MARCH9, a potential target for the treatment of acute pancreatitis, inhibits NLRP3 inflammasome-mediated pyroptosis

Purpose: To investigate the regulatory mechanism of membrane associated ring-CH-type finger 9 (MARCH9) in acute pancreatitis (AP). Methods: Fifteen AP patients admitted in Changzhou Second People's Hospital Nanjing, China and fifteen healthy individuals participated in this study. MARCH9 expression in serum samples taken from the subjects was assayed. To establish an AP cell model, rat pancreatic acinar (PA) cells were induced with ceruletide and then transfected with MARCH9 overexpression vector. The MARCH9 level and molecular structure, which are related to pyroptosis and inflammatory cytokines, were determined. Cell survival rate and caspase 1 activity was assessed. Results: MARCH9 was lowly expressed in AP patients’ serum and in ceruletide-induced PA cells. Overexpression of MARCH9 enhanced the survival rate of ceruletide-induced PA cells and reduced the levels of inflammatory cytokines and molecules associated with pyroptosis. MARCH9 overexpression led to a decrease in caspase 1 activity in ceruletide-induced PA cells. Additionally, the overexpression of MARCH9 had a negative regulatory effect on the levels of IL6, p-STAT3/STAT3 and p-JAK2/JAK2 in PA cells induced by ceruletide. Conclusion: Overexpression of MARCH9 suppresses NLRP3-induced pyroptosis in PA cells through the regulation of IL6/JAK/STAT3 pathway, thus offerimg insights for the potential management of AP.


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.
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.

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).

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 × 10 3 cells/well) for 12 h.Each well was mixed with CCK-8 solution (10 µL).One hour later, a microplate 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.

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).

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).

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).

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).

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 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 ceruletideinduced 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.