Neurological deficit was examined by a 6-point scale after ischemic stroke ( Figure 1 B). Relevant neurological deficit appeared in pMCAO group, such as less spontaneous activity and irregular posture. These indicators were remarkably relieved in Rhy-H group but not in Rhy-L group. Sham-operated rats did not show any neurological deficit. Ischemic brain edema was confirmed by assessing the cerebral water content of ischemic brain tissue. The cerebral water content was greatly relieved by Rhy-H treatment, not by Rhy-L treatment (Rhy-HpMCAO: 83.2% ± 1.1%85.6% ± 1.0%,< 0.05). Wortmannin, a PI3K inhibitor, abolished the protective effect of Rhy ( Figure 1 C).

Figure 1. Rhynchophylline attenuated the ischemic damage after pMCAO. ( A ) The bar graph represents the mean ± SE of the ischemic lesion volume. The microphotographs show representative brain slices ( * p < 0.05 vs. pMCAO); ( B ) The scatter graph shows the individual score for neurological deficits; ( C ) The graph shows the percentage of brain edema in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH), pMCAO + wortmannin (MW) and Rhy-H + wortmannin (RW) groups ( * p < 0.05 Rhy and Sham vs. pMCAO, # p < 0.05 RW vs. RH).

Figure 1. Rhynchophylline attenuated the ischemic damage after pMCAO. ( A ) The bar graph represents the mean ± SE of the ischemic lesion volume. The microphotographs show representative brain slices ( * p < 0.05 vs. pMCAO); ( B ) The scatter graph shows the individual score for neurological deficits; ( C ) The graph shows the percentage of brain edema in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH), pMCAO + wortmannin (MW) and Rhy-H + wortmannin (RW) groups ( * p < 0.05 Rhy and Sham vs. pMCAO, # p < 0.05 RW vs. RH).

We examined the effect of Rhy at two doses, 10 mg/kg and 30 mg/kg, on ischemic brain damage after pMCAO surgery. In the pMCAO group, we noticed a large brain infarction that encompassed the majority of the cortex in the brain hemisphere. The volume of the ischemic brain was considerably reduced in Rhy-H group compared to the control group (41.4% ± 3.7% and 28.5% ± 5.9%,< 0.05) ( Figure 1 A).

Figure 2. Effect of Rhy on the PI3K/Akt signaling and the downstream apoptosis-related proteins. ( A ) The expression of p-Akt, Akt, mTOR, p-mTOR, p-BAD, BAD, and cleaved caspase 3 in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH), Rhy-H + wortmannin (RW) and pMCAO + wortmannin (MW) groups; ( B ) Densitometry analysis; ( C ) Immunohistochemical staining of p-BAD and cleaved caspase 3 at 24 h after pMCAO; ( D ) Bar graph shows quantitative data from each group ( * p < 0.05 Rhy and Sham vs. pMCAO, # p < 0.05 RW vs. RH).

Figure 2. Effect of Rhy on the PI3K/Akt signaling and the downstream apoptosis-related proteins. ( A ) The expression of p-Akt, Akt, mTOR, p-mTOR, p-BAD, BAD, and cleaved caspase 3 in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH), Rhy-H + wortmannin (RW) and pMCAO + wortmannin (MW) groups; ( B ) Densitometry analysis; ( C ) Immunohistochemical staining of p-BAD and cleaved caspase 3 at 24 h after pMCAO; ( D ) Bar graph shows quantitative data from each group ( * p < 0.05 Rhy and Sham vs. pMCAO, # p < 0.05 RW vs. RH).

The immunohistochemical analysis confirmed the results obtained from western blotting. The immunopositive signals of cleaved caspase-3 in the infarct area were significantly elevated than the Sham group, whereas p-BAD expression was reduced ( Figure 2 C). Compared to the pMCAO group, Rhy treatment not only reduced the number of cells positive of cleaved caspse-3 but also increased that of p-BAD (< 0.05). Treatment of wortmannin partially abolished the protective effects of Rhy-H.

Western blotting analysis was used to estimate the activity of the PI3K/Akt signaling and the downstream apoptosis-related proteins ( Figure 2 A). In line with previous studies [ 18 ], up-regulated p-Akt and p-mTOR was observed in the pMCAO group compared to the Sham group. Compared to the pMCAO group and Sham groups, treatment with both high and low doses of Rhy considerably (< 0.05) increased the ratio of p-Akt/Akt and p-mTOR/mTOR. Cerebral ischemia decreased p-BAD expression and increased cleaved caspase 3 expression as compared to the Sham group. Rhy-H preconditioning greatly (< 0.05) prevented the p-BAD decrement and caspase-3 increment induced by the pMCAO. Wortmannin remarkably (< 0.05) eliminated p-Akt, p-mTOR and p-BAD elevation induced by Rhy.

The localization of MyD88 and NF-κB was further determined by immunohistochemistry after stroke. Few cells positive of MyD88 and NF-κB were observed in the Sham group. Cerebral ischemia increased the cells positive of MyD88 and NF-κB than the Sham group. Consistent with western blotting results, high dose Rhy treatment considerably reduced expression of MyD88 and NF-κB (< 0.05). Still, there were no significant differences between pMCAO and Rhy-L group ( Figure 3 C).

Figure 3. Effect of Rhy on the activation of TLR/NF-κB signaling. ( A ) The expression of TLR2/4/9, MyD88 and NF-κB at 24 h after pMCAO in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH) groups. ( B ) Densitometry analysis. ( C ) Immunohistochemical staining of NF-κB and MyD88 in the cerebral cortex. ( D ) Bar graph shows quantitative data from each group ( * p < 0.05 vs. pMCAO).

Figure 3. Effect of Rhy on the activation of TLR/NF-κB signaling. ( A ) The expression of TLR2/4/9, MyD88 and NF-κB at 24 h after pMCAO in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH) groups. ( B ) Densitometry analysis. ( C ) Immunohistochemical staining of NF-κB and MyD88 in the cerebral cortex. ( D ) Bar graph shows quantitative data from each group ( * p < 0.05 vs. pMCAO).

We analyzed the protein level of total TLR2, TLR4, TLR9, nuclear NF-κB and MyD88 with Western blot. In Sham group, the NF-κB was abundant in cytosolic fractions but scarce in nuclear extracts. While in pMCAO group, NF-κB level was expressively enlarged in nuclear fraction and poor in cytosolic fractions at 24 h after ischemia, signifying the translocation from the cytosol to the nucleus. TLR2, TLR4, TLR9 and MyD88 expressions were up-regulated 24 h post-pMCAO. Only high dose of Rhy was able to inhibit the expression of TLR2/4, MyD88 and NF-κB p65 translocation (< 0.05, Figure 3 A).

Figure 4. Effect of Rhy on the expression of claudin-5 and BDNF. ( A ). Expression of claudin-5 and BDNF at 24 h after pMCAO in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH) groups. ( B ). Densitometry analysis. ( C ). The mRNA expressions of claudin-5 and BDNF were measured in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH) groups ( * p < 0.05 vs. pMCAO).

Figure 4. Effect of Rhy on the expression of claudin-5 and BDNF. ( A ). Expression of claudin-5 and BDNF at 24 h after pMCAO in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH) groups. ( B ). Densitometry analysis. ( C ). The mRNA expressions of claudin-5 and BDNF were measured in Sham (S), pMCAO (M), Rhy-L (RL), Rhy-H (RH) groups ( * p < 0.05 vs. pMCAO).

We further investigated the levels of the tight junction protein claudin-5 and brain-derived neurotrophic factor (BDNF), which could alleviate damage following ischemia [ 19 20 ]. In pMCAO group, claudin-5 was significantly declined than in the Sham group. Only Rhy-H could reverse claudin-5 decrease at both protein and mRNA levels (< 0.05, Figure 4 ). The mean ratios of the BDNF densitometry data to those of β-actin in the Sham, pMCAO and Rhy-H groups were 0.55 ± 0.12, 0.31 ± 0.10, and 1.0 ± 0.14, respectively. The data above indicated the expression of BDNF was decreased after pMCAO surgery, while significantly upregulated after Rhy treatment. Similar results were obtained from RT-qPCR analysis ( Figure 4 C).

2.5. Discussion

pMCAO is a successful model to study cerebral ischemic damage in rats [ 21 ]. The cytotoxic responses, such as apoptosis, oxidative stress, proinflammation and neurological damage will initiate immediately upon the onset of cerebral ischemia. Anti-inflammation and anti-apoptosis therapies have attracted significant interest to combat ischemia-induced damage. Inhibiting inflammatory mediators displayed neuroprotection against ischemic brain damage [ 22 ]. Recent evidence suggests that TLRs may be the important targets for developing new treatment approaches for cerebral ischemia injury [ 23 24 ]. Accumulating evidence has demonstrated that TLR4 and TLR9 contribute to the inflammatory reaction [ 8 25 ]. Moreover, the expressions of TLR2 and TLR4 were enhanced after an ischemic stroke. Compared with wild-type mice, the neurological deficit and cerebral infarction induced by cerebral ischemia were remarkably attenuated in TLR2 or TLR4 knockout mice [ 26 27 ]. NF-κB, a key nuclear transcription factor, could combine with specific DNA sequence to trigger inflammation response and ischemic neuronal apoptosis [ 28 ]. TLRs were reported to induce NF-κB-associated pro-inflammatory cytokines through a MyD88-dependent pathway [ 29 30 ]. Cerebral ischemic injury activates TLR4-mediated signal transduction pathway and promote NF-κB translocation from the cytoplasm into nucleus. In our study, we observed the increment of TLR2, TLR4, nuclear NF-κB and MyD88 expressions at 24 h after ischemia, and high dose of Rhy considerably blunted the expression of these factors and inhibited p65 NF-κB transduction in ischemic brain. As an inflammatory mediator, TLR9 also involves in the process of ischemic damage [ 31 ], but Rhy fails to influence the expression of TLR9 in our study.

On the other hand, the PI3K/Akt pathway also renders neuroprotection after ischemic stroke [ 18 ]. Akt/mTOR signaling involves in cardiovascular disease and ischemic cardiomyocyte apoptosis [ 32 33 ]. In addition, Akt/mTOR signaling might relate to the protection against apoptosis in Parkinson’s and Alzheimer’s disease. Western blotting analysis revealed enhanced expression of p-mTOR in ischemia area in our study. The PI3K/Akt pathway promotes neovascularization and results in a noteworthy reduction in infarct size after ischemia [ 34 ]. However, there are inconsistent results about the level of phospho-Akt after ischemic injury. Osuka revealed that dysfunction of the Akt pathway was involved in ischemic damage [ 35 ], but Noshita and Shibata insisted that, within hours, pAkt transiently increase in neurons following cerebral ischemia [ 36 37 ], and this raise is regarded as a neuroprotective action. In Gao’s and Ishrat’s research, up-regulated pAkt was observed compared to sham group 24 h post-pMCAO [ 38 39 ]. Consistently, the same result was obtained in our manuscript.

Several reasons might contribute to the discrepancy in pAkt level. Firstly, p-Akt level depended on the timing after the injury/stress. After ischemia, pAkt was dephosphorylated immediately in the CA1 region and rephosphorylated after only 5 min of ischemia [ 40 ]. Secondly, the extent of ischemic injury also accounts-lethal damage downregulated pAkt in the CA1 region, but sublethal damage increased pAkt. Thirdly, the origin of tissue used in protein analysis also works. pAkt level was elevated in cortex in our manuscript or in peri-infarct cortical tissue in Ishrat’s research [ 39 ], or in the ipsilateral cerebral cortex as described by Pérez-Álvarez [ 41 ]. Both Gao and Shibata revealed that the enhanced pAkt occurred mainly in neurons located in the outer area of the middle cerebral artery territory (ischemic penumbra) [ 37 38 ], but Osuka reported that PI3K/Akt signaling was down-regulated in bilateral cortices adjacent to the hippocampus [ 35 ].

Cerebral ischemia motivates Akt and mTOR, which subsequently prevents BAD into the mitochondrial membrane, finally inhibiting death of neurons. Although BAD translocation into the mitochondrial membrane was inhibited by pAkt, phosphorylation of BAD was increased by activated Akt, thereby inhibiting the apoptotic activity and promoting cell survival [ 42 ]. As we know, p-BAD increment contributes to the inhibition of apoptosis. Apoptotic stimuli result in dephosphorylation of BAD, thus activating caspase-3 and Bax [ 43 ].

Our data show that Rhy reduced ischemic brain damage, probably by increasing the ratio of p-mTOR/mTOR, and triggering its downstream target p-BAD, thereby reducing ischemic neuronal death. These data resemble those reported by Ishrat that neuroprotective agents protect against stroke via mediating Akt/BAD phosphorylation [ 39 42 ]. Why there isn’t a direct and proportional relationship between pBAD and pAkt expression? It is reasonable that there is not enough pAkt to trigger pBAD at 24 h post-injury. Because the damage in the cortex region inflicted by the stroke model is relatively moderate. Herein, it is possible that Rhy treatment up-regulated pAkt in pMCAO rats and thereby increasing pBAD. Furthermore, in our study, wortmannin weakened the activation of BAD & Akt and thus abolished Rhy’s effects, confirming the critical role of PI3K/Akt pathway.