Iron metabolism abnormalities in autoimmune hemolytic anemia and Jianpishengxue keli can ameliorate hemolysis and improve iron metabolism in AIHA mouse models

Abstract

Objective: Autoimmune hemolytic anemia (AIHA) is rare heterogeneous disorder characterized by red blood cell (RBC) destruction via auto-antibodies, and after RBC is destroyed, proinflammatory danger-associated molecular patterns including extracellular hemoglobin, heme, and iron which causing cell injury. And oxidative stress represents one of the most significant effects of chronic hemolysis. Jianpishengxue keli can improve the symptoms of anemia patients with kidney disease and tumors and are beneficial in promoting recovery from chronic inflammation. Therefore, it is presumed that Jianpishengxue keli can improve the symptoms of AIHA. We aimed to investigate iron metabolism in AIHA and effects of Jianpishengxue keli on AIHA murine model.

Methods: Nineteen hemolytic episode AIHA patients, 10 remission patients and 10 healthy controls (HCs) were enrolled in this study. Serum hepcidin, ferritin and other related indicators of iron metabolism were measured. Mouse models of AIHA were established and received high, medium, or low doses of Jianpishengxue keli by gavage daily for 14 and 28 days respectively. The level of RBCs, Hb, bilirubin, LDH, hepcidin, and the expression level of hepcidin mRNA, and hepatic ferroportin 1(FPN1) protein were evaluated.

Results: Serum hepcidin in hemolytic episode AIHA patients and remission patients were significantly higher than that in HCs (p = 0.0083 and p = 0.0473, respectively). Serum ferritin in hemolytic AIHA patients was significantly higher than that in HCs (p = 0.008). Serum transferrin saturation levels are increased in patients with AIHA[ (57.21 ± 8.96) %]. EPO in hemolytic group was higher than that in healthy control (p＜0.05). In AIHA mouse models, IBIL decreased after 14 days of high dose drug intervention. After 28 days, TBIL and IBIL both significantly decreased in all dose groups and LDH significantly decreased in the medium-and high-dose groups. Body weight improved, and the level of RBCs, Hb and hepcidin in the high-dose group returned to normal. After 14 and 28 days of intervention, hepatic hepcidin mRNA in all dose group significantly decreased. Hepatic FPN1 protein which were significantly lower in the AIHA mouse models, increased in all dose groups after drug intervention for 28 days.

Conclusion: Iron metabolism abnormalities exists in AIHA patients and Jianpishengxue keli can ameliorate hemolysis and improve iron metabolism in AIHA mouse models.KEY MESSAGESIron metabolism abnormalities exists in hemolytic episode AIHA patients. Hepcidin and ferritin levels significantly elevated and also correlated with the severity of AIHA patients. Jianpishengxue keli can ameliorate hemolysis and prompt the recovery of AIHA.

Keywords: Autoimmune hemolytic anemia; Jianpishengxue Keli; hepcidin; immune inflammation; iron metabolic.

Figure 1.

(A) The plasma level of hepcidin for hemolytic episode AIHA patients (28888 ± 4546 pg/ml, p = 0.0083) and remission AIHA patients (19766 ± 3250 pg/ml, p = 0.0473) were both obviously higher than that of normal control (9482 ± 3572 pg/ml). (B) The plasma level of ferritin for hemolytic episode AIHA patients (551.2 ± 130.3 ng/ml, p = 0.008) were significantly higher than that of normal control (56.91 ± 11.56) ng/ml. (C) Transferrin saturation was significantly higher in patients in the hemolytic episode group, [57.21 ± 8.96) %] than in healthy controls[ (30.23 ± 10.2) %, p = 0.013]. (D) EPO levels were higher in both the hemolytic group [(265.7 ± 80.08) mIU/ml] and the hemolysis remission group [(105.2 ± 30.1) mIU/ml, p = 0.021] than in healthy controls[(19.16 ± 5.33) mIU/ml, p = 0.018]. *p<0.05; **p<0.01

Figure 2.

In AIHA patients, the level of IgM in peripheral blood was positively correlated with the plasma level of hepcidin (r = 0.5505, p = 0.0097) and the plasma level of TBIL, DBIL and CRP were positively correlated with the level of ferritin, respectively (r = 0.4113, p = 0.0330; r = 0.4338, p = 0.0238; r = 0.6270, p = 0.024). In AIHA patients, the level of hepcidin was positively correlated with the ferritin (r = 0.1546, p = 0.042), and but not with Hb, Ret, FHb and HP (p > 0.05).

Figure 3.

The expression of hepatic FPN1 protein. Hepatic FPN1 protein (62 kDa) were significantly lower in mouse models of AIHA than in the normal controls, but these increased in the models given any of the three doses of Jianpishengxue keli for 14 and 28 days. *p < 0.05.