LDN-193189 – Abcam Chem

Isoflurane post-conditioning attenuates cerebral ischemia/reperfusion injury by reducing apoptotic through activating the BMP7/SMAD signaling pathway in rats

Abstract

The expressions of different temporal patterns of bone morphogenetic proteins (BMPs) have changed after ischemic strokes, and ischemic preconditioning-induced neuroprotection was attenuated when BMP7 was inhibited. In the previous study, the neuroprotection of isoflurane postconditioning (ISPOC) against cerebral ischemia-reperfusion (I/R) injury has been addressed, with particular relevance to the role of BMP7. Conse- quently, in the present study, we continued to explore the mechanisms involved in the BMP7 signal mediated the neuroprotection of ISPOC. A rat model of the middle cerebral artery occlusion was used in this study. Rats were administered 1.5 % isoflurane, 60 min after 90 min of ischemia, followed by a 24 h reperfusion period. The 1.5 % ISPOC significantly ameliorated the cerebral infarct volumes, neurologic deficit scores, damaged neurons, and apoptotic neurons. Moreover, ISPOC unregulated the expressions of BMP7, p-Smad1/5/9, and p-p38. Whereas, the neuroprotective effect was weakened by LDN-193189 and SB203580, respectively, a BMP7/Smad1/5/9 and p38MAPK signaling pathway inhibitor. Furthermore, LDN-193189 downregulated the expression of p-p38. The present results of this study indicated that the neuroprotection of 1.5 % isoflurane postconditioning to cerebral ischemia-reperfusion injury is related to the activating of BMP7/Smad1/5/9 and p38MAPK signal pathway.

1. Introduction

Cerebral stroke is one of the most common causes of disability in adults and is the second-largest cause of death worldwide(Scheitz et al., 2016). Ischemic stroke is a transient or permanent reduction of blood supply resulting from the formation of a thrombus or an embolus that leads to middle cerebral artery occlusion (MCAO), which induces local cerebral tissue and neuron be short of oXygen and glucose, results in neuroinflammation, neuronal cell death and cerebral infarction(Liao et al., 2020). Reperfusion can cause serious brain lesions as a result of complex pathophysiological mechanisms, and apoptosis is a key mo- lecular pathway involved the cerebral ischemia-reperfusion injury (CIRI)(Awooda et al., 2015). Although tissue plasminogen activator (tPA) is commonly used to treat acute ischemic stroke within an appropriate therapeutic window, one of its side effects is an enhancement of inflammatory response in brain capillaries and subse- quently neuronal cell damage after stroke (Amani et al., 2019). Iso- flurane, a volatile anesthetic, that has been in use for clinical practice for the last three decades, has shown to alleviate brain injury during reperfusion both in vivo and in vitro(Lee et al., 2008; Lin et al., 2011; McMurtrey and Zuo, 2010). Our previous study has demonstrated that the neuroprotection isoflurane postconditioning (ISPOC) alleviate ce- rebral ischemia reperfusion(I/R) injury is related to the apoptosis and Transforming Growth Factor Beta superfamily(TGF-βs)(Zhang et al., 2019; Yin et al., 2020; Peng et al., 2019a, b; Peng et al., 2019c).Bone morphogenetic proteins (BMPs) are the largest subgroup of TGF-βs, several members of which function in the pathophysiological processes of neurological diseases (Xiao et al., 2010; Kang et al., 2014).
The expressions for different temporal patterns of BMPs have changed after ischemic stroke (Shin et al., 2012). Our previous study showed that 1.5 % of isoflurane postconditioning could regulate the expression of BMP4 and Aquaporins-4, inhibited the occurrence of brain edema, and reduce cerebral I/R injury (Yuan et al., 2018). In rats with ischemia, IPC-induced neuroprotection was attenuated when BMP7 was inhibited (Guan et al., 2013), and further studies have demonstrated that BMP7 contributes to IPC-induced ischemic tolerance by activating the p38 mitogen-activated protein kinase (MAPK) signaling pathway (Guan et al., 2014). There is also increasing evidence that MAPK is an impor- tant regulator of ischemic and hemorrhagic cerebral vascular disease, and it is a discovery target in stroke (Sun and Nan, 2016).
Therefore, in this study, we hypothesized that BMP7/Smad1/5/9and P38 is mediate the ISPOC-induced neuroprotection. To test this hy- pothesis, a transient focal brain ischemic model – MCAO in rats was used, either BMP7 inhibitor LDN-193189 and p38 inhibitor SB203580
was used as an inhibitor exploring the BMP7 and p38 signaling mech- anisms following isoflurane postconditioning.

2. Materials and methods
2.1. Animal

All male Sprague-Dawley rats were obtained from the EXperimental Animal Center of Shihezi University, each of these weighed between 250
g and 300 g. The animals were housed at controlled room temperature (20 ◦C–24 ◦C) and humidity (50%–60%), following strict 12 h light and 12 h dark cyclical conditions (Shihezi, China). The experiment was performed under strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rats were randomly divided into the following 5 groups: (1) Sham group (S), (2) MCAO group (I/R), (3) 1.5 %ISO MCAO group (ISPOC), (4) 1.5 %ISO MCAO LDN-193189 group (LDN), (5) 1.5 %ISO MCAO LDN-193189 SB203580 group (LDN SB203580). In the sham groups, included rats were subjected to the same procedure without occlusion. For the I/R group, 90 minutes of ischemia followed by 24 h of reper- fusion. For the ISPOC group, immediately at the beginning of reperfu- sion, rats were treated with 1.5 % isoflurane for 60 min. The concentration of isoflurane in the container was monitored using a gas monitor (Drager Vamos, Germany). Two inhibitors, in which rats were injected with either 1 mM in 1% DMSO SB203580 intra- cerebroventricularly or 3 mg/kg LDN-193189 intraperitoneally 30 min. before I/R(Zhao et al., 2020; Zhou et al., 2020), followed by ischemia, ISPOC, and reperfusion. The animal specimens were examined 24 h after surgery.

2.2. Construction of the rat MCAO model and ISOPC

According to previously reported procedures(Li et al., 2013), All the rats were anesthetized with a miXture of ketamine (60 mg/mL) and Xylazine (10 mg/mL) by intraperitoneal injection (0.15 mL/100 g). An incision was made in the neck ventrally in the midline exposing the left and right Common carotid arteries which were then separated from the surrounding tissue and the vagus nerve. A 4 0 nylon monofilament surgical suture was inserted into the isolated ICA lumen through the ECA stump and advanced approXimately 18–19 mm past the CCA bifurcation.

The ECA and inside nylon suture were tight, and the middle MCAO-induced-cerebral ischemic occurred. After a 90 min ischemic period, the suture was withdrawn to allow reperfusion, followed by either postconditioning or not postconditioning with 1.5 % isoflurane for 60 min in independent chambers. Sham group rats underwent similar surgery except for suture insertion. The incision was closed after ligating the external carotid stump. Throughout the experiment, the rectal and temporalis muscle temperatures were constantly monitored and main- tained between 36.5 and 37.5 by using an electrical heating pad and a temp controlled infrared heat lamp.

2.3. Neurological assessment

Neurological scores were evaluated 24 h after the MCAO (n 8 per group). Assessments were performed by a blinded investigator as pre- viously described (Camerlingo et al., 2006). Briefly, a score of 0 suggests no neurological symptoms, 1 suggests the inability to fully flex the forepaw, 2 suggests rotating while crawling and falling to the contra- lateral side, 3 suggests the inability to walk unaided, and 4 suggests unconsciousness.

2.4. Infarct size measurement

Rats were euthanized and intact brain removed 24 h after MCAO(n 8 per group). Removed brain tissues were sliced to 2 mm specimens on ice. Tissue was incubated in 2% 2,3,5-triphenyl tetrazolium (TTC, Solarbio, Beijing, China) at 37 ◦C in the dark for 30 min. 4% para-
formaldehyde PFA (Sigma-Aldrich, St.Loius, MO, USA) was used as a fiXative for the brain tissue, for 24 h at 4 ◦C. The non-infarcted brain
tissue appeared red and the infarcted area appeared white. Analysis of necrotic tissues was done using Image-Pro Plus 7.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

2.5. Hematoxylin-eosin (HE) staining

Rat brains were removed(n 4 per group), dehydrated and cut into coronal sections showing the hippocampus, and perfused with 4% paraformaldehyde in 0.1 mol L 1 PBS (pH 7.4) (Kaiji Biotechnology Development Company, China). The cerebral tissues in the ischemic area of the brain were collected and post-fiXed in 4% paraformaldehyde (Saiweier Biotechnology Development Company, China) overnight at 4 ◦ C.

The next day, the tissues were dehydrated, embedded in paraffin, and cut into sections (4 μm thickness) to adhere to the slice. Finally, the sections were stained with HE (Wuhan Servicebio Technology Co. Ltd, China). The cellular morphology was observed under a microscope (Nikon, Japan).

2.6. Nissl staining

Nissl staining was performed using the standard method. These paraffin sections were immersed in the Nissl staining solution at 56 ◦C
for 30 min (Sigma-Aldrich, St. Louis, MO, USA) (n 4 per group). Finally, the sections were dehydrated, fiXed, and sealed with neutral gum. The morphology was observed and analyzed by the Image-Pro Plus 6.0 image analysis software.

2.7. TUNEL staining

After 24 h reperfusion, the TUNEL staining performed, using the Insitu Cell Apoptosis Detection Kit (Wuhan Boster Bioengineering Co. Ltd, Hubei, China) following the manufacturer’s protocol. Hippocampus tissue slices were post-fiXed in a miXture of ethanol and acetic acid so-
lution, (in the ratio 2:1) for 5 min and rinsed Sliced brain tissues were post-fiXed in a miXture of ethanol and acetic acid solution(n = 4 per group). Tissue sections were then incubated in proteinase K (20 μg/mL) for 15 min, following a 10 min immersion in 3% H2O2at room temper- ature. Following three 10 min washes in PBS, the TUNEL reaction miXture was used to incubate them at 37 ◦C for an hour. Tissue sections were washed again in PBS three times for 10 min. each. A confocal fluorescent microscope was used to photograph the stained sections. The apoptotic neurons were counted; the results are expressed as TUNEL positive cells per mm2.

2.8. Immunofluorescence (IF)

The hippocampus tissue chips were dewaxed with Xylene and dehydrated with gradient alcohol (n = 4 per group). After routine dewaxing and gradient hydration, endogenous peroXidase was blocked by 3% hydrogen peroXide and antigen and was repaired by microwave. After blocking with PBS containing 0.3 % Triton X-100 and 20 % bovine serum albumin, slices were incubated overnight with primary antibodies (anti-BMP7, 1:100; Abcam, Cambridge, UK; anti-p-Smad1/5/9, 1:100, Abcam, Cambridge, UK;anti-p-p38, 1:200; Abcam, Cambridge, UK), followed by further incubation at 37 ◦C for one hour with secondary antibodies diluted at 1:100 in PBS. Then, the sections were stained with diamidinophenylindole (DAPI) and were sealed. Immunoreactivity was visualized and photographed using a Zeiss LSM510 laser-scanning confocal microscope (Zeiss, Jena, Germany).

2.9. Western blot analysis of protein expression

The ischemic cerebral hemisphere tissues were homogenized in Radio-Immunoprecation pit assay lysis buffer (Wuhan Boster Bioengi- neering Co. Ltd, Hubei, China) (n 3 per group). The concentration of protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Tissue samples were isolated on 10 % SDS-polyacrylamide for gel electrophoresis; separated proteins were transferred electrically onto polyvinylidene fluoride membranes (Solarbio, Beijing, China). 5% nonfat milk (BD, USA) was used to block the membranes at room tem- perature for 2 h, after which the membranes with proteins were incu- bated at room temperature for 2 h, followed by incubation with anti-
β-actin (1:1000, ZSGB-BIO, China), anti-BMP7 (1:1000, Abcam, Cambridge, UK), anti-phosphorylated-Smad1/5/9 (1:500, Abcam, Cam- bridge, UK), anti- phosphorylated-p38MAPK (1:500, Abcam, Cambridge, UK), anti-Bcl-2 (1:1000; Abcam, Cambridge, UK), anti-Bax (1:1000, Abcam, Cambridge, UK) at 4 ◦C overnight. Then, the mem- branes were washed thrice with Tris-buffered saline with Tween 20,protein loading. An image analysis software measured the intensities of the bands (Gel-pro analyzer; Media Cybernetics, USA).

3. Statistical analysis

All experiments were repeated at least 3 times independently in a randomized and blinded manner. SPSS 21.0 was used to analyze the data statistically and data were presented as the mean standard deviation (SD). The normality test by Kolmogorov-Smirnov method, P > 0.1 in-
dicates that the data is normally distributed. After confirming the normal distribution of the data, the Newman-Keuls method was used for post hoc analysis following analysis of the results with one-way ANOVA or non-normally distributed data were ranked by one-way ANOVA fol-
lowed by the Newman-Keuls method for post hoc analysis. P-value < 0.05 was considered statistically significant. 4. Results 4.1. Effects of 1.5 % ISPOC, BMP7 inhibitor LDN-193189, p38 inhibitor SB203580 on neurobehavioral function and infarct volume after focal cerebral I/R injury in rats TTC staining was used to expose necrotic zones of brain sections Fig. 1(a); the extent of brain injury following cerebral I/R was assessed by calculating the infarct volume and neurological score. The infarction rates in the I/R, ISPOC, LDN-193189, and LDN-193189 SB203580 groups were 0.29 0.05(P < 0.05 vs. sham), 0.07 0.03(P < 0.05 vs. IR),0.19 0.02(P < 0.05 vs. ISPOC), 0.23 0.03(P < 0.05 vs. ISPOC) Fig. 1(b). A significant reduction in the infarct volume and the neurological score was observed in the 1.5 % ISPOC(P < 0.05). Significant attenuation was observed in 1.5 % ISPOC-induced reduction of cerebral LDN-193189 and SB203580injection (P < 0.05) Fig. 1(b and c). 4.2. Effects of 1.5% ISPOC, BMP7 inhibitor LDN-193189, P38 inhibitor SB203580 on neuronal morphology in the hippocampal CA1 region after cerebral I/R injury in rats Normal clear cell outline and compact and abundant cytoplasm were observed in the pyramidal neurons of the hippocampal CA1 subfield in the sham grouping Fig. 2(a). The calculated pyknosis ratio was (5.25 2.61)% (P < 0.05 vs. I/R). In the I/R non-treated group, karyopyknosis was evident with the reduction in the number of neurons; the pyknosis ratio being (83.25 5.12)% (P < 0.05 vs. sham). In the ISPOC group, cell injury was significantly reduced; the pyknosis ratio being (15.38 4.96)% (P < 0.05 vs. I/R). By contrast, in the inhibitor group, ISPOC- induced reduction of hippocampal neuron karyopyknosis was significantly reduced by inhibitor drug injection; the pyknosis ratio being (48.38 3.62)% (P < 0.05 vs. ISPOC) and (62.50 5.29)% (P < 0.05 vs. ISPOC), respectively Fig. 2(c). Nissl staining analysis revealed that MCAO-induced several abnormal neurons with shrunken cytoplasm, pyknotic nuclei, and decreased Fig. 2(b). The numbers of intact neurons in Sham, I/R, ISPOC, LDN-193189 and LDN-193189 SB203580 groups were 67.13 5.62, 18.38 3.53, 51.25 3.11, 35.50 4.79 and 29.00 3.46 (P < 0.05), respectively Fig. 2(d). In the I/R group, The boundary was blurred, and the cell band was broken. The numbers of intact neurons were signifi- cantly reduced, and the Nissl body of numerous nerve cells was fragmented, which caused a significant reduction of Nissl bodies(P < 0.05). However, the cell gap was widened, and the cell band was thin, which indicated the neuroprotective effect of ISO postconditioning. Mean- while, when we pretreated the rats with LDN-193189 and SB203580 before reperfusion, the number of intact neurons was decreased, thus demonstrating that the neuroprotective effect of ISPOC was abolished(P < 0.05)Fig. 2(d). Fig. 4. Effect of 1.5 % Isoflurane Post-conditioning on rat hippocampus CA1 BMP7 expression by Immunofluorescence. (a) IF of BMP7 in the hippocampal CA1. (b) Mean fluorescence intensity of BMP7 in various groups. (c) BMP7 positive cells count. Data are expressed as means ± SEM (n = 4). *P < 0.05, respectively, compared to sham group; #P < 0.05, respectively, compared to I/R group; &P < 0.05, respectively, compared to 1.5 % ISPOC group. 4.3. Effects of 1.5 % ISPOC, BMP7 inhibitor LDN-193189, P38 inhibitor SB203580 on neuronal cell apoptosis and the expression of Bcl-2/Bax in the hippocampal after focal cerebral I/R injury in rats TUNEL staining revealed that neuronal apoptosis after CIRIoccurred in the hippocampal CA1 region Fig. 3(a). In the sham group, only a few TUNEL-positive cells were observed; however, 24 h after MCAO there was a significant increase in the TUNEL-positive cells. In contrast, compared to the I/R group (P < 0.05), 1.5 % of ISPOC significantly reduced the number of TUNEL-positive cells. On the contrary, admin- istering LDN-193189 and SB203580 before ISPOC inhibited these pro- tective effects(P < 0.05) Fig. 3(c). Western blotting was used to evaluate the expressions of apoptosis- related proteins Bcl-2 and Bax on the ischemic side of the hippocampus Fig. 3(b). Bcl-2 is a key protein related to cell survival. In the present study, the levels of Bcl-2 were reduced in the I/R group, compared with those in the sham group 24 h post-MCAO. However, the important apoptotic protein, Bax, was increased significantly in the I/R group. The ratio of Bcl-2/Bax was decreased significantly following I/R (P < 0.05), however, there was an increase in the Bcl-2/Bax ratio when Isoflurane was administered at a concentration of 1.5 % (P < 0.05). On the contrary, administering LDN-193189 and SB203580 before ISPOC inhibited these protective effects of ISPOC(P < 0.05)Fig. 3(d). 4.4. Effects of 1.5% ISPOC, BMP7 inhibitor LDN-193189,P38 inhibitor SB203580 on hippocampal CA1 subfield immunofluorescence(IF) IF staining showed that BMP7, p-Smad1/5/9, and p-p38 were located in the cytoplasm Figs. 4–6. Analysis of BMP7 optical density in the hippocampal CA1 showed that BMP7 significantly increased after I/ R injury, and ISPOC further promoted the expression (P < 0.05). How- ever, the BMP7 inhibitor LDN-193189 and p38MAPK inhibitor SB203580 attenuated the effect of 1.5 % ISPOC on the expression of BMP7 (P < 0.05) Fig. 4(a, b). Fluorescence expression in the hippocampal CA1 of p-Smad1/5/9 also showed the same trend with BMP7 Fig. 5(a, b). However, the BMP7 inhibitor attenuated the expression of p-Smad1/5/9 (P < 0.05). Besides, the expression level of p-Smad1/5/9 in the LDN193189 SB203580 group was the lowest among all groups (P < 0.05) Fig. 5(a, b). Analysis of p-p38 optical density in the hippocampal CA1 showed that the expression level of p-p38 was high in the Sham group Fig. 6(a, b). After I/R injury, the expression level of p-p38 in the I/R group significantly decreased, and 1.5 % isoflurane application increased the expression compared with the I/R and Sham group (P < 0.05) Fig. 6(a, b). However, the BMP7 inhibitor attenuated the expres- sion of p-p38 (P < 0.05). In addition, the expression level of p-p38 in the LDN-193189 SB203580 group was the lowest among all groups (P < 0.05) Fig. 6(a, b). 5. Discussion Ischemic stroke is a serious threat to human health, and preventive efforts have reduced the incidence of stroke and the associated mortality (Krishnamurthi et al., 2013), however, there are several failed large-scale clinical trials experiments (Moskowitz, 2010). Isoflurane as a widely used anesthetic, it has been studied by many researchers, including our previous study, we observed that isoflurane post- conditioning can significantly ameliorate neuronal apoptosis and brain edema, improve performance on neurobehavioral tests after cerebral I/R injury and brain infarct volumes(Zhang et al., 2019; Yuan et al., 2018). In this study, neurological score assessment and TTC staining were used in the present study to observe the effect of ISPOC on MCAO-induced brain injury. We found that at the same time of reperfusion, 60 min ISPOC after 90 min MCAO could immediately improve the neurological score and could reduce infarction. Cerebral I/R results in a characteristic necrotic zone, and a significant reduction in the infarct volume and the neurological score of the brain after 90 min cerebral ischemia and before reperfusion, was observed by 1.5 % isoflurane. Significant reduction in the number of nuclear pyknosis cells and a significant increase in the intact neurons cells in the hippocampal CA1 subfield in the ISPOC group were observed as compared with the I/R group. The previous study has demonstrated that apoptosis is one of the main pathophysiological mechanisms of cerebral ischemia-reperfusion injury and is promoted by the activation of the caspase family of cysteine proteases (Wang et al., 2020; Zheng et al., 2018). In the process of ischemia, apoptosis is most closely related to the anti-apoptotic Bcl-2 and the apoptotic Bax in the Bcl-2 family of proteins. Additionally, the incidence of apoptosis is determined by the proportion of Bcl-2 and Bax expression (He et al., 2019; Lian et al., 2020). In this study, we inves- tigated there was an increase in the Bcl-2/Bax ratio and a decrease in the number of TUNEL-positive cells when isoflurane was administered at a concentration of 1.5 %. On the contrary, administering LDN-193189 and SB203580 before ISPOC inhibited these neuroprotective effects. BMP7 is known to have a vital regulatory function in neuronal dif- ferentiation and growth and has demonstrated neuroprotection induced by IPC or 9-Cis-retinoic acid pretreatment in the MCAO model (Shen et al., 2009). In the present experiment, the relationship of BMP7 with ISPOC-induced neuroprotection was studied in detail. The highest value was observed at 24 h reperfusion. A common inhibitor of the BMP signaling pathway is LDN-193189, which is a small molecule inhibitor of BMP-type I receptor kinases (Malhotra et al., 2015).BMP7 signal trans- duction is known to induce activation and phosphorylation of Smad1/5/9 and is known to have a biological effect besides (Matsumoto et al., 2012). Despite LDN-193189 not being a BMP7 specific inhibitor, it was observed to suppress the BR-Smad-mediated signaling(Kanamori et al., 2016; Omi et al., 2019). Based on the results of this study, it was observed that by inhibiting ISPOC-induced drastic reduction in the number of nuclear pyknotic and TUNEL-positive cells, the LND-193189 inhibited the ISPOC-induced reduction of cerebral infarct volume, Bcl-2/Bax ratio and increased the neurological deficit score. On the other hand, TTC, TUNEL staining, and Bcl-2/Bax ratio results revealed that SB203580, an inhibitor of p38MAPK, combined with LDN-193189 also further suppressed the neuroprotective effects of ISPOC. These re- sults indicated that ISPOC-induced neuroprotection is mediated by BMP7-Smad1/5/9 and p38 MAPK signal transduction pathways by attenuating cell injury and other regulatory complex mechanisms. Immunofluorescence staining and western blot analysis were used to further investigate the mechanisms of BMP7-Smad1/5/9 and p38 MAPK signal in the neuroprotective effects of isoflurane postconditioning. 1.5 % ISPOC up-regulate both the BMP7, the phosphorylation of Smad1/5/ 9, and p38. LDN-193189 blocks BMP7 signaling, inhibits Smad1/5/9 phosphorylation and p38 phosphorylation, SB203580 also down- regulated the expression of BMP7 and phosphorylation of Smad1/5/9 after ISPOC, thus blocking isoflurane postconditioning from playing a neuroprotective role. As we know it, the phosphorylation of P38MAPK could affect the expression of BMP7 and phosphorylation of Smad1/5/9. We observed that LDN-193189 has not eliminated the neuroprotective effect of isoflurane postconditioning. This indicates that isoflurane postconditioning can play a neuroprotective role by up-regulating both the expression of BMP7 and then activating the phosphorylation of Smad1/5/9 and p38, although this is not the only mechanism of BMP7 induced neuroprotection.

In summary, this study provides potential evidence demonstrating BMP7/Smad1/5/9 and p38 MAPK signaling pathways, contributing to isoflurane post-conditioning to reduce apoptosis and ameliorate cere- bral I/R injury. Since the biological crosstalk between BMP7/Smad1/5/ 9 and p38 MAPK signaling pathways complicates the course of events, further research on the isoflurane postconditioning is required to elucidate the underlying mechanisms in its entirety.

Ethical statement