival rate of ceruletide-induced PA cells was boosted and the levels of 

inflammatory cytokines and components associated with pyroptosis were lowered when MARCH9 was 

overexpressed. In ceruletide-induced PA cells, caspase 1 activity was shown to be decreased when MARCH9 was 

overexpressed. Furthermore, when ceruletide was used to elevate PA cell levels of IL6, p-STAT3/STAT3, and p-

JAK2/JAK2, the overexpression of MARCH9 exhibited a negative regulatory impact. Results: MARCH9 

overexpression inhibits NLRP3-induced pyroptosis in PA cells via controlling the IL6/JAK/STAT3 pathway; this 

finding may have implications for the treatment of AP. Pyroptosis, acute pancreatitis, AR42J cells, Ceruletide. 

 

 

 INTRODUCTION  

Acute pancreatitis (AP) is an acute abdominal disease 

and the mortality rate can reach 47 to 69 % [1]. 

Blood amylase and lipase are significantly increased 

in AP patients, which can also be accompanied by an 

increase in blood sugar [2]. Systemic inflammation 

brings a second strike to the patients by increasing 

the burden on organs and exacerbating the severity of 

AP [3]. Therefore, seeking effective treatment for AP 

is currently a hot topic in research. Pyroptosis is a 

form of cell death involving members of the caspase 

family [4,5]. The release of pro-inflammatory 

cytokines induced by activating NLRP3 

inflammatome is one of the causes of pyroptosis. [6]. 

For instance, the activation of receptor-associated 

factor 6 induces pyroptosis through the caspase 1/3 

signaling pathway, thereby contributing to the 

progression of AP [7]. Therefore, inhibiting the 

occurrence of pancreatic cell pyroptosis is one of the 

ways to alleviate AP. Membrane associated ring-CH-

type finger (MARCH) includes 11 family members, 

from MARCH1 to MARCH11 and they play a role in 

immune regulation, cell polarity, and toll like 

receptor signal transduction [8]. MARCH9, a 

member of the MARCH protein family, may be 

related to cellular immune regulation [9]. In addition, 

it was recently shown that MARCH9 regulates the 

pancreatic cells pyroptosis induced by NLRP3 

inflammasome in AP [10]. This indicates that 

MARCH9 participates in the regulation of AP 



Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 5 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 5 

 

CTMJ | traditionalmedicinejournals.com  

 

occurrence. The study of MARCH9 in AP is still 

however immature.  

The potential function and possible mechanisms of 

MARCH9 in AP was discussed in the current study, 

with the intent being to provide new ideas for the 

treatment of AP.  

METHODS  

Clinical samples  

Venous blood (10 ml) were collected from patients 

with AP admitted in Changzhou Second People's 

Hospital Nanjing, China (AP group, n = 15, 6 

females and 9 males) and from 15 healthy individuals 

(normal group, n = 15, 8 females and 7 males) were 

collected. Healthy individuals were those who 

participate in physical examinations in outpatient 

clinics. AP patients were hospitalized and did not 

suffer from any disease other than AP. Serum was 

separated from blood sample and kept at - 80 °C. All 

participants signed informed consent. The study was 

approved by the Ethics Committee of The Affiliated 

Changzhou No. 2 People's Hospital with Nanjing 

Medical University (approval no. [2023]KY106-01) 

and complied with guidelines of World Medical 

Association Declaration of Helsinki [11].  

The amylase and lipase levels in the patient's serum 

were assayed using an amylase assessment kit and a 

lipase assessment kit. All kits were purchased from 

Nanjing Jiancheng Bioengineering Institute (Nanjing, 

Jiangsu, China).  

Cells culture  

Rat PA cells (AR42J cells, ATCC, Manassas, VA, 

USA) were cultured in DMEM medium (5 % CO2, 

37 °C). Streptomycin (100 μg/ml), fetal bovine serum 

(10 %) and penicillin (100 U/ml) (all reagents: Gibco, 

Waltham, MA, USA) were contained in the culture 

medium. AR42J cells were separated into ceruletide, 

control, ceruletide + NC, and ceruletide + MARCH9 

groups. To establish an AP cell model, AR42J cells 

were cultivated in the medium complemented with 

ceruletide (100 nmol/L, MCE, China) for 24 h. 

MARCH9 overexpression vector (MARCH9) and the 

negative control (NC) were got from GenePharma 

(Shanghai, China). The cells were harvested 24 h 

after transfecting by Lipofectamine 2000.  

Western blotting and RNA extraction and 

quantitative real time-PCR (qRT-PCR)  

The western blotting and qRT-PCR procedures are 

based on previous research. The relative expressions 

of interleukin-6 (IL-6), MARCH9, tumor necrosis 

factor α (TNF-α) and IL-1β were measured,and their 

primers sequences were listed in Table 1. The 

primary antibodies (Abcam) are as below: toll-like 

receptor 4 (TLR4, 1:1000), MARCH9 (1:1000), P65 

(1:500), NLRP3 (1:1000), p-P65 (1:500), IL-1β 

(1:1000), gasdermin D (1:1000), IL-6 (1:1000), 

caspase 1 (1:1000), IL-18 (1:1000), Janus kinase 2 

(JAK2, 1:5000), signal transducer and activator of 

transcription 3 (STAT3, 1:1000), p-JAK2 (1:500), p-

STAT3 (1:1000) and GAPDH 

(1:2000).

 

Immunofluorescence (IF)  

AR42J cells were fixed with paraformaldehyde 

solution (4 %, Sigma Aldrich, Darmstadt, Germany) 

and then blocked with PBS, Triton (0.3 %) and 

normal goat serum (5 %) for 1 hour. 

Samples were cultivated (4 °C) overnight with an 

anti-MARCH9 primary antibody (Abcam) diluted at 

a ratio of 1:100. After that, samples were cultivated 

with a cy3-conjugated secondary antibody (Abcam) 

diluted at a ratio of 1:500 and DAPI staining (1:1000, 

Sigma Aldrich) conducted for one hour at room 

temperature. The images were collected by a 

fluorescence microscope (UY203i; Aopu, 

Chongqing, Sichuan, China).  

Cell counting kit‐8 (CCK‐8) assay  

The cell survival rate was evaluated by CCK-8 

method and then planted in 96-well plates (1 × 103 

cells/well) for 12 h. Each well was mixed with 

CCK‐8 solution (10 μL). One hour later, a microplate 



Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 5 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 5 

 

CTMJ | traditionalmedicinejournals.com  

 

reader (A51119500C; Thermo Fisher) was used to 

read the absorbance at 450 nm.  

Determination of LDH level  

Lactic dehydrogenase (LDH) levels in the AR42J 

cells were determined using LDH assay kit (Sigma 

Aldrich).  

Flow cytometry  

To test pyroptosis in the AR42J cells, caspase 1 

detection kit (Beyotime, Shanghai, China) measured 

the active levels of caspase 1. The active caspase 1 

enzyme was labeled with FAM-YVAD-FMK and 

determined using flow cytometry. Pyroptosis was 

defined as being double positive for propidium iodide 

(PI) staining and FAM-YVAD-FMK.  

Statistical analysis  

The data was analyzed through SPSS 20.0 (Chicago, 

IL, USA) and displayed as SD. The statistical 

significance is assessed by one-way-ANOVA 

followed by Tukey post hoc test, t-test. P < 0.05 was 

deemed statistically significant. All assays were 

repeated as independent experiments in at least 

triplicate.  

RESULTS  

Low expression of serum MARCH9 in AP patients 

and AP cell models  

Serum samples from AP patients were collected and 

MARCH9 expression was determined. Figure 1 A 

demonstrated that AP patients showed higher levels 

of serum amylase and lipase. qRT-RCR and western 

blotting results revealed that MARCH9 was lowly 

expressed in serum from AP patients (Figure 1 B and 

C). Moreover, AP cell models were established using 

ceruletide and it was found that MARCH9 expression 

decreased in the ceruletide group (Figure 1 D and E). 

 

Figure 1: MARCH9 expression in serum of AP 

patients and AP cell models. (A) Levels of serum 

amylase and lipase. B) MARCH9 mRNA expression. 

(C) MARCH9 protein level. (D) MARCH9 mRNA 

expression. (E) MARCH9 protein expression. ***P < 

0.05, vs normal or control  

Overexpression of MARCH9 enhanced the 

activity of AR42J cells induced by ceruletide  

Here, AP cell models were transfected with 

MARCH9 overexpression vector. The results showed 

that MARCH9 level increased in AR42J cells 

induced by ceruletide after transfection with 

MARCH9 overexpression vector (Figure 2 A). In the 

ceruletide + MARCH9 group, there was a remarkable 

increase in cell survival rate (Figure 2 B), and a rapid 

reduction in LDH levels (Figure 2 C). 

 

Figure 2: The role of MARCH9 overexpressed in 

activity of AR42J cells induced by ceruletide. AR42J 

cells were induced with ceruletide and then 

transfected with MARCH9 overexpression vector. 

(A) Immunofluorescence (IF) images. (B) Cell 

survival rate. (C) the levels of LDH. ***P < 0.05, vs 

control; ##p < 0.05, vs ceruletide + NC Over-

expression of MARCH9 inhibited inflammation in 

AR42J cells induced by ceruletide  

As shown in Figure 3 A, the levels of inflammatory 

cytokinesin ceruletide + MARCH9 group were 

remarkably lower than ceruletide + NC group (p < 

0.05). TLR4 and p-P65/P65 expressions were down-

regulated in AR42J cells induced by ceruletide after 

transfection with MARCH9 overexpression vector 

(Figure 3 B). 



Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 5 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 5 

 

CTMJ | traditionalmedicinejournals.com  

 

 

Figure 3: Effect of MARCH9 overexpressed in 

inflammation in AR42J cells induced by ceruletide. 

(A) TNF-α, IL-6 and IL-1β levels. (B) TLR4 and p-

P65/P65 expressions. ***P < 0.05 or **p < 0.05, vs 

control; ##p < 0.05 or ###p < 0.05, vs ceruletide + 

NC  

Overexpression of MARCH9 inhibited the 

pyroptosis of AR42J cells induced by ceruletide  

Western blotting results revealed that MARCH9 

down-regulated the expressions of NLRP3, GSDMD-

N, caspase 1, IL-1β and IL-18 in AR42J cells induced 

by ceruletide (Figure 4 A). As shown in Figure 4 B, 

the overexpression of MARCH9 regulated the 

activity of caspase 1 in ceruletide + MARCH9 group, 

compared with ceruletide + NC group.  

Over-expression of MARCH9 inhibited IL-

6/JAK/SATA3 pathway  

To further investigate the regulatory mechanism of 

MARCH9 in the pyroptosis of AR42J cells induced 

by ceruletide, the expression of related proteins was 

determined using western blotting. The results 

showed that MARCH9 overexpression repressed the 

expressions of IL-6, p-JAK2/JAK2 and p-

STAT3/STAT3 in AR42J cells induced by ceruletide 

(Figure 5). 

 

Figure 4: Effect of MARCH9 overexpressed in the 

pyroptosis of AR42J cells induced by ceruletide. (A) 

Expressions of NLRP3, GSDMD-N, caspase 1, IL-1β 

and IL-18. (B) Activity of caspase 1. P < 0.05, vs 

control; ##p < 0.05, vs ceruletide + NC 

 

Figure 5: Effect of MARCH9 overexpressed in IL-

6/JAK/SATA3 levels. Expressions of IL-6, p-

JAK2/JAK2, and p-STAT3/STAT3. ***P < 0.05, vs 

control; ###p < 0.05 or ##p < 0.05, vs ceruletide + 

NC  

DISCUSSION  

Recent studies showed that inhibiting pyroptosis 

alleviates pancreatic injury and significantly affects 

the progression of AP [12]. This study showed that 

MARCH9 expression is downregulated in AP 

patients' serum and has the ability to regulate 

pyroptosis in PA cells.  

Research has shown that MARCH9 participates in 

cancer progression. In lung adenocarcinoma, the 

overexpression of MARCH9 attenuates the 

carcinogenic effect of intercellular adhesion molecule 

1 (ICAM-1) [13]. Furthermore, studies have found 

that MARCH9 is related to immune regulation in the 



Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 5 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 5 

 

CTMJ | traditionalmedicinejournals.com  

 

bod [9], and participates in innate immunity by 

catalyzing the polyubiquitination of key immune 

factors [14]. Another recent study found that 

MARCH9 was related to the occurrence of AP and 

the overexpression of MARCH9 inhibits the NLRP3 

inflammasome-dependent pancreatic cell pyroptosis 

by mediating the ubiquitination of NADPH oxidase-

2, which may regulate the progression of AP [10]. 

The NLRP3 inflammasome is involved in regulating 

cellular inflammatory responses through the 

modulation of downstream pro-inflammatory factors 

[15]. One way to effectively mitigate these 

inflammatory responses is to target NLRP3 [16]. In 

addition, NLRP3 is also closely related to the 

occurrence of pyroptosis. For example, isoliquiritin 

ameliorates depression symptoms by suppressing 

miRNA-27a-mediated pyroptosis induced via NLRP3 

inhibition [17]. Furthermore, cisplatin activates 

NLRP3/caspase 1 pathway, contributing to trigger 

pyroptosis in triple-negative breast cancer cells [18].  

In the present study, MARCH9 was lowly expressed 

in the serum of AP patients, which suggests that 

MARCH9 was related to the development of AP. To 

further explore the role of MARCH9 in AP, an in 

vitro AP cell model was established by inducing PA 

cells with ceruletide and investigated for its effect on 

PA cells pyroptosis by overexpressing MARCH9. As 

expected, the upregulation of MARCH9 suppressed 

the inflammation in ceruletide-induced PA cells, as 

well as NLRP3 inflammasome-mediated pyroptosis, 

leading to enhanced viability of the PA cells. In 

addition, this study revealed a potential association 

between MARCH9 and the IL6/JAK/STAT3 

pathway. However, this study is only a preliminary 

worK at the cellular level and has not been validated 

in animal models. We will supplement this in the 

follow-up study.  

CONCLUSION  

The results confirm that MARCH9 is low expressed 

in AP patients and its overexpression contributes to 

the inhibition of NLRP3-induced pyroptosis in PA 

cells by regulating IL6/JAK/STAT3 pathway. Thus, 

MARCH9 is a potential biomarker for AP treatment. 

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