Huanglian Jiedu Decoction Exerts Antipyretic Effect by Inhibiting MAPK Signaling Pathway

Aim The aim of this study was to explore the antipyretic effect and potential mechanism of Huanglian Jiedu Decoction (HLJDD) on LPS-induced fever in rats. Materials and Methods The fever rat model was established by LPS. Anal temperature of rats was measured every 1 hour after modeling. TNF-α, IL-6, PGE2, and cAMP in rat serum or hypothalamus tissue were detected by ELISA kit. In order to explore the potential active ingredients and mechanism of antipyretic effect of HLJDD, we predicted the underlying antipyretic mechanism by using network pharmacology and then verified its mechanism by Western Blotting. Results The results showed that HLJDD can alleviate LPS-induced fever in rats. The expression levels of TNF-α, IL-6, PGE2, and cAMP in the treatment group were significantly lower than those in the model group. Western Blotting results showed that the protein expression of p-ERK, p-JNK, and p-P38 was significantly inhibited. Conclusion The findings suggest that HLJDD has a good antipyretic effect on LPS-induced fever in rats, which may be closely related to the inhibition of MAPK signaling pathway.


Introduction
Fever is a complex physiological stress response characterized by a regulatory rise in body temperature in response to inflammation or infectious disease [1]. Normally, the body maintains a dynamic balance between heat production and heat loss. When this balance is upset, the body temperature becomes abnormal. Fever is a controlled increase in body temperature, a hypothalamic-mediated response caused by pathogenic injury or invasion [2,3]. is reaction promotes the synthesis of endogenous heat-producing factors, such as TNF-α, IL-6, PGE 2 , and cAMP, which will cause a series of biochemical and physiological changes in the body and eventually lead to elevated body temperature [4][5][6][7]. Hyperthermia is a treatment method that heats the temperature of a specific part of the body or the whole body to above the normal body temperature, so as to achieve the therapeutic effect. Traditional Chinese medicine (TCM) has heat therapy, such as sweat steaming, moxibustion, and cupping. Modern research shows that the combination of hyperthermia and chemotherapy is more effective than chemotherapy alone in the treatment of cancer diseases [8]. Studies have also found that increased body temperature regulation may be beneficial to the improvement of human immunity and reduce the sensitivity to infectious diseases [9]. However, in addition to being good for the body, fever can also be harmful [10]. For example, uncontrolled fever is associated with worse outcomes in patients with sepsis or neuronal damage [11].
TCM is a great treasure house, and it has unique advantages in the treatment of fever diseases. Chinese medicine classifies fever into two categories: fever due to external sensation and fever due to internal injury. External fever is caused by the feeling of external evil. Internal fever is caused by the imbalance of Yin, Yang, Qi, and blood in the internal organs. In external fever, the onset is rapid and the duration is short. In internal fever, the onset is slow and the duration is long, from weeks and months to years. According to the principles of TCM diagnosis and treatment, TCM treatment of fever includes the method of relieving symptoms and reducing fever, the method of dispelling dampness and reducing fever, and the method of nourishing Yin and reducing fever. Huanglian Jiedu Decoction (HLJDD), which originated from Medical Secrets of an Official (Wai Tai Mi Yao as named in Chinese) in the Tang Dynasty, was created by Tao Wang, a famous medical scientist in the Tang Dynasty. e original prescription of HLJDD is composed of Coptidis Rhizoma, Scutellariae Radix, Phellodendri Chinensis Cortex, and Gardeniae Fructus in the ratio of 3 : 2 : 2 : 3, which has the effect of clearing heat and removing toxicity. Clinical evidence suggests that HLJDD can be used in the treatment of a variety of diseases, which can relieve symptoms with a good clinical effect [12]. Pharmacological studies show that HLJDD has significant anti-inflammatory, antibacterial, and antiendotoxin activities [13,14]. It can be used to treat high fever in children [15], sepsis, and other diseases [16].
Lipopolysaccharide (LPS), commonly known as endotoxin, is an important cell wall component of Gram-negative bacteria [17]. e proinflammatory effects of LPS play an important role in inhibiting bacterial infection. However, dysregulation of the host response to LPS may lead to systemic inflammation, such as sepsis [18]. In recent years, LPS is often used to establish animal models of fever [19][20][21]. Studies have shown that MAPK signaling pathway plays a key role in regulation of the production of proinflammatory mediators in LPS-induced inflammatory responses [22][23][24]. However, the effects of HLJDD in LPS-induced fever and the relationship between HLJDD and MAPK signaling pathways are still unclear.
TCM has multicomponent, multitarget, and multipathway characteristics. e therapeutic mechanisms and material basis of many herbal medicines have not been elucidated. With the rapid development of bioinformatics and various medical databases, network pharmacology has strongly contributed to the understanding of the molecular mechanisms of TCM from a holistic and systemic perspective [25]. Meanwhile, it has great advantages in predicting the target of TCM components, discovering multitarget drugs and providing new insights for the study of TCM [26]. HLJDD is a classical Chinese medicine formula for relieving fever and is also commonly used in Chinese medicine clinics. Its antipyretic effect is remarkable, but its antipyretic mechanism has not been completely elucidated. In order to initially explore this problem, this study was conducted to comprehensively evaluate the antipyretic effect of HLJDD by establishing an animal model of fever, combined with modern bioinformatics technology, aiming to provide ideas for the development of new drugs for the efficient, safe, and rapid treatment of fever symptoms.
Meanwhile, it aimed to provide reasonable dosing guidance and lay an experimental foundation for the clinical application of HLJDD.

Ethics Statement.
is study was carried out in accordance with the recommendations of the Guidelines for the  ey were fed adaptively for 7 days under the conditions of temperature 25 ± 0.5°C, relative humidity 55 ± 5%, and alternating light (12 h light/dark cycle) and with free access to sufficient food and water. e anal temperature of rats was measured for 3 consecutive days, and the average value was taken as the baseline temperature of rats.

Establishment of LPS-Induced Fever Model and Drug
Administration.
e HLJDD groups were orally given different doses of HLJDD 1 h before the injection of LPS. At the same time, the control group were orally given with the same volume of normal saline. 1 hour after drug administration, the model group and each HLJDD group were intraperitoneally injected LPS (100 μg/ kg) to establish fever rat model. en the anal temperature of the rats was measured every 1 hour. e last anal temperature was measured at 7 hours, the rats were euthanized and blood was taken through the abdominal aorta, and then the hypothalamus tissue was collected for subsequent studies.

Measurement of the Serum and Hypothalamus Tissue Levels of Biochemical Indexes.
e ELISA kit (enzyme linked biology, Shanghai, China) was used for the detection of serum or hypothalamus tissue levels of TNF-α, IL-6, PGE 2 , and cAMP in accordance with the manufacturer's instructions.

Identification Targets of HLJDD and Fever.
Traditional Chinese Medicine Systems Pharmacology Database (TCMSP, https://old.tcmsp-e.com/tcmsp.php) was searched to collect the chemical components of the four herbs contained in HLJDD. e screening strategy used for potential active ingredients was oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18. Fever targets were collected from the GeneCard database (https://www. genecards.org/). e intersection targets of HLJDD and fever were obtained from Venn database (http://www. bioinformatics.com.cn/static/others/jvenn/example.html).

Construction of Compound-Target Network.
After the components and their corresponding target data were collected, the chemical components and potential targets of the abovementioned drugs in HLJDD were uploaded to Cyto-scape3.8.0 to construct the Compound-Target network.

Construction and Analysis of PPI Network.
e intersection targets of HLJDD components and fever were uploaded to STRING database (https://string-db.org/), and the screening conditions were with confidence score ≥0.7, so as to establish PPI interaction network. en, the interacting proteins screened by STRING were imported into Cyto-scape3.8.0 to collect the topological parameter values of each interaction target in PPI network, and then the core targets were screened and collected.

GO Enrichment and KEGG Pathway Analysis.
e database (DAVID) (https://david.ncifcrf.gov/) has annotation, visualization, and integrated discovery capabilities. We therefore applied the DAVID database for gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.

Western Blotting Analysis for Protein Expression.
e total protein in the hypothalamus tissue was extracted by using high-efficiency RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor. e hypothalamus tissue homogenate was centrifuged at 4°C and 12000 g for 10 min. Protein concentration was determined by BCA Protein Assay Kit (Solarbio, Beijing, China). e protein samples were separated by 10% SDS-PAGE of gel at 80 V for 30 min and 120 V for 1 h and then transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). After transmembrane, the PVDF membrane was removed and placed in Tris-buffered saline (TBS) containing 5% skim milk powder, which was sealed at room temperature for 2 h, and then incubated with primary antibodies at 4°C overnight; the detailed information is as illustrated in Table 1. After primary antibody incubation, wash with TBS-0.1% Tween 20 (TBST) buffer solution at room temperature for 3 times, 5 min each time. en the PVDF membrane was placed into the secondary antibody solution and incubated at room temperature for 1 h. GAPDH was used as internal reference. ImageJ software was used for quantitative analysis.
2.9. Statistics Analysis. All data was presented as mean-± standard deviation (‾X ± SD). e differences between the group means were calculated by one-way ANOVA and Duncan's multirange test with the SPSS computer program (version 26.0). GraphPad Prism software (version 8.2.1) was used to visualize the results. P < 0.05 was considered statistically significant and P < 0.01 was highly significant.

Effect of HLJDD on LPS-Induced Fever in Rats.
As shown in Figure 1, after LPS injection, the anal temperature increased in both model and HLJDD groups and reached the highest temperature at 6 h after LPS injection. e anal temperature of rats in each HLJDD treated group was lower than that of model group. e results showed that HLJDD had a certain antipyretic effect on LPS-induced rat fever.

Expression of Biochemical Indexes in Fever Rats.
In order to explore the antipyretic effect of HLJDD on LPS-induced fever in rats, we detected the expression levels of TNF-α and IL-6 in the serum and PGE 2 and cAMP in the hypothalamus tissue of fever rats (Figure 2). e expression of TNF-α, IL-6, PGE 2 , and cAMP in model group was significantly higher than that in control group (P < 0.01). It was shown that fever in the LPS-induced fever model in rats was closely associated with elevated levels of these indicators. Compared with model group, the expression of biochemical indexes in HLJDD intervention groups was significantly decreased (P < 0.05). Among them, HLJDD groups could significantly reduce the expression levels of PGE 2 and cAMP in a concentration dependent manner (Figures 2(c) and 2(d)).

Compound-Target Network and Analysis.
Due to the characteristics of multiple components and multiple targets, TCM compounds show a variety of pharmacological activities. erefore, we constructed a network to study the potential mechanism of TCM compounds treating diseases. As shown in Figure 3, there are 257 nodes with 792 edges. Among these active components, we screened 20 components with high degree value, such as quercetin (MOL000098, degree � 127), kaempferol (MOL000422, degree � 50), wogonin (MOL000173, degree � 37), and baicalein (MOL002714, Degree � 29). ese components with high degree value in the network are likely to be the main active components of HLJDD (Table 2).

3.3.2.
e PPI Network Construction of the Underlying Antipyretic Targets. A total of 64 chemical ingredients and 193 targets of HLJDD were collected and 939 targets of fever were obtained from GeneCard database (Figure 4(a)). ese 70 intersection targets may be potential targets of antipyretic effect of HLJDD. e intersection targets were imported into STRING database. After that, PPI network of the underlying antipyretic targets was constructed by Cytoscape3.8.0 with 63 nodes and 455 edges (Figure 4(b)).

GO Enrichment and KEGG Pathway Analysis.
e top 10 significantly enriched terms in biological process (BP), cellular component (CC), and molecular function (MF) categories are shown in Figure 5, which indicated that HLJDD may exert its antipyretic effect by regulating positive regulation of nitric oxide biosynthetic process, cellular response to organic cyclic compound, extracellular space, and identical protein binding. In order to explore the potential pathways involved in the antipyretic effect of HLJDD, we conducted KEGG pathway analysis, as shown in Figure 6, with 15 top signaling pathways. Among these 15 signaling pathways, MAPK signaling pathway plays a crucial role.

Experimental Verification on the Mechanism of HLJDD against LPS-Induced Fever.
To further evaluate the underlying antipyretic mechanism of HLJDD, MAPK signaling pathway was determined. As shown in Figure 7, the expression of ERK, p-ERK, JNK, p-JNK, P38, and p-P38 was detected and the results of Western Blotting were quantified by ImageJ. e results showed that, compared with the control group, the protein expression levels of p-ERK, p-JNK, and p-P38 were significantly increased after LPS injection (P < 0.05 or P < 0.01). HLJDD could downregulate their protein expression levels, which indicated that HLJDD has antipyretic effect on LPS-induced fever in rats through MAPK signaling pathway suppressing.

Discussion
Our study shows that HLJDD has a good antipyretic effect on LPS-induced fever in rats, and this effect may be carried out by inhibiting the MAPK signaling pathway. HLJDD is a classic Chinese medicine prescription for clearing heat and detoxifying [28]. Previous studies have shown that HLJDD significantly reduces the levels of inflammatory factors such as IL-2, TNF-α, and IFN-c and inflammatory mediators such as PGE 2 and NO and suppresses immune and inflammatory responses [29,30]. We studied the pharmacological effects of HLJDD (composed of Coptidis Rhizoma, Scutellariae Radix, Phellodendri Chinensis Cortex, and Gardeniae Fructus at 3 : 2 : 2 : 3) on LPS-induced fever in rats. At present, there are many methods to replicate the rat model of fever; the common ones are dry yeast, 2,4-dinitrophenol, and lipopolysaccharide. LPS, the outer membrane of Gram-negative bacteria, is a common febrifuge in animal experiments and stimulates macrophages and neutrophils to produce the endogenous pyrogens [31]. In this experiment, the rat fever model was established by intraperitoneal injection of LPS (100 μg/kg). After the administration of HLJDD, it was found that different doses of HLJDD could reduce the temperature rise of rats, and the antipyretic effect was obvious. e biochemical indexes of inflammation were detected by enzyme-linked immunoassay kit, and HLJDD was found to reduce the secretion of LPS-induced inflammatory factors TNF-α, IL-6, PGE 2 , and cAMP. is finding suggests that HLJDD reduces body temperature, possibly by reducing proinflammatory cytokines and by inhibiting PGE 2 and CAMP synthesis.
is study is the first to investigate the antipyretic activity of HLJDD. e multicomponent, multitarget nature of Chinese medicine makes its pharmacodynamic mechanism more Evidence-Based Complementary and Alternative Medicine complex. erefore, with the help of modern science and technology, we combined network pharmacology to predict the main pharmacodynamic components of HLJDD, further predict the targets corresponding to the components, construct a component target network, and then screen the core targets and predict the possible pathways. rough network pharmacology analysis, we found that quercetin, kaempferol, wogonin, and baicalein might be the main pharmacodynamic components of HLJDD. Baicalin and its aglycone, baicalein, are the main components in Scutellariae Radix.
e antioxidant and anti-inflammatory effects of baicalein have been demonstrated in a variety of disease models, including cardiovascular disease, inflammatory bowel disease, gout and rheumatoid arthritis, asthma, neurodegenerative diseases, liver and kidney diseases, and cancer [32]. For future research, we plan to select a compound from the prediction results of network pharmacology and explore the antipyretic effect and mechanism of a compound in HLJDD.
MAPK signaling pathway widely exists in all kinds of animal cells and participates in the regulation of cell proliferation, differentiation, transformation, and apoptosis through phosphorylated nuclear transcription factors, cytoskeleton proteins and enzymes, and is closely related to the occurrence of many diseases such as inflammation and tumor [33]. e MAPK pathway has four major branching routes, including ERK, JNK, p38/MAPK, and ERK5. Each MAPK signaling pathway has a relatively independent function. An important role of MAPK signaling pathway is to regulate cellular responses in response to changes in the extracellular environment. ERK pathway is mainly involved in cell proliferation and differentiation, while JNK pathway and p38MAPK pathway are mainly involved in cellular inflammatory response, stress response, and apoptosis [34]. Since MAPK signaling pathway is a classical inflammatory pathway, in addition, we referred to the predicted results of this network pharmacology and selected MAPK signaling pathway, using WB experiments as experimental validation. It was found that the administration of HLJDD reduced the expression levels of phosphorylated proteins of ERK, JNK, and p38 in different dose groups. In particular, the high dose group of HLJDD significantly inhibited the expression of related proteins in MAPK signaling pathway. In summary, HLJDD inhibits the protein expression level in MAPK signaling pathway, thus playing the role of antipyretic and relieving inflammation.

Phellodendri Chinensis Cortex
Evidence-Based Complementary and Alternative Medicine 7    Evidence-Based Complementary and Alternative Medicine

Conclusion
In this study, we investigated the antipyretic effects of HLJDD at the overall animal level by constructing a febrile rat model and combined with network pharmacology techniques to detect the serum levels of the pyrogenic factors IL-6 and TNF-α and the biochemical indicators PGE 2 and cAMP in the hypothalamus of the model animals. e expression of ERK, p-ERK, JNK, p-JNK, P38, and p-P38, which are related proteins of MAPK signaling pathway, was analyzed in the hypothalamus. e results of this study suggest that HLJDD has antipyretic effect on LPS-induced fever in rats, and its potential mechanism may be related to the inhibition of MAPK signaling pathway. is study lays a theoretical foundation for further study of HLJDD in the treatment of fever.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.