Utilizing network pharmacology to explore the underlying mechanism of Cinnamomi Ramulus in Zika Fever induced by Zika Virus

Background: Zika fever is a mosquito-borne infection induced by Zika virus (ZIKV),effective drug and vaccine against ZIKV infection is still unavailable. Cinnamomi ramulus (CR) is a traditional Chinese herb with a long history ,can stimulate secretion of sweat and relieve exterior syndrome. The aim of this research was to evaluate the effect of CR against ZIKV and uncover its mechanism of action by network pharmacology . Methods: Cell viability assay, qRT-PCR assay and western blot assays were perfermed to evaluate anti-ZIKV activity in vitro. Survival rate, body weight were observed and viremia was detected in AG6 mice. Drug- target-disease networks, GO enrichment, and KEGG pathway analysis was established to clarify the therapeutic mechanism. Results :In this study, we find that CR can alleviate cytopathic effect( CPE) after ZIKV infection. In the premixed administration mode, CR showed superior activity to inhibit viral RNA replication and protein expression in cells. Orally administered CR effectively protected AG6 mice infected with lethal doses of ZIKV, conferring 50% or 20% survival rate at a dosage of 900mg/ml or 450mg/ml, reducing body weight loss, inhibiting viral RNA replication. Beta-sitosterol, α-Longipinen, (-)-alpha-cedrene, ()-alpha-Longipinene may the main active compound and PTGS2, GABRA1, PTGS1, PTGES and CCR5 may the main targets. 176 biological processes （ BP ） , 16 cell components(CC), 27 molecular functions(MF), and 37 pathways were significantly identified in GO enrichment and pathway analysis. Conclusions: These results reveal that CR treatment of Zika fever is mainly related to PTGS1 and PTGS2 regulated prostaglandin(PG) release, Concentration; EC 50: The 50% effective inhibitory concentrations ; OB: oral bioavailability ； DL: drug-likeness ； BBB: blood- brain barrier ； SI: Select Index.

includes natural malformations, fetal death and microcephaly are caused by pregnant women infected with ZIKV (2). Guillain-Barre syndrome is an autoimmune disease, mainly affects the peripheral nervous system and can cause muscle paralysis and severe peripheral nerve disease,even death (3,4). ZIKV is a positive-sense, single-stranded enveloped RNA virus, and dengue virus(DENV), West Nile virus (WNV), yellow fever virus (YFV), and Japanese encephalitis virus are belonging to the genus Flavivirus of the Flaviviridae family, has emerged in many countries and widespread in human population mainly by infected female Aedes aegypti and Aedes albopictus (5). Beside the major source of transmission mosquito bites, the virus can also be passed on through sexual contacts and blood transmissions , such as blood transfusion and share syringe (6). ZIKV is first isolated from three owl monkey in the Zika forest of Uganda in African by American scientists in 1947 and isolated from the blood of a Nigerian patient in 1956, even though the first human infection with ZIKV was reported (7). From the 1960s to the 1980s, only 15 ZIKV infection cases were scattered sporadically Africa and South Asia including Uganda, Egypt, Tanzania and Indonesia, Malaysia, Thailand, Vietnam District. In April, 2007, ZIKV broke out in Asia and Africa, firstly occurred in Micronesia United Endemic to Bang Yap Island, and caused about 70% of local residents were infected with ZIKV (8).In November 2015, ZIKV also triggered wide-scale outbreaks in Central and South America (9).On February 1, 2016, the World Health Organization (WHO) announced Fetal microcephaly and other related diseases caused by ZIKV have constituted "International Emergency Public Health events "and call on the international community to work together to respond (10). Currently, there are no vaccines and specific drugs to against ZIKV, the most effective way to prevent mosquito bites.
Since WHO announced ZIKV as an international public health issue in 2016, ZIKV vaccines and drugs have become the hotpot of research. In the last two years,several candidate vaccines and anti-ZIKV drugs are under Phase I and Phase II clinical trials experimental evaluation (11)(12)(13), the absence of vaccines and drugs highlights an urgent need for new classes of anti-ZIKV agents to combat potential human ZIKV pandemics. Cinnamomi ramulus is the well-known Traditional Chinese medicinal herb from the dried branch and skin of the cinnamon family Cinnamomum cassia.
Based on the current research status of ZIKV and related to the experience of TCM against epidemic infection disease, we surprisely find that extracts from Cinnamomi Ramulus have obviously anti-ZIKV effects and never has seen reported.
In this study,we examined the efficacy of CR against ZIKV in Vero-E6 cells and Hela cells based on the cytotoxicity assays. The CPE assay, qRT-PCR analysis and Western blot analysis methods were used to explored the anti-ZIKV activity in vitro. For the animal experiment, AG6 mice (ifnar1 -/-ifngr1 -/-) is highly susceptible to ZIKV and thus the strain mice for the testing of CR antivirals activity in vivo (20). The survival rate of mice and viral RNA level expression in blood were observed in our study.
TCM is a complex system of multiple components, multiple targets and multiple pathways. Network pharmacology is a new discipline emerging in recent years.
Through network analysis, the interaction effects and potential molecular mechanisms among disease-target-drug active components are studied at the molecular level. In this study, network pharmacology was used to study the mechanism of CR for Zika fever and to provide a reference for the further research.

Preparation of the plant and plant extract
Cinnamomi ramulus was purchased from Guangxi Provinces in South China.
The authenticity of these twigs was identified as the shape according to Chinese Pharmacopoeiaby Liangwen Yu professor of Guangzhou University of TCM. 50g Cinnamomi ramulus were pulverized and extracted with 300ml water for 30min using a heating reflux method, the liquid was filtered and drug residues were extracted again with same methods. The liquid was combined and concentrated into 1g/ml(about 50ml ) using a rotary evaporator, the extracted was stored to -20℃ overnight and then dried with vacuum freeze dryer，the powder was diluted with culture medium before using during the experiment.

Cells and viruses
African

Antiviral activities of CR by Western blot analysis
Vero-E6 cells and Hela were plated at 2×10 5 cells/well in a 6-well plate incubated at 37°C in 5% CO2 for 18h. Permixed administration, simultaneous administration and therapeutic administration modes were performed and cells were collected and lysed with lysis buffer, total protein was collected by centrifugation at 10,000 ×g for 10 min at 4°C, the denatured protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto a polyvinylidene difluoride membrane, blocked with 5% skim milk, and sequentially incubated with anti-NS5, anti-NS3, anti-E, anti-prM and GAPDH primary antibodies (GeneTex，San Antonio, USA), followed by incubation with a secondary antibody. The tegrated bands were analyzed using FluorChem R multifunctional imaging machine(protein simple, USA).

Antiviral activities of CR by Survival rate of mice and viral RNA replication
AG6 mice were divided into 4 groups(n=6) used for the experiment, provided by the Laboratory Animal Services Center, Guangzhou University of Chinese Medicine, female aged 5~6 weeks, housed in individually ventilated cages. The temperature was maintained at 22±2℃ and humidity at 50±10%,12h/12h light/dark cycles. The mice were challenged by injecting with 3.3×10 6 pfu of ZIKV or 0.9% sterile saline 50μl.
CR was orally administered with 900mg/kg and 450 mg/kg, the orbital blood was collected on day 3 and day 5 post-infectionon and the qRT-PCR assay as previously described (28).The mice were monitored the weight, number and time of death daily until day 15. The experiment was carried out in animal class II biosafety safety cabinets.

Collection targets of candidate compounds and Zika fever
The compounds of CR were collected by the TCMSP database (TCMSP, http://tcmspw.com/tcmsp.php). Considering that Zika fever is related with bloodbrain barrier, so ADME (absorption, distribution, metabolism, excretion) parameters selected OB(oral bioavailability) ≥ 30%, DL(drug-likeness)≥ 0.1, BBB(Blood-brain barrier)≥ 0.3 as the filter conditions of the active ingredients. In addition, Some compounds were classified into the candidate compound even not meet the screening criteria, but were essential pharmacological ingredients and with significant antiviral effects on the human body.

Drug-Target-Disease Network
The targets of compounds and Zika fever disease were input Draw Venn

GO enrichment and KEGG pathway enrichment
In order to elucidate the role of targets that interact with the candidate compound of CR in gene function and signaling pathway, the overlapping compound targets for GO enrichmentand KEGG pathway enrichment analysis using STRING datebase. The potential targets involved in the BP, CC, MF, and the pathways were also described.

Statistical analysis
The results were analyzed by one-way analysis of variance (ANOVA) or Dunnett's T3 when the data were not normally distributed.The date showed as the mean ± standard, P-values of less than or equal to 0.05 were considered statistically significant.

CR can represses ZIKV infection in Vero-E6 cells
The 50% Cytotoxic Concentration of CR(CC50) was ＞ 4500μg/ml in Vero-E6 cells and 3396μg/ml in Hela cells( Fig.1a and 1b). CR displayed different anti-ZIKV activities under different modes of medication. CPE was monitored by direct microscopic observations (Fig.1c). Compared with the virus control, far less CPE than in CR treated groups. The cell viability rate was deterninated by using the MTT, CR displayed a clear anti-ZIKV activity in Vero-E6 cells. Compared with other medication modes, the premixed administration modes showed a superior antiviral activity, the inhibition rate was up to 100% at the concentration of 100~600μg/ml ( Fig.1d-1f).

CR can inhibit ZIKV replication in Vero-E6 cells and Hela cells
Based on the result of the CPE reduction assay, qRT-PCR assay was carried out to determine the level of viral RNA in Vero-E6 cells and Hela cells at different medication modes. The result showed that CR can inhibit viral RNA synthesis, the premixed medicine therapy mode was the better mode than other modes and the 50% effective inhibitory concentrations (EC50 )was＜25 μg/ml in Vero-E6 cells (Fig.2a-2c) and 74±1.50μg/ml in Hela cells (Fig.2d-2f). The specific EC50 and selection index(SI) of CR was displayed in the (Table1).

CR can inhibit the expression of ZIKV protein in Vero-E6 cells
In order to further explored the antiviral activity of CR, the Western blot assay was performed under the different medication modes. The experiment result showed that CR can almost completely inhibit the expression of NS5, NS3, prM and E protein of ZIKV with the concentration of 25~500 μg/ml at the premixed administration modes (Fig.2g). However,the simultaneous and therapeutic administration modes didn't disply a good inhibition activity of ZIKV protein as the premixed therapy( Fig.2e and 2f). Under the therapeutic administration mode, CR can't inhibit the expression of proteins with the concentration 25~300 μg/ml, but can inbit the NS5 and E protein expression at the 500 μg/ml and only can inhibit E protein expression at the 400 μg/ml. In the summary, the result indicated the premixed administration modes playing a significant role in the antiviral process.

CR can reduces the mortality of mice infected ZIKV
AG6 mice were used to test the efficiency of CR and the normal group mice were survived healthily and no death during the 15 observation. The ZIKV-infected mice presented signs of illness symptoms, inactivity,hind limb paralysis, weight loss and lack of appetite on the fourth day, the body weight of all the survival mice were gained again on the twelves days (Fig.3b). The model group mice were began to die at days 7 post-infection and all the mice were dead on ninth days, loss 30% body weight was considered dead. CR treated mice were obviously protected and the survival rate were 50% ( 900mg/kg ) and 30% ( 450mg/kg ), respectively (Fig.3a). The viral RNA level was measured by qRT-PCR alalysis on day 3 and day 5 post-infection.
Compared with ZIKV group, the mice of viral RNA copies all reduced after the dose of 900mg/kg and 450 mg/kg administered, however,the dose of 450 mg/kg had no difference with ZIKV model group on the day 3 post-infection but had difference on the day 5 post-infection. Comparison of ZIKV infected mice on day 3 and ZIKV infected on day 5, the viremia has statistically significant, the viral RNA level in blood was higher on day 3 than day 5, the reason may be virus was through the blood conveyed to the tissues (Fig.3c) .On the whole, the result presented that CR can inhibit ZIKV in vivo by the inhibit the viral RNA replication.

Screening for Active Compounds and fishing targets
Considered CR mainly take effects by oral administration, OB, DL and BBB parameters were used to screen the active compounds of Zika fever. 664 targets were identified from 24 compounds and 5 mainly pharmacological ingredients, 557 known therapeutic targets were obtained from the disease Zika fever. 20 targets in CR were associated with Zika Fever (Fig.4a), we matched 24 compounds with the the overlapping targets. and average node degree were 3, the color of line indicates the type of interaction evidence (Fig.4b). 48 nodes and 136 edges constructed Drug-Target -Disease Network (Fig.4c). The network was evaluated by network centralization and network heterogeneity, the valus were 0.429 and 0.888 respectively, the result indicated that some nodes are more concentrated in the network than others and means that the compound target space has a tendency for certain compounds and targets. We can know PTGS2(degree=20), GABRB3(degree=15), PTGS1(degree=14) ，

GO enrichment and KEGG enrichment analysis
To analyze the functional characteristics of 20 identified targets, 219 terms were

Discussion and Conclusion
At present study and clinical diagnosis reported, Zika fever is usually asymptomatic or mild in adults by ZIKV infection, but ZIKV can quickly resolve blood viremia and persist for months in saliva, urine, semen, breast milk and the central nervous system and further developed Zika syndrome and Guillain-Barre syndrome (21,22). Therefore, outbreaks and epidemics of ZIKV are caused a huge threats to human health, especially pregnant women and newborns. The absence licensed drugs highlight the importance to discover and develop more novel antiviral agents.
As previously reported, Vero-E6 cell strain is easily infected by ZIKV (23) and Hela cell strain is also susceptible to ZIKV (24). For the reason that we choose Vero-E6 cell strain and Hela cell strain for our experimental research. First of all, we found that CR had a limited cytotoxicity by detecting the cell viability using MTT in surprisingly, CR can effectively reduce cytopathy and protect the cell from ZIKV, the results were showen in Fig.1b. Different drugs have different antiviral action modes due to the stage of viral replication, premixed, simultaneous and therapeutic admonostration modes were performed according to the previously described (25).
The result showed that CR had a significant effect at premixed administration mode to inhibit the replication of ZIKV in Vero-E6 cells and Hela cells, the EC50 values were 45.75μg/ml in Vero-E6 cells and 74.02μg/ml in Hela cells, the Select Index(SI) values were more than 98.36 and 45.88, the data was showed in Fig.1c and Table 1. NS5, NS3, E and prM protein were the main proteins of ZIKV and their expression levels are essential for ZIKV to reflect the extent of viral protein synthesis. Compared with the other treatment modes, the premixed administration mode showed a better inhibitory effect in Fig.2b. The result is completely consistent with qRT-PCR assays and showed obvious inhibition in viral protein synthesis. Taken together, our results verified that CR has superior against ZIKV activity in vitro.
AG6 mice were can be effectively used as murine models for Zika virus research. In order to further confirm whether CR has an inhibitory influence on ZIKV-induced mouse model, AG6 mice were used in our study, the result showed CR exhibited a significant mortality protection of mice against ZIKV infection, the infected mice were trated with CR showed a obviously delay in mortality and protected the body weight loss in Fig. 3a and 3b, the survival rate is up to 50% when the mice were treated at the high dose of 900 mg/kg and 30% survival rate at the low dose of 450mg/kg. In addition, the viremia was monitored at day 3 and day 5 post-infection, the date showed that CR can reduce the viremia at 5 post-infection in has been reported that PTGS1 can regulate lipopolysaccharide-induced inflammation and brain damage (27) and PTGS2 can promote the occurrence of inflammatory neuropsychiatric disorders (28). PTGS1 and PTGS2 oxidize arachidonic acid to PG and regulate the body's homeostasis and inflammation during PG synthesis (29). PG were widely distributed in the central nervous system and can regulate the release and activity of neurotransmitters, has a regulatory effect on autonomic nerve media and regulate brain growth and physical activity, enhance the sensitivity of pain receptors to pain-causing substances and their pain-causing effects (30). The release of PG can also amplify inflammatory pain and induce inflammation-redness, swelling, heat and pain (31). Fever is a common symptom of infectious and inflammatory disease. It is well-established that prostaglandin E2 is the final mediator of fever, which by binding to its EP3 receptor subtype in the preoptic hypothalamus initiates thermogenesis (32).The GO enrichment and KEGG enrichment analysis result showed that CR relieved Zika fever and condition deterioration by regulating the expression of PTGS2 and PTGS1 to further inhibit PG release and regulate metabolism to reduce inflammation response.

Conclusion
In conclusion, Zika fever, as an infectious diseases have strong ability to spread in endemic areas and cause a serious harm to pregnant women and fetus. Our findings demonstrated that CR shows potentially effective against ZIKV in vitro and vivo, the data provides reference basis for the prevention and treatment of Zika fever.
Beta-sitosterol, DBP, α-Longipinen, (-)-alpha-cedrene, ()-alpha-Longipinene may the main active compound of CR to play an antiviral role and PTGS2, GABRA1, PTGS1, PTGES and CCR5 may the main targets related to Zika fever induced by ZIKV. The mechanism of CR treatment of Zika fever may be related to PTGS1 and PTGS2 mediated PG release, metabolism and inflammation response, but the detailed verification about active compounds and potential targets experiments need further research and performed.   the CR, red nodes denote the disease-Zika fever,orange nodes denote the active ingredients and blue nodes denote the potential targets,respectively. Node size is relative to the degree, and the edges demonstrate the interaction between nodes.