In our study, the plasma levels of C3 and C4 between diabetic stroke and non-diabetic strokes were analyzed to explore the similarities and differences between groups. The main results are as follows: (1) in the diabetic stroke group, high plasma C4 level at admission showed an association with patient prognosis independently of traditional risk factors; (2) in AIS patient, with or without diabetes, high plasma C3 levels were associated with concurrent infection. To our knowledge, this study was the first time to analyze the relationship between C4 and clinical outcomes in diabetic stroke patients.
Complement, as an important branch of the innate immune system, plays important roles in development, homeostasis, and regeneration in the central nervous system (CNS)(Carpanini et al. 2019). Given the roles of complement, enhancing complement activity may be of benefit in some certain situations(Hammad et al. 2018). However, in the context of CNS pathology, complement dysregulation may lead to over-activation and contribute to neuroinflammation(Carpanini et al. 2019; Hammad et al. 2018; Mellbin et al. 2012; Szeplaki et al. 2009).During neuroinflammation, not only microglia and astrocytes, but also neurons, oligodendrocytes, and endothelial cells in the brain, can express complement components and receptors, many of which are up regulated by inflammatory signals(Carpanini et al. 2019; Lubbers et al. 2017). In addition, as a result of increased blood brain barrier permeability, circulating neurotransmitters and large proteins such as complement are infiltrated into the injured brain during pathology(Carpanini et al. 2019; Ueno et al. 2016).
C3 and C4 are acute phase proteins and important components of the complement pathways of the immune system(Ritchie et al. 2004). Even though many cell types express these proteins including adipose tissue and vascular cells, hepatic production is the main source of C3 and C4(Lubbers et al. 2017; Ritchie et al. 2004).Previous animal models and clinical studies have revealed that plasma C3 and C4 were increased in patients with acute stroke(Alawieh et al. 2015; Cervera et al. 2010; Lin et al. 2018).Meanwhile, higher plasma complement levels were associated with an unfavorable outcome, and the predictive value of these markers may depend on stroke subtype(Stokowska et al. 2011; Stokowska et al. 2013). In line with previous research results, our analyses also showed higher C3 was associated with concurrent infection. The relationships had remained significant after adjustment for other risk factors. However, it is difficult to determine the causal relationship among enhancing complement activity, cerebral ischemia injury and concurrent infection on the available evidence. We can only preliminarily infer that the complement activity may be closely related to concurrent infection in stroke patients.
Complement factors have their different roles within the complement cascade(Carpanini et al. 2019). Studies of human populations have revealed that C3 and C4 are associated with increased levels of cardiovascular risk factors, like obesity, hypertension, and diabetes(Copenhaver et al. 2019; Engstrom et al. 2005; Nilsson et al. 2014). However, high C4 levels may be associated with the incidence of cardiovascular disease, independently of traditional cardiovascular risk factors(Engstrom et al. 2007). Baseline C4 level was an independent predictor of stroke in a broad population of patients referred for coronary angiography(Cavusoglu et al. 2007). These findings are partly consistent with our main results that high plasma C4 levels, but not C3, in the diabetic stroke patients were independently related with concurrent infection and unfavorable outcomes. It is interesting to note that this association was lost in non-diabetic stroke group.
A recent study with a large sample size by Mellbin et al. showed that not only the artery disorder but also diabetes per se is associated with complement activation and inflammation(Mellbin et al. 2012). Some of the complement regulatory proteins could be affected by glycation in diabetic stroke(Mellbin et al. 2012). However, the high admission levels of C4 may, at least in part, be a result of the activation of the complement cascade due to acute ischemia(Carpanini et al. 2019). Meanwhile, different parts of the complement system may play different roles in the setting of cardiovascular disease and diabetes(Lau et al. 2019). Although there were few studies on the C4 and related mechanisms in patients with diabetic stroke, some reasonable deductions have been put forward. Considering that C3 and C4 is expressed and produced in abdominal adipose tissue, cytokines that stimulate the hepatic production of them may also stimulate the production of lipids and reduce insulin sensitivity(Engstrom et al. 2007). Increased C3 and C4 levels are both associated with the metabolic syndrome including abdominal obesity, independently of inflammatory activity. Moreover, in a Hungarian study, they reported a positive correlation between serum C4 levels and BMI, independently of C3 in regression analyses(Phillips et al. 2009). In the diabetic mouse models, a marked association between the increased serum complement activity and the ischemic brain injury was demonstrated(Lin et al. 2018). Despite the fact that plasma C3 and C4 are both increased, complement activation in ischemic stroke occurs predominantly by the classic pathway, which involves cleavage of C4 to C4b(Cavusoglu et al. 2007; Pedersen et al. 2009). In a word, C4 is involved in both cerebral ischemia injury and metabolic events and may play a more important role in the pathogenesis of diabetic stroke(Cavusoglu et al. 2007; Cojocaru et al. 2008). However, further studies are needed to explore the mechanism of plasma levels of C4 in the incidence of diabetic stroke.
Our data should be interpreted with some caution due to limitations of the study. Since the participants in this study were recruited only from one clinical unit, there may have retrospective bias inherent due to the insufficient sample size. NIHSS was used to assess the patient's neurologic function and our study did not explore the relationship between cerebral infarct volume and poor prognosis. If infarct volume per se has a major implication on complement components levels, this may contribute to our findings of a strong association with outcome. However, as results had shown, we did not find strong correlation between NIHSS score at admission and plasma complement levels in the acute phase, which spoke against any major effect of infarct size on these parameters. In addition, DM types were not distinguished. However, considering that people with type 1 DM usually show symptoms in early life and all of our patients are middle-aged adults, all cases of DM in this study are very likely to be type 2 DM(Li et al. 2017).Further studies should be performed to expand the sample size and investigate the relevant immune pathways.