




































 Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 6 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 6 

 

CTMJ | traditionalmedicinejournals.com  

 

By inhibiting NLRP3 inflammasome via autophagy activation, 

carnosol improves sevoflurane-induced cognitive impairment in old 

rats. 

Dr.V.Ravikumar 1, Dr.P.Aravinda Reddy 2,Shilpadas 3, Ashaful alom 4,  
Assistant professor 1,2,3,4, 

Department of Pharmacy, 
Samskruti College of Pharmacy, 

Kondapur (V), Ghatkesar (M) Medchal Dist, Telangana, India. 
 

Abstract:  

Specifically, we want to learn how carnosol works and how it affects postoperative cognitive dysfunction 

(POCD). Methods: The sevoflurane (SEV) paradigm of cognitive impairment in rats was developed. We 

used a dihydroethidium (DHE) test to measure the impact of carnosol on ROS levels in rats that had been 

produced by sevoflurane. The impact of carnosol on mitochondrial damage was evaluated using 

adenosine triphosphate (ATP) generation and immunoblot tests. Cognitive impairment in the rats was 

assessed by a water maze experiment. The immunoblot test was used to evaluate the action mechanism. 

The results showed that carnosol inhibited ROS generation and mitochondrial damage caused by 

sevoflurane in rats, and it also triggered autophagy. Carnosol also inhibited SEV-induced cognitive 

impairment via activating autophagy and SEV-induced NLRP3 inflammasome activation, respectively. 

Conclusion: Carnosol, by inhibiting the NLRP3 inflammasome, improves SEV-stimulated cognitive 

impairment and may one day be used as a treatment for cognitive impairment. Carnosol, Sevoflurane 

(SEV), NLRP3 inflammasome, and autophagy are some of the terms used to describe this phenomenon. 

 

INTRODUCTION  

Postoperative cognitive dysfunction (POCD) is a 

type of complication with long-term 

consequences, defined as impaired memory, 

attention and information processing that occurs 

after anesthesia [1]. Studies show that about 10 

% of surgery patients as well as 40 % of older 

patients over the age of 65 develop POCD [2]. 

Compared to patients without POCD, patients 

with POCD have significantly higher mortality 

rates and higher dependence on social security 

[3]. Despite extensive research efforts, the 

pathogenesis of POCD remains unknown [4]. 

New therapeutic agents still need to be 

developed to treat POCD. Rosemary (Salvia 

rosmarinus) is a Mediterranean plant that is now 

widely available in several countries [5,6]. 

Rosemary extract can be used to treat 

inflammation-related diseases [7]. Carnosol is a 

phenolic diterpenoid which exists in rosemary 

extract and has been shown to have anti-

inflammatory and antioxidant effects [8]. 

Carnosol treatment inhibited the eosinophils in 

the bronchoalveolar lavage fluid of mice after 

ovalbumin treatment [9]. Previous studies have 

shown that carnosol alleviates 

sevofluranestimulated cognitive dysfunction in 

aged rats via NF-κB pathway. It also causes the 

activation of autophagy in breast cancer [8]. 

However, the potential effect and mechanism of 

action of the compound on POCD remains 

unknown. There is growing evidence of a causal 

relationship between SEV-stimulated cognitive 

impairment and NLRP3 inflammasome [10]. In 

response to cellular stress, NLRP3 recruits ASC 

as well as pro-caspase-1, which causes the 

cleaved caspase-1 to activate and process the 

maturation of IL-1β and IL-18 [11]. NF-κB is 

involved in the control of many cellular 

processes [11]. The activation of NF-κB family 

is a key step in regulating pyroptosis. However, 



 Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 6 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 6 

 

CTMJ | traditionalmedicinejournals.com  

 

the effects of carnosol on autophagy and its 

relationship with NLRP3 is unclear. The aim of 

this study was to investigate the role of carnosol 

in POCD progression using a sevoflurane-

induced cognitive impairment rat model of rats.  

 

EXPERIMENTAL  

POCD rat model Male Sprague– 

Dawley rats (12 months old, 250 – 280 g) were 

purchased from Vital River (Beijing, China). 

The procedures in this study were approved by 

the Ethics Committee of Xi'an International 

Medical Center Hospital, Shanxi, China 

(approval no. 202315), and conducted in 

accordance with the guidelines of Declaration of 

Helsinki in Biomedical and Behavioral Research 

[12]. For the establishment of POCD model, 

sevoflurane (2 % SEV) was inhaled by old rats 

(8 months old) for 5 h, and carnosol 

administered to the rats in doses of 5 and 10 

mg/kg every 2 days, respectively. The rats were 

divided into the following groups: control, SEV, 

SEV + carnosol (5 mg/kg), as well as SEV + 

carnosol (10 mg/kg) groups.  

Morris water maze (MWM) tests  

The MWM tests were performed in a swimming 

pool with four quadrants and the activity of the 

rats was recorded. The rats were trained for 4 

days in order to determine the location of the 

platform; if the rats could not find the location 

within 90 s, they were placed on the platform. 

Finally, the platform was removed. The time and 

number was recorded.  

Determination of ROS  

For ROS staining, the cells were mounted and 

stained with Dihydroethidium (DHE). DHE 

fluorescence of 20 cells per field in five random 

fields was quantified by the use of AxioVision 

software.  

JC-1 staining  

To analyze mitochondria damage, samples were 

placed in 12-well plates and kept for 24 hours 

for cell adhesion. After rinsing, the cells were 

incubated with 2 μM JC-1 for 15 min at 37 °C. 

After rinsing in phosphate buffer (PBS) 3 times, 

the cells were photographed and the JC-1 

positive cells were counted per field.  

Determination of ATP production  

For the determination of ATP production, an 

ATP detection kit (Beyotime, Beijing, China) 

was utilized in accordance with the 

manufacturer's protocol.  

Immunoblot assay  

The samples were electrophoresed and 

transferred onto PVDF membranes and then 

blocked with 5 % fat-free milk. Subsequently, 

the membranes were conjugated with antibodies 

targeting LC3 (ab192890, 1:1000, Abcam), p62 

(ab109012, 1:1000, Abcam), NLRP3 (ab263899, 

1:1000, Abcam), ASC (ab283684, 1:500, 

Abcam), Caspase-1 (ab207802, 1:500, Abcam), 

IL-18 (ab243091, 1:1000, Abcam), IL-1β 

(ab254360, 1:1000, Abcam), and β-actin 

(ab8226, 1:3000, Abcam) for 2 h at room 

temperature. Subsequently the membranes were 

incubated with secondary antibodies (1:2000, 

Abcam) for 1 h at room temperature. The blots 

were then analyzed with ECL kit.  

Statistics  

Statistical analysis was performed with 

GraphPad 7.0 software using t-test, and the data 

are shown as mean ± SD. P < 0.05 was 

considered statistically significant.  

RESULTS  

Carnosol suppressed sevoflurane-induced ROS 

production of rats The results showed that 

carnosol treatment decreased DHE expression 

levels in sevofluraneinduced hippocampal 

neurons, suggesting the inhibition of ROS 

production (Figure 1). Therefore, Carnosol 

suppressed sevofluraneinduced ROS production 

of rats. 



 Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 6 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 6 

 

CTMJ | traditionalmedicinejournals.com  

 

 

Figure 1: Carnosol suppressed sevoflurane 

induced ROS production of rats. DHE was used 

to detect ROS levels in hippocampus from 

control, SEV, SEV+carnosol (5 mg/kg), and 

SEV+carnosol (10 mg/kg) groups. Scale bar 

indicates 100 μm  

Carnosol suppressed mitochondrial damage 

induced by sevoflurane, and activated 

autophagy  

Immunostaining showed the degree of 

mitochondrial damage of the hippocampus from 

each group. The aggregates and monomers 

reflected the degree of mitochondrial damage. 

The data showed that the monomers were 

enhanced and the aggregates decreased in SEV 

hippocampal neurons (Figure 2 A). In addition, 

carnosol reduced the monomers and increased 

the aggregates in SEV-induced hippocampal 

neurons, suggesting the inhibition of 

mitochondrial damage (Figure 2 A). Similarly, 

carnosol decreased ATP production in SEV 

hippocampal neurons but induced the production 

of ATP in SEV hippocampal neurons (Figure 2 

B). Western blot results showed that the ratio of 

LC3BII/LC3BI in SEV hippocampal neurons 

increased, while carnosol decreased the ratio in 

SEV hippocampal neurons, suggesting the 

inhibition of autophagy (Figure 2 C). 

Interestingly, increased expression of p62 was 

found in SEV hippocampal neurons but carnosol 

decreased p62 expression in SEV hippocampal 

neurons, further confirming the previous data 

(Figure 2 D). Therefore, carnosol alleviated 

mitochondrial damage induced by sevoflurane 

and activated autophagy. 

 

Figure 2: Carnosol suppressed mitochondrial 

damage induced by sevoflurane and activates 

autophagy. (A). Immunostaining showed the 

degree of mitochondrial damage of hippocampus 

from the indicated groups. Green indicates 

monomers; Red indicates aggregates. Scale bar 

indicates 50 μm; (B). ATP production was 

shown in hippocampus from the indicated 

groups; (C). Immunoblot assays showed the 

expression levels of LC3B in hippocampus from 

the indicated groups. The ratio of 

LC3BII/LC3BI was calculated; (D). Immunoblot 

assays showed the expression levels of p62 in 

hippocampus from the indicated groups. Data 

are presented as mean ± SD; ***P < 0.001, SEV 

vs control, ##p < 0.01, ###p < 0.001, 

SEV+carnosol vs SEV  

Carnosol suppressed sevoflurane-induced 

NLRP3 inflammasome activation  

To determine the potential mechanism of 

carnosol-mediated effect in sevoflurane induced 

rats, the role of carnosol in the NLRP3 pathway 

was assessed after treatment with SEV, carnosol 

and the inhibitor of autophagy, 3-MA. Carnosol 

increased the levels of NLRP3 in SEV group 

was found, and reversed the increase of NLRP3 

expression caused by SEV treatment (Figure 3 

A). However, 3-MA treatment further increased 

the expression of NLRP3 in carnosol-treated 

rats, suggesting the regulation of autophagy in 

NLRP3 pathway (Figure 3 A). Furthermore, 

NLRP3 signaling pathway was activated 

following the establishment of SEV-induced 

model as demonstrated by increased levels of 



 Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 6 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 6 

 

CTMJ | traditionalmedicinejournals.com  

 

ASC, IL-1β and IL-18 (Figure 3 B). The 

decreased levels of ASC, IL-1β and IL-18 levels 

were caused by carnosol treatment (Figure 3 B). 

3-MA treatment increased further the expression 

of NLRP3 in carnosol-treated rats. Thus, the 

data suggest that carnosol suppressed 

sevofluraneinduced NLRP3 inflammasome 

activation by regulating autophagy. 

 

Figure 3: Carnosol suppressed sevoflurane 

induced NLRP3 inflammasome activation by 

activating autophagy. (A). Immunoblot assays 

showed the expression levels of NLRP3 in 

hippocampus from the indicated groups; (B). 

Immunoblot assays showed the expression levels 

of indicated proteins in the hippocampus from 

the indicated groups. Data are presented as mean 

± SD, ***P < 0.001, SEV vs control, ###p < 

0.001, SEV+carnosol vs SEV, $$$p < 0.001, 

SEV+carnosol+3-MA vs SEV+carnosol 

 

Figure 4: Carnosol suppressed sevoflurane 

induced cognitive dysfunction by inhibiting 

NLRP3 inflammasome activation. (A). Water 

maze experiment reveals rat movement track 

from the indicated groups; (B-E). Escape latency 

(B), time of across the stealth platform (C), time 

in target quadrant (D), and distance in target 

quadrant (E) of rats from the indicated groups. 

Data are presented as mean ± SD; ***p < 0.001, 

SEV vs control, #p < 0.05, ##p < 0.01, ### p < 

0.001, SEV+carnosol vs SEV, $p < 0.05, $$$p < 

0.001, SEV+carnosol+3-MA vs SEV+carnosol  

Carnosol suppresses sevoflurane-induced 

cognitive dysfunction by inhibiting NLRP3 

inflammasome activation  

As expected, the cognitive ability of rats in SEV 

group was impaired significantly with poor 

ability to escape latency, locate the target, long 

latency time and longer time in the target 

quadrate (Figure 4 A - E). Carnosol treatment 

improved the performance of rats (Figure 4 B - 

E). Interestingly, NLRP3 treatment further 

impaired the performance of rats, suggesting that 

carnosol alleviated cognitive dysfunction 

(Figure 4 B - E). Therefore, carnosol suppressed 

SEV-induced cognitive dysfunction by 

suppressing NLRP3 inflammasome activation.  

DISCUSSION  

With the different types of surgery and the 

duration of general anesthesia, the degree of 

POCD varies. In recent years, there has been 

great progress in anesthesia technology, 

monitoring instruments and surgical operations, 

and these have greatly improved the safety of 

surgery, POCD is still common and its 

pathogenesis and mechanisms have attracted 

wide attention [10]. POCD can usually be 

treated with medication [10]. If the patient has 

cognitive dysfunction after surgery, it can be 

treated with drugs, as the main purpose is to 

improve the patient's condition and facilitate 

recovery [10]. With the aid of intravenous drip, 

nutritional neurodrugs and other treatments to 

ameliorate brain tissue ischemia and hypoxia, 

and postoperative recovery, using mecobalamine 

tablets, adenosine monophosphate and other 

drugs, may improve the cognitive ability of 

patients [13]. However, it is still necessary to 

find more effective drugs. Here, carnosol was 



 Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 6 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 6 

 

CTMJ | traditionalmedicinejournals.com  

 

shown to ameliorate the sevoflurane-induced 

cognitive impairment in aged rats. Therefore, 

carnosol may serve as a drug for the 

management of POCD.  

Carnosol is a diterpenoid phenolic compound, 

which has antioxidant, anti-inflammatory, 

antiproliferation and anti-tumor effects [14,15]. 

Due to its remarkable pharmacological activity, 

more and more scholars at home and abroad 

have paid more attention to it in recent years [8]. 

It has a potential role in the prevention and 

treatment of neuropathic diseases [8]. Carnosol 

is a potent ribosome S6 kinase (RSK2) inhibitor 

and also an carnosol is also an Nrf2 activator, 

increasing Nrf2 levels and promoting heme 

oxygenase-1 (HMOX1) expression [16]. It 

alleviated mitochondrial damage induced by 

SEV and also activated autophagy and increased 

SEV-induced NLRP3 inflammasome activation 

by activating autophagy. Carnosol ameliorated 

sevoflurane-induced cognitive impairment. 

Previous studies have shown that it alleviated 

sevoflurane-induced cognitive dysfunction in 

aged rats by mediating NF-κB pathway [16].  

In the present study, carnosol alleviated 

sevoflurane-induced cognitive dysfunction by 

regulating NLRP3 inflammasome as well as 

autophagy. However, further in vivo studies to 

elucidate the mechanism of action are still 

needed. Autophagy is vital in the elimination of 

protein aggregates and abnormal organelles [10]. 

Enhanced autophagy disrupts neuronal 

homeostasis [17]. Activation of autophagy 

suppresses the formation of NLRP3 

inflammasome [10]. NLRP3 inflammasome is 

highly expressed and over-activated in aged rats, 

and is vital in the central inflammatory response 

[18]. Over-activation of NLRP3 inflammasome 

leads to a variety of diseases, including POCD 

[10]. NLRP3 inflammasomes are involved in the 

important mechanisms of POCD [10]. It also 

reduced cognitive impairment in older rats. 

Interestingly, carnosol further improved 

SEVinduced NLRP3 inflammasome activation 

by activating autophagy, thus ameliorating 

POCD. However, the precise mechanism of 

action needs to be investigated further.  

CONCLUSION  

Carnosol ameliorates SEV-induced cognitive 

impairment in aged rats by suppressing NLRP3 

inflammasome, as well as autophagy. Therefore, 

carnosol is a promising drug for the management 

of POCD. 

REFERENCES  

1. Li X, Gao Y, Han X, Tang S, Li N, Liu 

X, Ni X. Maresin1 ameliorates 

postoperative cognitive dysfunction in 

aged rats by potentially regulating the 

NF-kappaB pathway to inhibit 

astrocyte activation. Exp Gerontol 

2023; 176: 112168.  

2.  Wang G, Shen J, Zhai L, Lin Y, Guan 

Q, Shen H. TL1A promotes the 

postoperative cognitive dysfunction in 

mice through NLRP3-mediated A1 

differentiation of astrocytes. CNS 

Neurosci Ther 2023; 29(11):3588- 

3597.  

3. Wang X, Chen X, Wu F, Liu Y, Yang 

Y, Chen W, Pan Z, Hu W, Zheng F, He 

H. Relationship between postoperative 

biomarkers of neuronal injury and 

postoperative cognitive dysfunction: A 

meta-analysis. PLoS One 2023; 18(4): 

e0284728.  

4. Zhao W, Li J, Wang J, Liu M, Yu D. 

Effect of dexmedetomidine on 

postoperative cognitive dysfunction and 

the T helper 17/regulatory T cell 

balance in geriatric patients 

undergoing orthopedic surgery: a 

randomized controlled study. Am J 

Transl Res 2023; 15(4): 2634-2644.  

5. Farouk SM, Abdel-Rahman HG, 

Abdallah OA, El-Behidy NG. 

Comparative immunomodulatory 

efficacy of rosemary and fenugreek 

against Escherichia coli infection via 

suppression of inflammation and 

oxidative stress in broilers. Environ Sci 

Pollut Res Int 2022; 29(26): 40053-

40067.  

6.  Ma W, Shi H, Wei G, Hua M, Yu H. 

Loureirin B attenuates amiodarone-

induced pulmonary fibrosis by 

suppression of TGFβ1/Smad2/3 



 Chinese Traditional Medicine Journal | 2020 | Vol 3 | Issue 6 

ISSN : 2693 - 6356 

2020 | Vol 3 | Issue 6 

 

CTMJ | traditionalmedicinejournals.com  

 

pathway. Trop J Pharm Res 2020; 

19(7): 1371-1376.  

7.  Bai R, Yuan C, Wang T, Liu L, Li J, 

Lai Y, Li H, Chen Z, Li C, Ke D et al. 

Apple pomace and rosemary extract 

ameliorates hepatic steatosis in 

fructose-fed rats: Association with 

enhancing fatty acid oxidation and 

suppressing inflammation. Exp Ther 

Med 2020; 20(3): 1975-1986.  

8.  Gonzalez-Cardenete MA, Gonzalez-

Zapata N, Boyd L, Rivas F. Discovery 

of Novel Bioactive Tanshinones and 

Carnosol Analogues against Breast 

Cancer. Cancers (Basel) 2023; 15(4): 

1318.  

9. Lee JE, Im DS. Suppressive Effect of 

Carnosol on Ovalbumin-Induced 

Allergic Asthma. Biomol Ther (Seoul) 

2021; 29(1): 58-63.  

10. Zhou J, Zhang C, Fang X, Zhang N, 

Zhang X, Zhu Z. Activation of 

autophagy inhibits the activation of 

NLRP3 inflammasome and alleviates 

sevoflurane-induced cognitive 

dysfunction in elderly rats. BMC 

Neurosci 2023; 24(1): 9.  

11. Han QQ, Le W. NLRP3 

Inflammasome-Mediated 

Neuroinflammation and Related 

Mitochondrial Impairment in 

Parkinson's Disease. Neurosci Bull 

2023; 39(5): 832-844.  

12. World Medical A. World Medical 

Association Declaration of Helsinki: 

ethical principles for medical research 

involving human subjects. J Postgrad 

Med 2002; 48(3): 206-208.  

13. Wang W, Zhao B, Gao W, Song W, 

Hou J, Zhang L, Xia Z. Inhibition of 

PINK1-Mediated Mitophagy 

Contributes to Postoperative Cognitive 

Dysfunction through Activation of 

Caspase-3/GSDME-Dependent 

Pyroptosis. ACS Chem Neurosci 2023; 

14(7): 1249-1260.  

14. Habtemariam S. Anti-Inflammatory 

Therapeutic Mechanisms of Natural 

Products: Insight from Rosemary 

Diterpenes, Carnosic Acid and 

Carnosol. Biomedicines 2023; 11(2): 

545.  

15. Luan Y, Jiang L, Luan Y, Xie Y, Yang 

Y, Ren KD. Mitophagy and Traumatic 

Brain Injury: Regulatory Mechanisms 

and Therapeutic Potentials. Oxid Med 

Cell Longev 2023; 2023: 1649842. 

16. Li X, Zhang Q, Hou N, Li J, Liu M, 

Peng S, Zhang Y, Luo Y, Zhao B, 

Wang S et al. Carnosol as a Nrf2 

Activator Improves Endothelial Barrier 

Function Through Antioxidative 

Mechanisms. Int J Mol Sci 2019; 

20(4): 880.  

17. Pajarillo E, Kim SH, Digman A, 

Dutton M, Son DS, Aschner M, Lee E. 

The role of microglial LRRK2 in 

manganese-induced inflammatory 

neurotoxicity via NLRP3 

inflammasome and RAB10-mediated 

autophagy dysfunction. J Biol Chem 

2023; 299(7): 104879.  

18. Wu Y, Hu A, Shu X, Huang W, Zhang 

R, Xu Y, Yang C. Lactobacillus 

plantarum postbiotics trigger 

AMPKdependent autophagy to 

suppress Salmonella intracellular 

infection and NLRP3 inflammasome 

activation. J Cell Physiol 2023; 238(6): 

1336-1353. 

 


