Objective To explore the therapeutic role of dihydroartemisinin on MRL/lpr lupus mice autoimmunity and its regulatory effect on the differentiation of T follicular helper cells (TFH).

Methods 12 female MRL/lpr lupus mice (12 weeks old) were randomly divided into dihydroartemisinin treatment group and vehicle treatment group. They were then intragastric-administered with 150 mg/kg dihydroartemisinin and PBS solution once a day for 4 weeks, respectively. At the end of the treatment, urine was collected and assayed to detect the level of 24-hour urine protein by Coomassie blue staining. At the age of sixteen weeks, the MRL/lpr mice were then sacrificed and the kidneys were resected and stained with hematoxylin and eosin (H-E) staining for the kidney impairment evaluation. Serum anti-ds-DNA antibody and antinuclear antibody (ANA) level were measured by ELISA. Additionally, the spleens were also resected and then weighed. The percentage of TFH cells in the mononuclear cells isolated from the spleens were analyzed by flow cytometry. Naive CD4⁺ T cells were isolated from the spleens of vehicle group by naive CD4⁺ T cell isolation kit for in vitro experiments. For TFH differentiation, sorted naive CD4⁺ T cells were cultured with 10 ng/mL IL-21, 20 ng/mL IL-6 and reagents of T cell activation/expansion kit with various dose of dihydroartemisinin for 5 d (0, 0.1, 1.0, 10 μM). Flow cytometry was performed to determine the percentage of TFH cells. The concentration of IL-21 secreted by TFH cells in cell culture was detected by Il-21 ELISA kit.

Results The dihydroartemisinin group (1 536±150.1) μg/24 h showed reduced 24-hour protein in urine level compared with the vehicle group (2 548±93.30) μg/24 h, P < 0.01. Dihydroartemisinin treatment reduced the renal pathological scores compared with vehicle group (2.410±0.535) vs (5.713±0.662), P < 0.01. Dihydroartemisinin treatment significantly reduced serum anti-ds-DNA antibody (345.3±29.00 vs 477.8±20.07IU/mL, t＝7.514, P < 0.01) and ANA (514.5±19.64 vs 726.3±16.27 IU/mL, t＝8.302, P < 0.01) titers; and the treatment significantly decreased the proportion of TFH cells from the spleens (12.40%±2.726%) compared with vehicle group (36.33%±2.750%), P < 0.01. The study further demonstrated that dihydroartemisinin could dose-dependent inhibit TFH cells differentiation. Along with the increase of dihydroartemisinin concentrations (0, 0.1, 1.0, 10 μM), there were gradually decreasing trends in the proportion of TFH cells (29.43%±1.94%, 21.07%±1.19%, 11.27%±1.40%, 6.28%±1.02%, respectively, P < 0.01), as well as the concentration of IL-21 (87.07±5.49, 63.63±3.70, 47.67±4.02, 34.37±5.10 pg/mL, respectively, P < 0.01).

Conclusion Dihydroartemisinin is an effective drug for the treatment of lupus autoimmunity, can ameliorate lupus nephritis and reduce autoimmune-antibody titers, which may be related to the inhibition of TFH cells differentiation.