Effects of rosuvastatin combined with fasudil therapy on rabbits with dyslipidemia
Li et al. Lipids in Health and Disease
Effects of rosuvastatin combined with fasudil therapy on rabbits with dyslipidemia
Zhiming Li 2
Hua Lian 0
Qin Liang 0
Fanfang Zeng 1
Dongdan Zheng 0
0 Department of Cardiology, The Easten Hospital of the First Affiliated Hospital of Sun Yat-sen University , 58 Zhongshan Road 2, Guangzhou 510080 , China
1 Department of Cardiology, Shenzhen Sun Yat-sen Cardiovascular Hospital , Shenzhen 518000 , China
2 Department of Cardiology, Huizhou Municipal Central Hospital , 41st Eling North RD, Huicheng District, Huizhou 516000 , China
Background: Present study was conducted to investigate the effects of rosuvastatin combined with fasudil on rabbits with dyslipidemia. Methods: Dyslipidemia model of rabbits were produced by prescribing atherogenic diet for 2 weeks. Thereafter, 40 rabbits with dyslipidemia were randomly and evenly divided into four groups as follow: untreated group (orally prescribed 3 ml of normal saline), rosuvastatin group (orally prescribed 3 mg/kg body weight daily, dissolved in 3 ml of normal saline), fasudil group (intravenously prescribed 0.5 mg/kg body weight daily, dissolved in 3 ml of normal saline), and combined group (the same doses of rosuvastatin and fasudil as aforementioned). At baseline, 2 weeks of dyslipidemia establishment and 2 weeks of medical therapy, fasting venous blood was drawn for laboratory examination. Results: After 2 weeks' atherogenic diet treatment, lipid disorders and impaired fasting glucose were observed. Systemic inflammation and oxidation were also promoted as revealed by increased serum levels of high sensitive C-reactive protein (Hs-CRP) and malondialdehyde (MDA). Notably, endothelial function has been impaired significantly as reflected by decreased nitric oxide (NO) production and increased serum asymmetric dimethylarginine (ADMA) level. RhoA associated kinase (ROCK) activity was also profoundly enhanced (P < 0.05). Inter-group comparisons showed that when compared to untreated group, modest improvements of endothelial function, inflammation and oxidation were observed in rosuvastatin and fasudil groups (P > 0.05). These benefits were improved more prominently in combined group (P < 0.05). Intra-group comparisons also showed that when compared to 2 weeks of dyslipidemia, slight improvement of endothelial function, inflammation and oxidation in rosuvastatin and fasudil groups were observed (P > 0.05). The improvements were more prominent in the combined groups (P < 0.05). Conclusion: Rosuvastatin combined with fasudil conferred synergistic effects on endothelium-protection and inflammation- and oxidation-amelioration in the setting of early stage of dyslipidemia.
Dyslipidemia; Endothelial function; RhoA-associated kinase
Dyslipidemia, featured by increased serum level of low
density lipoprotein cholesterol (LDL-C), is one of the most
important risk factors for atherosclerosis and atherosclerotic
cardiovascular disease (ASCVD) worldwide [1, 2]. In
the early stage of dyslipidemia, endothelium becomes
dysfunction in accompany with systemic inflammation
and oxidation [3, 4]. Therefore, effectively treating
dyslipidemia at an early stage by lifestyle modification or
medicines should be beneficial for restoring endothelial
function and preventing atherosclerosis progression .
With regard to lipid-lowering medicines, statins is the
most commonly used currently. Other than lowering
cholesterol, statins has other efficacies now universally
known as pleiotropic effects which are largely dependent
upon its potent effects on inhibiting isoprenylation of the
small GTP-binding proteins during cholesterol biosynthesis
[5, 6]. RhoA is the key member of small GTP-binding
proteins and through activating its main downstream
effector, named Rho associated kinase (ROCK), RhoA
exerts multiple adverse effects on cardiovascular system
[7, 8]. Fasudil, the specific inhibitor of ROCK, has been
found potential to protect endothelium and inhibit
inflammatory cells infiltration in the basic researches [9, 10].
However, no consistent clinical benefits have been achieved
in previous studies with the use of fasudil on ASCVD
therapy. Regarding the potent efficacies of statins and
fasudil on vascular system, it is reasonable and rational to
postulate that statins combined with fasudil may render
synergistic effects on restoring endothelial function and
preventing atherosclerosis progression. However, the
evidence is lacking. Therefore, we conducted a basic
research using rabbits with dyslipidemia model and giving
different therapeutic strategies to address our hypothesis.
The protocol for dyslipidemia model production was
approved by the Ethic Committee of the First Affiliated
Hospital of Sun Yat-sen University. Totally 50 male New
Zealand White rabbits, weighing 1.3–1.5 kg and 5–8
weeks old, were used in present study. After 1 week’s
accommodation, according to previous research , 40
rabbits, used as dyslipidemia model production, were
randomly selected and treated with 50 g per kilogram
body weight per day of standard chow diet enriched with
0.5 % cholesterol (Sigma Aldrich, St. Louis, MO, EUA) for
2 weeks. The other 10 rabbits, used as the control group,
were given standard chow of 50 g per kilogram body
weight per day. All animals received water ad libitum.
After dyslipidemia model was successfully established as
revealed by LDL-C elevation, 40 rabbits with dyslipidemia
were randomly and evenly divided into four groups:
untreated group (orally prescribed 3 ml of normal saline),
rosuvastatin group (orally prescribed 3 mg/kg body weight
daily, dissolved in 3 ml of normal saline), fasudil
group (intravenously prescribed 0.5 mg/kg body weight
daily, dissolved in 3 ml of normal saline), and combined
group (the same doses of rosuvastatin and fasudil as
aforementioned). The therapeutic duration was 2 weeks.
At baseline, 2 weeks of dyslipidemia production and
2 weeks of medical therapy, fasting venous blood was
drawn for laboratory examination. Lipid profiles including
triglyceride (TG), total cholesterol (TC), high density
lipoprotein cholesterol (HDL-C) and LDL-C, fasting blood
glucose (FBG), liver enzymes including alanine
aminotransferase (ALT) and aspartase aminotransferase (AST) were
detected by Automatic Biochemistry Analyzer (Beckman
coulter UniCel DxC 800 Synchron). Parameters of
endothelial function including nitric oxide (NO) production (ELISA
kit, Nanjing Jiancheng Bioengineering Institute) and serum
level of asymmetric dimethylarginine (ADMA, ELISA kit,
Shanghai Ying-gong Industrial Company) were assessed in
accordance to the manufacture’s introduction. Serum levels
of high sensitive C-reactive protein (Hs-CRP Assay
Kit, Immune-turbidimetry method, Nanjing Jiancheng
Bioengineering Institute) and malondialdehyde (MDA
Assay Kit, TBA method, Nanjing Jiancheng Bioengineering
Institute) were measured to evaluate the changes of
systemic inflammation and oxidation over time. Serum
ROCK activity was detected by enzyme-linking
immuneabsorbent assay (ELISA kit, Yuping BioMedical Company,
Data were expressed as means ± S.E.M., and inter-group
and intra-group comparisons were analyzed by one-way
ANOVA followed by Dunnett’s multiple comparison test
or by student t-test when appropriately using SPSS 19.0
statistical analysis program. P < 0.05 was considered as
Changes of parameters before and after dyslipidemia
After 2 weeks of atherogenic diet treatment, inter-group
differences were compared. As presented in Table 1,
after 2 weeks of atherogenic diet treatment, lipid disorders
and impaired fasting glucose were observed as indicated by
increased serum levels of TG, TC, LDL-C and FBG.
Systemic inflammation and oxidation were also promoted
by atherogenic diet treatment as revealed by increased
serum levels of Hs-CRP and MDA. Notably, with 2 weeks
of dyslipidemia, endothelial function has significantly
impaired as reflected by profoundly decreased NO
production (Fig. 2) and significantly increased serum
level of ADMA. ROCK activity was also profoundly
enhanced as shown in Fig. 1. Compared to the control
group, all the between-group differences were statistically
significant (P < 0.05).
Inter-group comparison of parameters after medical
After 2 weeks of medical therapy, inter-group differences
regarding the effects of different therapeutic strategies
were evaluated and compared. As shown in Table 1, Figs. 1
and 2, lipid profiles were improved in the rosuvastatin and
combined groups but not in the untreated and fasudil
groups (P < 0.05). Serum level of FBG was similarly
declined in the 3 medical therapy groups, however, no
significant between-group difference was observed when
compared to the untreated group. Modest between-group
differences were observed in the parameters of Hs-CRP,
MDA, NO production, ADMA and ROCK activity in the
rosuvastatin and fasudil groups when compared to the of Hs-CRP, MDA, NO production, ADMA and ROCK
untreated group (P > 0.05). Notably, rosuvastatin combined activity were slightly improved in the rosuvastatin and
with fasudil therapy had synergistic effects as reflected by fasudil groups when compared to those at 2 weeks of
the more prominent improvement in the parameters dyslipidemia (P > 0.05). However, the improvement in
of Hs-CRP, MDA, NO production, ADMA and ROCK the parameters of Hs-CRP, MDA, NO production,
activity when compared to the untreated group (P < 0.05). ADMA and ROCK activity were more prominent in
the combined groups when compared to those of 2 weeks
Intra-group comparison of parameters after medical of dyslipidemia (P < 0.05), suggesting that rosuvastatin
therapy combined with fasudil therapy had synergistic effects on
Intra-group comparisons of parameters were also com- improving endothelial dysfunction, inflammation and
pared. As shown in Table 1, Figs. 1 and 2, the parameters oxidation induced by dyslipidemia.
Fig. 1 Inter- and intra-groups comparisons of ROCK activity. Denote: * P < 0.05 versus other groups at the same time point; & P < 0.05 versus the
Untreated groups at the same time point; ※ P < 0.05 versus 2 weeks of dyslipidemia in the same group
Dyslipidemia is prevalent worldwide and is one of the
most important risk factors for multiple diseases such as
ASCVD. Endothelial dysfunction is implicated in the
early stage of dyslipidemia as well as in the process of
atherosclerosis development [12, 13]. Therefore, treating
dyslipidemia effectively is of clinical importance. Results
from present research show that rosuvastatin not only
could ameliorate dyslipidemia but also could improve
endothelial function as well as systemic inflammation and
oxidation, which strongly supporting the cardio-protective
effects of rosuvastatin therapy. Moreover, although no
effects on lipid-modification, fasudil therapy is effective on
improving endothelial function, inflammation and
oxidation. Expectedly and importantly, fasudil combined with
rosuvastatin therapy confers synergistic effects on vascular
system in the early stage of dyslipidemia which should
have important clinical relevance.
Knowingly , LDL-C elevation is detrimental to
endothelium and could promote systemic inflammation
and oxidation through multiple mechanisms. Therefore,
decreasing LDL-C level by statins therapy is of clinical
importance in preventing cardiovascular events. Other
than lipid-lowering effects, statins has other efficacies
which are predominantly associated with its effects on
attenuating small GTP-binding proteins isoprenylation
during cholesterol biosynthesis. As is well known that
RhoA isoprenylation, the most commonly studied small
GTP-binding protein, could lead to its downstream target
ROCK activation thereby eliciting multiple adverse effects
Fig. 2 Inter- and intra-groups comparisons of NO production. Denote: * P < 0.05 versus other groups at the same time point; & P < 0.05 versus
the Untreated groups at the same time point; ※ P < 0.05 versus 2 weeks of dyslipidemia in the same group
such as vessel constriction, inflammatory cells migration
and infiltration, platelet activation, and endothelial
dysfunction [15, 16]. Therefore, not only decreasing
RhoA isoprenylation is critical, inhibiting ROCK activity is
also crucial in preventing atherosclerosis development
and reducing cardiovascular events. For example,
Naoki Sawada et al. reported that Y-27632, a specific
ROCK inhibitor, might be an effective therapeutic
strategy for treating vascular proliferative disorders and
hypertension . In addition, results from Anju Nohria
showed that inhibition of the Rho/ROCK signaling
pathway by fasudil should provide a useful strategy to restore
NO bioavailability in humans with atherosclerosis .
Taken together, we postulated that statins combined with
ROCK antagonist could provide synergistic effects in
treating dyslipidemia and its associated inflammation and
oxidation, and results from our preliminary research
supported this hypothesis. Of note, in our present
research, although fasudil had no benefit on dyslipidemia
modification, nonetheless, we observed that endothelial
function as well as systemic inflammation and oxidation
were all improved after 2 weeks of fasudil therapy, which
strongly suggesting that the benefits derived from fasudil
therapy was independent of lipid-lowering. Notably,
these benefits were further enhanced by combined
therapy which we considered was associated with further
attenuation of ROCK activity. In light of previous reports
[6, 19–21], we considered that the two following
mechanisms might at least partially explain our findings. In the
first place, by reducing RhoA isoprenylation during
cholesterol biosynthesis, rosuvastatin could robustly inhibit
ROCK activation thereby enhancing endothelial function
and ameliorating systemic inflammation and oxidation.
On the other hand, since fasudil is a specific and potent
antagonist for ROCK, therefore, as adjunctive to inhibiting
ROCK activation by rosuvastatin therapy, fasudil
therapy could block the downstream effects provoking by
already-activated RhoA/ROCK signaling pathway. Taken
together, through different and complemented
mechanisms, rosuvastatin combined with fasudil therapy
conferred synergistic and protective effects on vascular system
in rabbits with dyslipidemia. O-linked N-acetylglucosamine
(O-GlcNAc) is a reversible post-translational modification
of serines/threonines substrate and has been found
associated with the development of dyslipidemia and other
cardiovascular diseases [22, 23]. Therefore, detecting the
change of O-GlcNAc should provide mechanisms
regarding the additive benefits of combined therapy on rabbits
with dyslipidemia. Thus, it is the potential limitation of
our present research for not investigating the change of
O-GlcNAc before and after therapy and in the future
it is important and warranted to further investigate the
relationship between O-GlcNAc and the application of
rosuvastatin and fasudil therapy.
Interestingly, we observed that dyslipidemia resulted
in fasting blood glucose elevation, and with rosuvastatin,
fasudil or combined therapy, fasting blood glucose was
modestly reduced. Whether this finding had clinical
implications regarding the development of metabolism
syndrome or diabetes mellitus in the setting of dyslipidemia
needed further investigation. Moreover, concerning the
safety of rosuvastatin and fasudil therapy, liver enzyme
was serially detected and no significant elevations of
ALT and AST were observed, which indicated that
2 weeks of rosuvastatin and fasudil therapy was no harm
to liver function.
Preliminary data from our present research revealed
that rosuvastatin combined with fasudil therapy
conferred synergistic effects on endothelium-protection
and inflammation- and oxidation-amelioration in the
setting of early stage of dyslipidemia. Further study is
warranted to investigate whether these efficacies could
translate into clinical benefits.
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