The effectiveness of ruxolitinib and cyclophosphamide combination on T helper 17 and regulatory T cells in rat experimental membranous glomerulonephritis

Abstract

The progression and pathogenesis of membranous glomerulonephritis (MGN) are inextricably linked to chronic inflammation. Despite improving clinical remission rates due to the application of cyclophosphamide (CYC), treatment of MGN still requires further exploration. Ruxolitinib (Ruxo) negatively affects the signaling pathways participating in the production of pro-inflammatory cytokines. Hence, we investigated whether the combination of CYC and Ruxo can modulate inflammation through influencing T helper 17 (Th17) lineages and regulatory T cells (Tregs). Passive Heymann nephritis (PHN), an experimental model of MGN, was induced in a population of rats. Then, the animals were divided into five groups: PHN, CYC-receiving, Ruxo-receiving, CYC-Ruxo-receiving PHN rats, and healthy controls. After 28 days of treatment, biochemistry analysis was performed and splenocytes were isolated for flowcytometry investigation of Th17 cells and Tregs. The correlative transcription factors of the cells, alongside their downstream cytokine gene expressions, were also assessed using real-time PCR. Furthermore, serum cytokine signatures for the lymphocytes were determined through ELISA. The combination of CYC and Ruxo significantly reduced the serum values of urea in rats versus the PHN group (24.62 ± 7.970 vs. 40.60 ± 10.81 mg/dL). In contrast to Treg's activities, the functionality of Th17 cells noticeably increased not only in PHN rats but also in CYC or Ruxo-receiving PHN animals when compared with the control (10.60 ± 2.236, 8.800 ± 1.465, 8.680 ± 1.314 vs. 4.420 ± 1.551 %). However, in comparison to the PHN group, the incidence of Th17 cells notably fell in rats receiving CYC and Ruxo (10.60 ± 2.236 vs. 6.000 ± 1.373 %) in favor of the Treg's percentage (5.020 ± 1.761 vs. 8.980 ± 1.178 %), which was verified by the gene expressions and cytokine productions correlative to these lymphocytes. The combination of CYC and Ruxo was able to decline Th17 cells in favor of Tregs improvement in PHN rats, suggesting an innovative combination therapy in MGN treatment approaches.