Piperine reduces spleen lymphocytes proliferation by inhibiting DHODH
Human DHODH is a critical enzyme for de novo pyrimidine synthesis, and it has been pharmacological targeted for the therapy of autoimmune diseases with self-reactive T lymphocytes(Goodnow, 2007). Multiple sclerosis is an immune-mediated disease and blocking autoreactive T cells is considered to be a promising strategy(Jelcic et al., 2018; Löffler, Fairbanks, Zameitat, Marinaki & Simmonds, 2005). To test the immunosuppressive effects of piperine, we investigate the anti-proliferative potential of piperine by using concanavalin A (Con A)-stimulated mouse spleen lymphocytes expansion (Figure 4A). Known DHODH inhibitor A771726 was used as a positive control. As shown in Figure 4B, piperine markedly impairs ConA-induced T lymphocytes proliferation in a dose-dependent manner. Considering that DHODH is the rate-limiting enzyme in the endogenous biosynthesis of uridine monophosphate, we asked whether uridine treatment could rescue piperine-induced anti-proliferation on T lymphocytes. As speculated, uridine treatment completely eliminates piperine’s inhibition capacity on ConA-induced lymphocytes proliferation (Figure 4B). The mixed lymphocyte reaction (MLR) is a classical model for T-cell reactivity. Similarly, we found that piperine exhibits strong suppressive activities on MLR in a dose-dependent manner, while uridine supplement significantly recovers lymphocytes proliferation (Figure 4C-D).
Then, we turn to investigate whether cellular DHODH activity is affected by piperine. Cultured Jurkat T cells were treated with piperine and cellular orotic acid was quantified by a fluorogenic probe 4-trifluoromethyl-benzamidoxime(Yin, Kabashima, Zhu, Shibata & Kai, 2017) (Figure 4E). As shown in Figure 4F, piperine treatment significantly reduces DHODH activity of Jurkat T cells in a dose-dependent manner with an IC50 value of 0.75 μM . Taken together, these results suggest that piperine reduces lymphocytes proliferation via directly inhibiting DHODH.
Piperine reduces clinical score and severity of EAE
MS is an inflammatory and autoimmune disease. We next sought to determine whether pharmacological inhibition of DHODH by piperine would attenuate experimental autoimmune encephalomyelitis (EAE), a valuable model for human MS(Dendrou, Fugger & Friese, 2015b). For EAE induction, C57BL/6 mice were immunized with MOG35-55 followed by pertussis toxin (PTX) administration (Figure 5A). EAE mice was administered daily i.p. injections with vehicle, A771726, or piperine (10 or 30 mg/kg) from day 7 after MOG’s induction and clinical sign was evaluated using a 5-point scoring system. As shown in Figure 5B, therapy with piperine suppresses the manifestation of clinical symptoms over the course of the study. Meanwhile, the lower sum of scores for each individual mouse indicated a reduced disease burden for most mice in the piperine-treated group (Figure 5C). In the EAE disease progression, body weight loss is considered as a quantitative surrogate marker for disease severity(Mardiguian et al., 2013). Figure 5D shows a significant recovery in animal body weight following piperine treatment during the EAE period. This schedule is referred to as preventive treatment.
We then sought to investigate whether piperine treatment introduced at the peak of the EAE clinical symptoms could reduce the severity of disease (Figure 5E). Remarkably, piperine treatment starting at the peak of EAE (day 20) lead to an accelerated recovery from the EAE clinical signs when compared to the vehicle group (Figure 5F). Furthermore, the sum of scores for individual mouse and average body weight loss was largely improved (Figure 5G-H). H&E analysis of organs sections from vehicle- or piperine- treated EAE mice suggest that piperine is well tolerant and safe following oral dosage of 30 mg/kg every day in EAE mice (Figure S1). Taken together, these results strongly suggest that piperine alleviates the disease severity of EAE in mice as administrated in both preventive and therapeutic regimens.