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.