Postoperative cognitive dysfunction (POCD) is a clinical condition that is characterized by impairment in multiple cognitive domains including memory, concentration, language comprehension and learning difficulties [1]. Although acute and mild cognitive decline commonly occurs after surgery, some patients develop more severe and enduring forms of cognitive dysfunction that maybe associated with increased mortality [2]. The underlying mechanism of POCD is still unclear but postoperative neuroinflammation has been shown to be involved with the development of this condition [3]. Previous studies have demonstrated that both surgical trauma and anesthesia could initiate systemic and neuroinflammation in postoperative models [3]. For instance, the general anesthetic agent isoflurane up-regulates pro-inflammatory cytokines brain of mice [4]. Macrophages penetrate the brain and trigger glia activation and up-regulates inflammatory cytokines in the hippocampus after surgery [5,6]. This was associated with cognitive impairment but the degree of dysfunction was reduced by inhibiting macrophages activation and neuroinflammation in the brain [5]. Neuroinflammation also plays a critical role in synaptic dysfunction and tauopathy, in which tau hyperphosphorylation is observed under neuroinflammatory states [7]. Abnormal tau phosphorylation and accumulation are shown to interfere with synaptic function and results in cognitive impairment [7]. Collectively, these data highlight the pivotal role of neuroinflammation in the pathogenesis of POCD and the therapeutic potential of anti-inflammatory strategies to minimize POCD by inhibiting inflammation. These strategies include pharmacological pretreatment that renders an organism more resistant to a subsequent significant insult, a concept that is similar to the practice of Traditional Chinese Medicine (TCM) that emphasizes the “preventive measure”.

Siegesbeckiae Herba (SH, also called Xixiancao in Chinese) is a traditional Chinese medicine (TCM) commonly used for eliminating symptoms including wind-damp, limbs weakness and detoxification as first recorded in the Newly Compiled Materia Medica. Being one of the main plant sources of SH, the dried aerial part of Siegesbeckia Orientails L. (S. Orientails) is usually used for management of chronic inflammatory diseases such as rheumatoid arthritis (RA). In the last few decades, numerous studies have focused on the anti-inflammatory effect of S. Orientails and/or its derivative compounds in experimental RA models, with its use improving both the scores and inflammatory markers of the animal with arthritis [8]. In view of the anti-inflammatory properties of S. Orientails, we hypothesized that S. Orientails could alleviate postoperative cognitive deficits by attenuating systemic inflammation and neuroinflammation. The purpose of current study is to explore the therapeutic potential of S. Orientails in postoperative animals, especially in terms of cognitive performance which is correlates with the presence of inflammation and tau modulation.

Substantial evidence suggest that surgery induces cognitive dysfunction in patients in whom a continuous inflammatory response is present [3]. Apart from various physical barriers, perioperative inflammation was once considered as the second line of defense mechanism to protect human from pathogens after surgery. Nevertheless, it is increasingly recognized that several postoperative complications are related to inflammation including atrial fibrillation [12], hemorrhage [13] and cognitive dysfunction [14]. Various therapeutic strategies targeting inflammation throughout the surgical period have been suggested to combat these complications. In agreement with previous reports, our results suggest that surgery induces systemic and neuroinflammatory response in postoperative animals [3]. Furthermore, activation of pro-inflammatory pathways was observed, which was associated with an increase in tau phosphorylation and cognitive impairment, thus showing that treatment using S. Orientails extract before surgery partially prevented these pathological changes and demonstrated the therapeutic potential of S. Orientails in preventing POCD.

It is well established that peripheral inflammation could trigger a neuroinflammatory response in the brain [15]. Chronic neuroinflammation has been shown to accompany deterioration in cognitive function. For instance, inflammation cytokines induce the hyperactivation of astrcotyes [16], which may trigger the release of other cytokines or gliotransmitter such as GABA that affect neuronal activity and plasticity in the AD model [17]. Inhibiting neuroinflammation in turn resulted in amelioration of astrogliosis and improved subsequent cognitive impairment in AD [18]. On the other hand, therapeutic strategies targeting systemic inflammation may improve neuroinflammation and ameliorate neuronal damage, synaptic dysfunction and cognitive performance in different models, including POCD [19]. Traditional Chinese medicine has been used over many centuries and mounting evidence has demonstrated anti-inflammatory properties. S. Orientails has been widely used in treating arthritis through inhibiting systemic inflammation [20, 21]. From previous studies, it was shown to improve paw edema in experimental RA animal model, accompanied with the reduction of IL-6 in serum [22]. From in vitro assay, S. Orientails was shown to reduce IL-6, TNF-α and NO in RAW264.7 cells. Topical application of S. Orientails active components reduces inflammatory cytokines in carrageenan and complete Freund's adjuvant induced inflammatory models [23]. These data demonstrated that S. Orientails is a potent natural anti-inflammatory herb which may exert broad range of beneficial effects by ameliorating inflammatory responses.

In view of these anti-inflammatory properties, we examined whether extract from S. Orientails can improve the cognitive function in postoperative animal. We have shown a significant increase in the novel object exploration time in postoperative animals with 14 days of extract pretreatment compared with surgery only group, implying an improvement in hippocampal dependent recognition memory. Based on this observation, we examined if the extract could reduce systemic inflammation and neuroinflammation in postoperative mice. Along with better preservation of cognitive function, there was a reduction of pro-inflammatory cytokines IL-6 and IL-8 in the liver and serum of postoperative animals with extract pretreatment in a dose dependent manner (Fig. 3). At the same time, extract pretreatment reduced the mRNA of IL-1β and IL-6 in the hippocampus (Fig. 4). These results demonstrated that extract pretreatment reduced inflammatory responses by suppressing pro-inflammatory cytokines in both the periphery and in the hippocampus, a result similar to that found in experimental RA models.

To further elucidate how the extract could reduce the production of pro-inflammatory cytokines in brain, we investigated if the activation of pro-inflammatory intracellular signaling was modulated. JNK and NF-κB are major pathways involve in inflammation [24,25] and they respond to external stimulus such as reactive oxygen species, bacteria or virus. In neurodegeneration, activation of JNK and NF-κB are observed in the brains of Alzheimer's Disease models [26,27]. Both molecules are phosphorylated and translocated to the nucleus, binding to the promoter and enhancing the transcriptional activities of the genes, thus increasing the synthesis of pro-inflammatory cytokine [24,25]. Over-activation of JNK and NF-κB in the brain are shown to worsen inflammatory responses while inhibition of these molecules ameliorates neuroinflammation and subsequent cognitive dysfunction. Our results demonstrated that surgery leads to the phosphorylation of both JNK and NF-κB in the brain, which was in line with the increase in pro-inflammatory cytokine gene expression. In contrast, extract pretreatment significantly reduced the phosphorylation of both JNK and NF-κB (Fig. 4), thus further confirming that the extract reduces the neuroinflammatory response by impeding the activation of pro-inflammatory pathways in the hippocampus.

One of the possible explanations for the anti-inflammation effect of S. Orientails is the modulation of peripheral immune activities. Previous reports have shown that surgery-induced neuroinflammation was partially triggered by the activation and penetration of peripheral immune cells into the central nervous system [28]. These cells could activate inflammatory pathways and stimulate the production of inflammatory cytokines in the brain. On the other hand, previous studies also indicated that S. Orientails could modulate the activities of peripheral immune cells [29,30], which implies the that S. Orientails may also inhibit surgery-induced neuroinflammation through regulating peripheral immunity. However, apart from interlukins, we could not observe significant modulation of other cytokines such as TNF-α in the brain. It may be accounted by the differential expression time of respective cytokines. For example, from in vitro studies, up-regulation of TNF-α mRNA was observed in lipopolysaccharide (LPS) challenged bone marrow derived dendritic cells as early as 1 hr and returning to basal levels within 24 hrs [31], which may explain the failure of observing TNF-α modulation in our current study. It needs further investigation to confirm if other cytokine expression will be modulated at earlier time points in the postoperative period.

We further asked how the attenuation of neuroinflammation by the extract may modulate tau phosphorylation in the hippocampus. Tau is an important microtubule associated protein in which abnormal phosphorylation and aggregation of tau protein is a pathological hallmark in cognitive dysfunction model [7]. Upon phosphorylation, tau was shown to be dissociated from microtubule which undergo aggregation and oligomerization and exert neurotoxicity or inhibit the function of synapse, leading to synaptic and neuronal dysfunction. It has been shown that inflammatory cytokines such as IL-6 could induce tau phosphorylation which deteriorate neuronal degeneration [32]. In our model, tau phosphorylation was up-regulated in the hippocampus after surgery. With extract pretreatment , reduction of phosphorylated tau was observed compared with surgery alone group (Fig. 5). These results imply that S. Orientails extract may have beneficial effect on postoperative animals through anti-inflammation and a reduction in the tau phosphorylation burden in the hippocampus, which is consistent with the previous findings that reducing tau phosphorylation improved neuronal and synaptic deficit in neurodegeneration such as Alzheimer's disease [33]. For future studies, other tau phosphorylation sites and the aggregation of tau protein in the hippocampus maybe required to give a full spectrum of how S. Orientails can modulate tauopathy in postoperative animals.

Despite the promising effect of S. Orientails in the current study, some issues would still need to be addressed. For example, pulmonary toxicity was reported for the water extract of Herba Siegesbeckiae (HS) in mice [34], and the toxic compounds were identified to be presented mainly in the aqueous-soluble part of the extract [35]. The median lethal dose (LD50) for oral acute toxicity of the water extract of HS in mice was reported to be 146.7 g (herb)/kg, and the toxic effects was observed when the dosage was higher than 24.4 g (herb)/kg in a 2-week sub-acute test. Moreover, the acute toxicity of the 70% ethanol extracts of HS (LD50: 267.00 g (herb)/kg) was determined to be lower than that of the water extract (LD50: 147.91 g (herb)/kg). Although the maximum dosage of S. Orientails was set at 10 g (herb)/kg in this study, which was much lower than the LD50 of the acute and sub-acute dosages of the water extract (LD50: 24.4 g (herb)/kg), the potential toxicity of the S. Orientails should be carefully considered and further investigation is needed in future.

To conclude, this study presented in vivo data on the neuroprotective effect of S. Orientails extract against surgery induced cognitive dysfunction. With pretreatment using higher doses of S. Orientails extract, surgery induced systemic and neuroinflammation were ameliorated, as shown by the inhibition of inflammatory pathway JNK and NF-κB and the reduction of interlukin inflammatory cytokines expression respectively. This is associated with the attenuation of tauopathy in the hippocampus that may underlie the neuroprotective mechanism in our model. Taken together, we have demonstrated that S. Orientails may have beneficial effect in postoperative subjects.