Berberine Exhibits Neuroprotective Effects Through Reduced Autophagy and Mediated Microglial Polarization in a Rat Model of Intracerebral Hemorrhage

Introduction: Intracerebral haemorrhage (ICH) induces autophagy excessive activation and microglia mainly switched into proinammatory M1 type, which can cause severe secondary injury. Current evidence has implied that berberine has a protective effect against ischaemic stroke through mediated autophagy and microglial polarization. However, the neuroprotective effect of berberine in intracerebral haemorrhage (ICH) remains unclear. Methods: In this study the effect of berberine on a rat model of intracerebral haemorrhage was investigated through Immunouorescence, qPCR, ELISA, and western blot. Results: Berberine administration signicantly reduces cellular apoptosis, brain edema and neurological decits via inhibited autophagy, promoted M1 type microglia to M2 type, and subsequently exerts anti-inammation effects in a rat model of ICH. Conclusions: These results suggest that berberine reduced secondary injury and improved neurological outcomes in ICH model.


Introduction
Intracerebral haemorrhage (ICH) was a devastating type of stroke with approximately 40% mortality at one month and high disability after hospital. Mass effect from hematoma induced the primary brain damage, while secondary brain injury, including toxic effects of hemolytic products, oxidative stress and in ammatory activation, was an important factor that leaded to neurological outcome progress [1].
Currently, Effective treatment in ICH is limited, hence it is urgently to carry out related studies.
Autophagy is self-degradation of damaged organelles and misfolded or injured protein in order to maintain cellular hemostasis. Autophagy participated in many pathophysiological processes, including cell survival, differentiation, proliferation and in ammation [2]. A variety of studies focus on the role of autophagy in neurological disease. Research presented that promoting autophagy had protective effect in animal models of subarachnoid hemorrhage [3], traumatic brain injury [4] and Parkinson diseases [5], while the effect of promoting autophagy is controversial in ischemic stroke. However, recent studies presented that excessive autophagy occurs after ICH which possibly contributes to brain injury and cell apoptosis.
Microglia were cerebral resident macrophages and were originated from myeloid progenitor cells.
Microglia were activated into two phenotypes: M1 microglia was pro-in ammation and M2 microglia is anti-in ammation. In the early phase of ICH, microglia were activated and mainly switched into M1 microglia polarization. M1 type released proin ammatory cytokines and chemokines, which lead to secondary brain injury [6]. Studies demonstrated that inhibition of autophagy could switched M1 microglia to M2 phenotype in ischemic stroke, which exerted anti-apoptosis effect [7].
Berberine is a nature alkaloid collected from herb Coptis chinensis, which is widely used as an antiin ammatory, antidiabetic and antihyperlipidemic drug. Berberine exert protective effect in ischemic stroke via mediating autophagy and microglia polarization [8,9]. Dent et al found that berberine could inhibit autophagy through downregulating the expression of BECN1 [10]. Previous studies presented that inhibition of autophagy ameliorated neurological outcome in ICH [11]. However, the role of Berberine in ICH remained uncleared. Hence, we initially explored that berberine possibly exerted neuroprotection via autophagy inhibited and promoting M1-to-M2 microglia phenotype.

Animals and groups
The protocols of animal usage were approved by the Institutional Animal care and Use Committee of the Second Xiangya hospital of Central South University in compliance with NIH guidelines. Sprague-Dawley adult male rats with 250 to 300g were employed in our study. Animals were kept at a constant temperature and a 12/12h light/dark cycle. Rats (n = 54, 2 sacri ced) is randomized divided into three groups: (1) ICH + Berberine (Beri group, n = 17), rats were administered with berberine intragastrically (sigma, 50mg/kg) daily for 10 consecutive days before ICH and 3 days after ICH; (2) ICH + vehicle (vehicle group,n = 17); (3) sham-operated group (sham group, n = 18).

ICH models
Rats were anaesthetized with chloral hydrate and placed on a stereotaxic frame. 0.2U in 2.0 µL collagenase type IV was injected into the right hemisphere at 3.0 mm lateral to the midline, 0.2 mm anterior to bregma and 6 mm deep by micro pump with 5 minutes. The needle was maintained in brain for 5 minutes after injection. The sham group carried out the same procedures with no collagenase type IV injection.

Behaviour analysis
Neurological de cit scores were evaluated by modi ed neurological severity score (mNSS, n = 8) 24 and 72 hours after ICH. The mNSS is an 18-point scale and maximal de cit is 18. Rats from each group were assessment.
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining Perihematomal para n section (4µm, n = 4) was dewaxed with xylene and ethyl alcohol. Apoptosis was examined with a TUNEL staining kit (Roche, Swit) according to the manufacture's instructions. The slide was examined with a uorescence microscope (Olympus, Japan).

Immuno uorescence
Perihematomal para n section (4µm, n = 4) was dewaxed with xylene and ethyl alcohol. Brain section was antigenically repaired with EDTA buffer, added with spontaneous uorescence quenching reagent and serum. Primary and secondary antibody were added. Antibody were included LC3B (Proteintech Inc, US, 18725-1-AP). Cell nucleus was stained with DAPI. Immuno uorescence was observed with a uorescence microscope (Olympus, Japan).
qPCR Total RNA (n = 5) was collected from perihematomal brain tissue 72 hours after ICH. The speci c primer was used to synthesize the complementary DNA. PCR conditions were performed according the manufacturer's instructions. The analysis of qPCR was used by a PCR instrument. Data was normalized to actin. mRNA expression was relative to sham group. The primer sequences were as follows: iNOS

Statistical Analysis
GraphPad Prism 5.01 software was used for statistical analyses. All data were presented as the mean ± standard deviation (mean ± SD). Comparisons between two groups were analyzed with Mann-Whitney test or ANOVA followed by Bonferroni test. Statistical signi cance was set as P<0.05.

Results
Berberine reduced mNSS scores and apoptosis in ICH This study found that Beri group had signi cantly lowered mNSS scores (Fig. 1A) at 72 hours after ICH compared with the vehicle group. Meanwhile, Berberine obviously reduced cerebral edema 72 hours after ICH (Fig. 1B). Additionally, berberine markedly reduced apoptotic cells in the perihematoma (Fig. 2) which was analysis with TUNEL staining. These were suggested that berberine exerted obviously protective effects in ICH.
Berberine declined excessively activated autophagy in ICH LCII and beclin-1 are two markers to determine the activation of autophagy. The LC3II staining was signi cantly increased in perihematomal area after ICH (Fig. 3A). The LC3-II/LC3-I ratio and expression of beclin-1 were markedly augmented after ICH (Fig. 3B). These results indicated that autophagy was evidently activated in perihematomal area after ICH. Beri group show signi cantly lower LC3II staining, LC3-II/LC3-I ratio and expression of beclin-1 compared with vehicle group (Fig. 3). These results implied that berberine signi cantly reduced autophagy compared with vehicle in ICH.

Berberine promoted M1 microglia switched into M2 microglia after ICH
The makers of microglia M1 and M2 were determined by qPCR, expressions of M1-related markers including CD32 and iNOS was obviously reduced in Beri groups while M2-related markers including CD206 and Arg1 was signi cantly increased in Beri groups compared with Vehicle group (Fig. 4).
Meanwhile, levels of M1-related cytokines including IL-6, IL-1β and TNF-α, investigated by Elisa, were signi cantly reduced after berberine treatment in ICH, while M2-related cytokines, the levels of IL-10, were signi cantly increased in Beri group compared with vehicle group (Fig. 5). Therefore, these indicated that berberine signi cantly facilitated M1 microglia transformed into M2 microglia, which could exert antiin ammation effect after ICH.

Discussion
Previous studies demonstrated that berberine could reduce apoptosis in neurological diseases. Liang et al stated that berberine exhibited neuroprotective effect via against amyloid β-protein induced apoptosis [12]. Studies presented that berberine could improve brain traumatic brain injury against neuronal damage via anti-in ammation and Sirt1/P38 signaling [13]. Meanwhile, berberine inhibited nigrostriatal dopaminergic neuronal loss and protected against hippocampal apoptosis in mice model of Parkinson's disease [14]. Berberine signi cantly attenuated neuronal apoptosis in status epilepticus [15].
Additionally, berberine could signi cantly reduce cell apoptosis after ischemic stroke via phosphoinositide 3-kinase signaling pathway [16]. Simultaneously, our study initially found that berberine could decrease cell apoptosis in perihematomal area after ICH, eventually improved neurological outcome.
Berberine ameliorated variety of diseases through mediating the autophagy [17]. Previous study presented that berberine decreased NLRP3 in ammasome activation by promoting autophagy in macrophages[18].
Jin et al found that berberine alleviated high glucose-induced podocytes injury through enhancing autophagy [19]. However, it is reported that berberine could protect kidney from cisplatin-induced damage through inhibition of autophagy [20]. Berberine signi cantly protected nucleus pulposus cell from oxidative stress damaged through reducing autophagy [21]. Jia et al found berberine improved hypoxiainduced myocytes damage through attenuating excessive autophagy [22]. Additionally, berberine exerted protective effects in ischemic stroke through alleviating autophagy [9]. In this study, we found that berberine play neuroprotective role in ICH by inhibiting autophagy.
Autophagy was possibly associated with microglia polarization. Jin et al found that enhancement of autophagy signi cantly promoted M2 microglia polarization and inhibited neuroin ammation in Parkinson's diseases [25]. Augment of autophagy facilitated M1-to-M2 phenotypic shift in LPS-induced microglia [26]. Nevertheless, inhibition of autophagy markedly promoted M1 switch into M2 microglia polarization and reduced pro-in ammatory cytokines products in ischemic stroke [7]. In ischemic white matter damage, suppression of autophagy could accelerate M1-to-M2 microglia polarization [27]. Therefore, our study showed berberine promoted M1-to-M2 microglia transformation possibly through reduced autophagy.

Conclusions
Our study initially revealed that berberine treatment in ICH exerted neuroprotective effects through inhibited autophagy and promoted M1 transferred to M2 polarization, which signi cantly ameliorated neuroin ammatory injury. The study provides evidence that berberine is probably a novel therapy for ICH. Treatment with Beri signi cantly reduced mNSS and brain water content of Ipsilateral cerebral. A. mNSS was evaluated 72 hours after ICH. B. Brain water content was examined 72 hours after ICH. Values are presented as the mean±SD; *p<0.05, **p<0.01 in all graphs.

Figure 2
Treatment with Beri markedly attenuated apoptosis in the perihematomal area after ICH. Representative photographs showing immunostaining for TUNEL+ (Green) in the perihematomal area 72 hours after ICH (bar=20 µm) and DAPI (blue). Values are presented as the mean±SD; *p<0.05, **p<0.01 in all graphs.  Treatment with Beri attenuated M1 markers expression and increased M2 markers expression in the perihematomal area after ICH. qPCR was used to examined the M1 markers expression (iNOS and CD32) and M2 markers (CD206 and Arg1). Values are presented as the mean±SD; *p<0.05, **p<0.01 in all graphs.