Targeting PAD4: A Promising Strategy to Combat β-Cell Loss in Type 1 Diabetes

Abstract

Peptidylarginine deiminase 4 (PAD4) catalyzes protein citrullination, a post-translational modification implicated in type 1 diabetes mellitus (T1DM). This study examined PAD4 expression and activity in the pancreas of streptozotocin (STZ)-induced diabetic Wistar rats. Animals were divided into three groups: (A) STZ-induced diabetic rats (60 mg/kg, i.p.), (B) non-diabetic controls, and (C) diabetic rats treated with Cl-amidine (5 mg/kg), a pan-PAD inhibitor, from week six post-induction. Analyses included PAD4 mRNA and protein expression, citrullinated histone H3 (CitH3), calcium concentration, and neutrophil elastase activity. Diabetic rats exhibited increased PAD4 expression, CitH3 levels, and NETosis markers, alongside reduced pancreatic calcium, suggesting calcium consumption during PAD4 activation. Cl-amidine treatment attenuated NETosis. These results implicate PAD4 in T1DM pathogenesis via NETosis and support the utility of STZ-induced diabetic rats as a model for PAD4-targeted studies. Cl-amidine may represent a promising therapeutic approach to reduce pancreatic inflammation in T1DM.

Keywords: PAD4; STZ; Wistar rat.

Figure 1

The expression of TNF- α in the plasma, liver, and pancreas. According to the comparison, over the first six weeks, STZ may only damage DNA in the pancreas through GLUT2. Neither the liver nor the other organs were damaged. (n = 10), * p < 0.05.

Figure 2

PAD4 mRNA levels in the pancreas. Comparison of PAD4 mRNA expression between control and T1DM groups. * p < 0.05. Results are presented as mean ± S.E.M. (Control, n = 7; T1DM, n = 10).

Figure 3

(A) PAD4 protein expression in the pancreas. PAD4 protein level between control and T1DM groups against internal control (Beta-actin); PAD4 expression in the T1DM group was greater than in the control in the pancreas (* p < 0.05). Results are presented as mean ± S.E.M. (Control, n = 7; T1DM, n = 10) (B) CITH3 levels in the pancreas. Change in the expression of citrullinated histone-3 (CITH3) between control and T1DM groups. The result indicated the successful induction of T1DM according to the citrullinated level (* p < 0.05). Results are presented as mean ± S.E.M. (Control, n = 8; T1DM, n = 6).

Figure 4

Ca²⁺ content in the pancreas. Change in the expression of calcium ion between control and T1DM groups; Ca²⁺ is a cofactor for PAD4 and our measurement presents a profound impact on the pancreas between control and T1DM groups (* p < 0.05). Results are presented as mean ± S.E.M. (Control, n = 5; T1DM, n = 6).

Figure 5

Blood glucose level before termination. (A) In the 6th week, by confirming blood glucose levels, T1DM was successfully induced. (B) In experiment B, we used a PAD inhibitor (PADI) to further examine PAD4 expression. After ten days of dosing, we also checked the blood glucose level. *** p < 0.001 (all groups, n = 10).

Figure 6

The expression of PAD4 mRNA level in the pancreas in exp B. The relative mRNA level is significant between vehicle–T1DM and PADI-T1DM (* p < 0.05, all groups n = 10).

Figure 7

NETosis level in the serum and pancreas in exp B. A component of extracellular trap formation is neutrophil elastase (NE). We employed Cl-amidine, one of the PAD inhibitors (PADI). * p < 0.05, *** p < 0.001, NS: non-significant (A) vehicle–control, n = 5; PADI–control, n = 7; vehicle–TIDM, n = 5; PADI-T1DM, n = 8; (B) vehicle–control, n = 5; PADI-control, n = 7; vehicle–TIDM, n = 5; PADI-T1DM, n = 7.

Figure 8

Experiment A and B designs (exp A and exp B). (A) Verification that the STZ dosage of 60 mg/kg was feasible for creating diabetic rats. (B) Cl-amidine, a PAD inhibitor, is frequently used to study PAD4 expression in mice. Here, we also evaluated the expression of PAD4 in rats by using Cl-amidine.