Gastrodia-Uncaria Water Extract Inhibits Endoplasmic Reticulum Stress and Matrix Metalloproteinase in Protecting against Cerebral Ischemia

Stroke is the second leading cause of death in worldwide, in which cerebral ischemia accounts for 87% of all cases. The building up of endoplasmic reticulum stress in cerebral ischemia contributes to the disruption of blood brain barrier and neuronal cell death. The only FDA-approved drug, recombinant tissue plasminogen activator, is still of limited use due to the narrow window period and lack of neuroprotective effect. Therefore, it is necessary to explore alternative treatment on cerebral ischemia. Tianma-Gouteng decoction is a traditional Chinese Medicine prescription used to treat brain diseases in China. In this study, we investigated the neuroprotective effect of a water extract consisting of Gastrodia elata and Uncaria rhynchophylla, which are the two main herbs in the decoction. Cerebral ischemia was induced in rats using middle cerebral artery occlusion. GUW-treated rats have significantly reduced infarction volume and recovered neurological functions. The number of protein aggregates and caspase-12 positive cells were significantly inhibited. In vitro oxygen-glucose deprivation / reoxygenation stroke model demonstrated that the unfolded protein response proteins GRP78 and PDI were upregulated by GUW. Less ubiquitin puncta and normalized ubiquitin distribution indicated the reduction in endoplasmic reticulum stress. Furthermore, a lower Evan blue signal and MMPsense signal was observed, suggesting that GUW may preserve the blood brain barrier integrity through inhibiting MMP activity. Taken together, this suggested that GUW protected ischemic neurons and the blood brain barrier through inhibiting endoplasmic reticulum stress.


INTRODUCTION
Stroke is the second leading causes of death in worldwide, in which 87% of the stroke cases 48 were cerebral ischemia [1]. After stroke, more than 60% of patients would experience 49 neurological impairments and loss in locomotor functioning [1]. Recombinant tissue 50 plasminogen activator (tPA) is the only prescribed medicine in worldwide. However, the 51 shortcoming of tPA treatment are the narrow therapeutic window and increasing risk of 52 intracerebral hemorrhage. Therefore, it is a challenge to seek for alternative treatment methods. 53 Cerebral ischemia is defined by the disruption of blood flow towards the brain tissue. Such a 54 blockage is caused by the formation of thrombus and embolus, a plaque that could obstruct the 55 blood vessel [2]. After the removal of the blood clot through mechanical thrombectomy or by 56 using recombinant tissue plasminogen activator, blood supply is restored and reperfusion phase 57 proceeds. During this phase, the calcium stores in the ER will be depleted, resulting in a 58 decrease in protein folding efficiency and the accumulation of unfolded protein aggregates [3]. 59 Thus, endoplasmic reticulum stress is built up. The unfolded protein response (UPR) is a 60 mechanism to counteract ER stress [4]. Chaperones such as GRP78, protein disulfide 61 isomerase (PDI) are upregulated to assist in proper folding of the protein aggregates to attenuate 62 ER stress [5]. Nonetheless, if ER stress persists for a long period of time, caspase-12 is 63 activated to trigger cell death [6]. 64 The increase in ER stress leads to the activation of matrix metalloproteinases (MMP), causing 65 the disruption of blood brain barrier [7]. The activation of MMP during cerebral ischemia can 66 cause irreversible loss of blood brain barrier (BBB) integrity, edema formation, hemorrhagic 67 transformation, and further neuronal cell death [8,9]. Inhibition of MMP may protect BBB 68 disruption through reducing brain microvascular endothelial cell damage [10]. The above 69 indicated inhibiting ER stress may be beneficial in protecting against BBB dysruption.

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Gastrodia elata (GE) and Uncaria rhynchophylla (UR) are the two main herbs in Tianma- 71 Gouteng decoction, a Chinese Medicine prescription that is used to treat head related disease 72 such as stroke. We have previously found that GE and UR in the Gastrodia-Uncaria water 73 extract (GUW) work synergistically to protect against cerebral ischemia through inhibiting 74 oxidative stress and apoptosis [11]. Metabolomics analysis of the cerebral spinal fluid revealed 75 changes in the amount of endoplasmic reticulum stress-related metabolites in GUW-treated 76 ischemic rats [12]. In this study, we investigated the effects of GUW in protecting against 77 cerebral ischemia-induced apoptosis through reducing ER stress and preserving blood brain 78 barrier integrity. suture for 2 h. The suture was then removed and the external carotid artery was cauterized.

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The operated rats were caged individually and warmed with a heating lamp. The same 98 operation procedures were carried out on the shams rats but without inserting the nylon suture 99 to occlude the middle cerebral artery.

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GUW treatment and assessment 101 GUW powder was dissolved in ddH2O and was orally administered to the MCAO rats with a 102 human equivalent dosage of 288.6 mg/kg GUW once daily for seven consecutive days [13].

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One group of rats subjected to MCAO received GUW treatment. The rest of the MCAO rats 104 and sham rats are fed with ddH2O. After treatment, neurological deficit score and triphenyl 105 tetrazolium chloride (TTC) staining were performed to assess the brain infarct volume and 106 neurological functioning as previously described [11].    Fluorescence microscopy was carried out using ApoTome.2 fluorescence microscopy.  plate was incubated at 37 o C under air with 21% O2 and 5% CO2 at 80% humidity for 24hr. 164 After 24 hr, the wells were aspirated and 100 µl of 0.5 mg/ml MTT was added to each well. 165 After incubation for 3 h, the solution was drawn out and 100 µl DMSO was added to each well. 166 An addition lane was added with DMSO to serve as the solvent control. The plate was then    One-way ANOVA post hoc Tukey's test was used in all data sets besides from cytotoxicity 203 test and OGD/R cell viability assessment. One-way ANOVA post hoc Dunnet's test was used 204 on the above mentioned two data set. Student's t-test was used in Evan's blue and MMPsense 205 assay. p < 0.05 was considered to be statistically significant.

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GUW protected against cerebral ischemia 208 The neurological deficit score was assessed on day 1 and 7 post-operation (Fig. 1a). At day 1, 209 a significant increase in neurological deficit score was observed in MCAO (p < 0.001) and 210 GUW rats (p < 0.001) when compared with sham group rats; insignificant difference (p > 0.05) 211 was found between MCAO and GUW rats. GUW rats had significant improvements in 212 neurological deficits on day 7 when compared with the score on day 1 (p < 0.001) and MCAO 213 rats on day 7 (p < 0.001). While the mean neurological deficit score didn't significantly 214 improve in the MCAO rats at day 7 (p > 0.05) and remained significantly higher than the sham 215 rats (p < 0.001), insignificant difference was found between GUW rats and sham rats on day 7 216 (p > 0.05). TTC staining was performed to examine the infarction volume (Fig. 1b). The brain 217 was significantly infarcted by 45.6% after MCAO (p < 0.001, Fig. 1c). GUW treatment rats, 218 the infarct volume was significantly reduced by 55.7% when compared with MCAO rats (p < 219 0.001).

GUW reduced ER stress 221
To investigate the effect of MCAO and GUW on neurons, cresyl violet staining was used.

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Under a higher magnification, two types of staining patterns could be observed (Fig. 2a). The (p < 0.001), yet it is significantly fewer than that in the sham group rats (p < 0.001).

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Immunohistochemistry was used to study the amount of protein aggregates and protein 229 expression of caspase-12 at the infarct region. In MCAO rats, ubiquitinated protein aggregates 230 were present in the cell body and the ubiquitin distribution was retracted to the soma (Fig. 2c).

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For GUW group, the number of aggregates was significantly reduced by 88.3% (p < 0.001; Fig.   232 2d). Furthermore, a normal ubiquitin distribution was observed. The caspase-12 expression 233 was upregulated at the infarct region in the MCAO rats when compared to the sham rats (Fig.   234 2e). The number of caspase-12 positive cells was reduced significantly by 46.6% in GUW-235 treated rats (p < 0.001; Fig. 2f).

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More unfolded proteins were present in the OGD/R neurons (Fig. 4a). Moreover, a retraction 253 of ubiquitin from the neurites to the cell was observed, indicating the occurrence of 254 neurodegeneration. After treating with GUW, the amount of unfolded proteins was reduced 255 significantly by 26.4%, while ubiquitin was normally distributed throughout the neuronal cell 256 (Fig. 4b). Western blot showed that GUW led to an increase in expression of chaperone 257 proteins GRP78, PDI expression (Fig 4c). A downregulation of XBP-1(u) was detected. We 258 further investigated the expression of caspase-12, the initiator caspase of ER stress-induced 259 apoptosis. An upregulation of pro-caspase-12 expression was observed in GUW treatment 260 groups.

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GUW treatment reduced BBB impairment 262 Evans blue signal was detected at the left hemisphere after the induction of ischemic insult 263 at both control and GUW treatment group (Fig. 5a). TBR of Evans blue signal in GUW 264 treatment group was 42.3% lower than MCAO group (p<0.001; Fig. 5b).

GUW inhibited near-infrared fluorescence signal of matrix metalloproteinases activity
266 NIRF images were obtained at day 3, 5 and 7 of post-operation. A lower fluorescence 267 intensity was observed in the ischemic lesion of GUW treatment group rats compared with 268 MCAO control group (Fig. 6a & 6b). The treatment with GUW led to significant reduction 269 of the fluorescent intensity of matrix metalloproteinases by 25.1 (p<0.001), 21.6 (p<0.01), 270 and 24.3% (p<0.05) at day 3, 5 and 7 respectively (Fig. 6c).

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Ex vivo brain NIRF imaging was conducted to exclude any possibility of interference caused 272 in vivo. At day 7 of post-operation, MMP activities were found in the left hemisphere of 273 MCAO rats (Fig. 7a). The TBR of MMP in MCAO rats was 1.34 fold of sham group 274 (p<0.001). GUW treatment led to a significant reduction in the TBR by 26.2% when 275 compared with MCAO rats (Fig. 7c and 7e). NIRF imaging of the brain slices ( Fig. 7b and   276 7d) and from the corresponding brains showed that TBRs in GUW-treated rats were 32.7% 277 lower than that in MCAO group (Fig. 7f; p <0.001).

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Brain stroke is the second leading causes of death in worldwide. Yet, there is currently no 280 available FDA-approved medication in treating cerebral ischemia at reperfusion phase. In this 281 study, we showed that GUW may significantly protect the brain reperfusion injury followed by 282 ischemic insults through reducing ER stress and preserving BBB integrity.

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Seven days of reperfusion was allowed for observing the effect of GUW on recovery.   In vitro study was carried out to visualize the intracellular events in OGD/R. GE and GUW 307 extract led to a higher SK-N-SH viability. Furthermore, neurodegeneration may occur in CNS 308 neurons after damages, which may lead to neuronal dysfunction [25,26]. After addition of 309 GUW, the dendritic length and connections were maintained as compared with OGD/R group.

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From western blotting results, it was found that GRP78 and PDI were upregulated by GUW.

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Chaperones GRP78 and PDI were studied because these two enzymes played important roles