Betahistine alleviates benign paroxysmal positional vertigo (BPPV) through inducing production of multiple CTRP family members and activating the ERK1/2-AKT/PPARy pathway

Abstract

Background: Betahistine is a clinical medication for the treatment of benign paroxysmal positional vertigo (BPPV). Otolin, a secreted glycoprotein with a C-terminal globular domain homologous to the immune complement C1q, has been identified as a biomarker for BPPV. However, the role of complement C1q/TNF-related proteins (CTRPs) with a C-terminal globular domain in BPPV is unclear, so we explored the change of CTRPs in betahistine treated BPPV.

Methods: We treated BPPV patients with Betahistine (12 mg/time, 3 times/day) for 4 weeks and observed the clinical efficacy and the expression of CTRP family members in BPPV patients. Then, we constructed a vertigo mice model of vestibular dysfunction with gentamicin (150 mg/Kg) and a BPPV model of Slc26a4^loop/loop mutant mice. Adenoviral vectors for CTRP expression vector and small interfering RNA were injected via the intratympanic injection into mice and detected the expression of CTRP family members, phosphorylation levels of ERK and AKT and the expression of PPARγ. In addition, we treated mice of vestibular dysfunction with Betahistine (10 mg/Kg) and/or ERK inhibitor of SCH772984 (12 mg/Kg) and/or and PPARγ antagonist GW9662 (1 mg/Kg) for 15 days, and evaluated the accuracy of air righting reflex, the time of contact righting reflex and the scores of head tilt and swimming behavior.

Results: After treatment with Betahistine, the residual dizziness duration and the score of the evaluation were reduced, and the expression of CTRP1, 3, 6, 9 and 12 were significantly increased in BPPV patients. We also found that Betahistine improved the accuracy of air righting reflex, reduced the time of contact righting reflex and the scores of head tilt and swimming behavior in gentamicin-treated mice and Slc26a4^loop/loop mutant mice. The expression levels of CTRP1, 3, 6, 9 and 12, phosphorylation levels of ERK and AKT, and PPARγ expression were significantly increased, and the scores of head tilt and swimming behavior were decreased in vestibular dysfunction mice with overexpression of CTRPs. Silencing CTRPs has the opposite effect. SCH772984 reversed the effect of Betahistine in mice with vestibular dysfunction.

Conclusion: Betahistine alleviates BPPV through inducing production of multiple CTRP family members and activating the ERK1/2-AKT/PPARy pathway.

Keywords: BPPV; Betahistine; CTRP family; The ERK1/2-AKT/PPARy pathway; Vestibular dysfunction.

Fig. 1

The clinical effect of Betahistine on BPPV and the expression of multiple CTRP family members. BPPV patients were treated with Betahistine (12 mg/time, 3 times/day) for 4 weeks, and blood was collected and serum was separated. The duration of residual dizziness (A), DHI score (B) and BBS score (C) were recorded. The expression of CTRP1 (D), CTRP 3 (E), CTRP 6 (F), CTRP 9 (G) and CTRP 12 (H) in serum was detected by using ELISA. N = 5, tatistical differences were performed by using the Student’s t-test. Compared with BPPV patient, *P < 0.05, **P < 0.01

Fig. 2

Construction of a vertigo model of vestibular dysfunction in mice. Mice were subcutaneously injected with gentamicin (150 mg/Kg) and/or Betahistine (10 mg/Kg) for 15 days, twice a day. We evaluated the accuracy of air righting reflex (A), the time of contact righting reflex (B) and the scores of head tilt and swimming behavior (C) in vertigo mice of vestibular dysfunction. N = 7, Statistical differences were performed by using a one-way ANOVA. *P < 0.05, **P < 0.01

Fig. 3

Overexpression of CTRP1, 3, 6, 9 and 12 in mice with vestibular dysfunction vertigo. Mice of vestibular dysfunction vertigo were injected with pSB-T-CTRP1, 3, 6, 9, 12 or pSB-T empty vector, respectively. RT-qPCR was used to detect the mRNA expression of CTRP1 (A), CTRP3 (B), CTRP6 (C), CTRP9 (D) and CTRP12 (E). F and G Western blotting was used to evaluate the protein expression levels of CTRP1, 3, 6, 9 and 12. N = 4, Statistical differences were performed by using the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 4

Overexpression of CTRP1, 3, 6, 9 and 12 reduces vestibular dysfunction in mice. The accuracy of air righting reflex and the scores of head tilt and swimming behavior were detected in mice treated with pSB-T-CTRP1 (A and B), pSB-T-CTRP3 (C and D), pSB-T-CTRP6 (E and F), pSB-T-CTRP9 (G and H) and pSB-T-CTRP12 (I and J), respectively. N = 3, Statistical differences were performed by using the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 5

Silencing CTRP1, 3, 6, 9 and 12 exacerbates vestibular dysfunction in mice. Mice of vestibular dysfunction vertigo were injected with si-CTRP1, 3, 6, 9, 12 or their scrambled, respectively. RT-qPCR was used to detect the mRNA expression of CTRP1 (A), CTRP3 (B), CTRP6 (C), CTRP9 (D) and CTRP12 (E). F and G Western blotting was used to evaluate the protein expression levels of CTRP1, 3, 6, 9 and 12. The scores of head tilt and swimming behavior were detected in mice treated with si-CTRP1 (H), si-CTRP3 (I), si-CTRP6 (J), si-CTRP9 (K) and si-CTRP12 (L), respectively. N = 3, Statistical differences were performed by using the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 6

Overexpression of CTRP1 and 12 promotes phosphorylation of ERK1/2 and AKT and expression of PPARγ. Mice of vestibular dysfunction vertigo were injected with pSB-T-CTRP1, 12 or pSB-T empty vector, respectively. A and B We detected the protein levels of p-ERK1/2 (C) and p-AKT (D) by using western blotting and evaluated the mRNA (E) and protein expression levels of PPARγ (F). N = 3, Statistical differences were performed by using the Student’s t-test. *P < 0.05, **P < 0.01

Fig. 7

Betahistine reduces vestibular dysfunction through inducing the expression of CTRP family members and activating the ERK1/2-AKT/PPARγ pathway in mice. Fifty-six mice were randomly divided into four groups and treated with normal saline, gentamicin, gentamicin + Betahistine, gentamicin + Betahistine + SCH772984, respectively. Then, CTRP1 (A) and CTRP12 mRNA (B) and protein levels (C and D), p-ERK1/2 protein levels (E and F), p-AKT protein levels (G) and PPARγ mRNA (H) and protein levels (I) were detected with RT-qPCR and western blotting, respectively. We also evaluated the accuracy of air righting reflex (J), the time of contact righting reflex (K) and the scores of head tilt and swimming behavior in mice (L). N = 7, Statistical differences were performed by using a one-way ANOVA. *P < 0.05, **P < 0.01

Fig. 8

The expression of CTRPs in Betahistine treated Slc26a4^loop/loop mutant mice. Slc26a4loop/loop mutant mice were subcutaneously injected with Betahistine (10 mg/Kg) for 15 days, twice a day. We performed auditory brainstem response (ABR) testing (A) and behavioral testing (B) in Slc26a4^loop/loop mutant and wild-type mice. Next, we evaluated the accuracy of air righting reflex (C), the time of contact righting reflex (D) and the scores of head tilt and swimming behavior in vertigo mice of vestibular dysfunction (E). The expression of CTRP1 (F), CTRP 3 (G), CTRP 6 (H), CTRP 9 (I) and CTRP 12 (J). In serum was detected by using ELISA. N = 7, Statistical differences were performed by using one-way ANOVA. *P < 0.05, **P < 0.01