STOCKHOLM, May 10(Greenpost) — Swedish Radio och Aftonbladet rapporterade den 6 maj att svenska Tulleverket och Läkeverket hade testat den kinesiska traditionella örtinnehållsmedicinen Lianhuaqingwen som de konfiskerade och gav en slutsats att läkemedlet inte fungerar och endast innehåller mentol i det.
Denna rapport sprids till Kina och kinesiska medicinska akademiker och relevanta organisationer reagerade omedelbart på denna nyhet och förklarade att den svenska slutsatsen inte var komplette. Nyheterna sa att svensk tjänsteman sa att han inte hittade tillräckligt med bevis för att medicinen är användbar eftersom han bara hittade mentol i den, men nämnde inte något annat och hävdade att det inte fanns några relevanta artiklar om det. Denna slutsats visar att han inte hittade artikeln om Lianhuaqingwen eller läste den. Han fann varken annat i medicinen eller så hittade han något men han visste inte vad de var. Nyheter från kinesiska Dr. Li sa att det faktiskt fanns 13 ingredienser eller komponenter i medicinen. Men bara mentol har ett välkänt kemiskt format medan de andra kinesiska örterna inte hade kemiskt format. Till exampel, Nobel Prisvinnare Tu Youyou hittade kemiskt form i kinesiska örter och sedan kan man tillverka massiva läkemedel.
De 13 ingredienserna är Lianqiao som är Forsythoside E, Jinyinhua som är kaprifol, liknande maskros, luding är Rutin, mahuang, dahuang, som är Epherdra, bitter aprikoskärna, gips, banlangen som är ett välkänt antivirusmedicin i Kina. Banlangen uppfanns i mitten av 1990-talet.
De 13 ingredienserna är Lianqiao som är Forsythoside E, Jinyinhua som är kaprifol, liknande maskros, luding, Rutin, mahuang, dahuang, som är Epherdra, bitter aprikoskärna, gips, banlangen som är en välkänd antivirusmedicin i Kina. Banlangen uppfanns i mitten av 1990-talet.
Jag minns att jag en gång blev ombedd att översätta specifikationerna för läkemedlet eftersom mina radiofolk ofta ombads att göra den här typen av översättningsjobb. Men jag vägrade att göra det eftersom jag kände att jag visste så lite om medicinkunskap. Men med åren har Banlangen blivit en daglig medicin som Alvedon i Sverige. Om människor har en influensatyp kan människor ta Banlangen. Om de har bakterietyp förkylning, kan de bara ta ganmaochonji en slags liten partikel typ av pulver och bara lägga det i en kopp varmt vatten och dricka det.
Mianmaguanzhong, jag vet inte vad det är, men Yuxingcao är också ett välkänt namn för att hantera ögon, Huoxiang är också en mycket vanlig men väl effektiv kinesisk medicin. Hongjingtian en växter i Tibet och är känd för sin funktion för att hjälpa dig att få tillräckligt med oxegon. När vi var i Tibet kände en kollega att han saknade oxegon, han gick för att köpa oxegonflaska och Hongjingtian. Gancao var också en kinesisk medicin både i tablettform eller i rå örtform. Man kan svälja tablett, eller dricka vatten som suger gancao örten. Den sista är exakt mentol.
Mentol är verkligen en bra komponent för olika typer av kinesisk medicin. Mintsocker i Lidl hjälper dig faktiskt om du har ont i halsen. Akademiker Zhong Nanshan som var nyckelpersonen i att leda kampanjen mot Covid-19. President Xi Jinping ledde kampanjen genom att skicka sjukhus, läkare och sjuksköterskor och alla förnödenheter genom olika provinser, Zhong Nanshan var en symbol för teknisk del, mycket information och kunskap om covid-19 skickades ut genom Zhongs namn. Akademiker Zhang Boli som har gått hemma, men han kallades för att leda i kampanjen med sin rika kunskap om kinesisk medicin. De båda är auktoritativa medan Zhong var i västerländsk medicin och Zhang var i kinesisk medicin. Men båda rekommenderade Lianhuaqingwen. Under kinesiska krig emot covid 19, både kinesiska medicine och västra medicin samarbetade bra.
Låt oss titta på koronaviruset igen. Det är ett virus, men symptomen när människor får det är hosta, feber, rinnande eller torkande näsa och trötthet. Således symptom liknar en allvarlig förkylning. Endast när människor antingen är för svaga eller för friska för att kämpa mot viruset utan styrka eller för mycket styrka, hårt, så skadar det dina organ, särskilt lungan. Det mest typiska symtomet i slutändan är andningsproblem. Men i början blev du bara förkyld.
Och alla dessa komponenter eller ingredienser i Lianhuaqingwen är användbara i kampen mot kyla. Således kan det nästan behandlas som en mest kraftfull typ av medicin vid behandling av förkylning.
Enligt en gemensam studie av forskare vid Nanjing Chinese Medicine University och Shanghai Drug Research Institute under Chinese Academy of Sciences är Lianhuaqingwen hittills den mest funktionella medicinen för att hämma koronavirus från reproduktion och bota patienterna.
Enligt professor Fang Bangjiang från Longhua Hospital vid Shanghai Chinese Medicine University var det bara 20-30 procent av patienterna som hade feber. De flesta av dem hade inte feber. Han skickades till Leishenshan sjukhus i Kina som byggdes inom tio dagar efter stängningen av Wuhan den 23 januari.
Fang var ansvarig för en enhet som hade 108 patienter med lätta symtom. Han fann att patienterna hade ett enhetligt symptom, det var panik och rädsla, oro och svaga nerver. Efter att ha provat västerländsk medicin i en vecka fann han att den inte bara inte gav positiva resultat utan gav negativa effekter på levern. Således föreskrev han hela tiden kinesisk medicin efter en vecka. Den kinesiska medicinen lugnade huvudsakligen patienterna, befriade dem från panikskänslor och fick dem att sova bättre. Som ett resultat blev ingen mer allvarlig och ingen död hände i hans enhet. Som jämförelse, de som behandlades med västerländsk väg eller medicin, många av dem kunde bli värre och skickades till ICU. Enligt forskningsdokumentet användes Lianhuaqingwen också för svåra patienter och genom att lindra deras feber eller andra symtom räddades liv. Men hittills erkänner experter att det inte fanns något botemedel mot alla typer av medicin mot covid-19. Men som en stödjande medicin eller hälsosam produkt, de som bekräftas att bli covid-19 positiva, kan de ta Lianhuaqingwen. Oavsett om det fungerar eller inte, kan man känna det efter tre dagar. Om det efter tre dagar inte fungerade, känner du dig värre, måste du ringa läkaren igen.
Sverige har försökt sitt bästa för att hantera covid-19 och undvikit problemet Kina hade i början av att bli sjukhuset för trångt och nästan kollapsa. Sverige ville låta covid-19 hända långsamt, vilket är en bra idé. Men viruset älskar människor och kan snabbt spridas från en person till två personer om du har nära kontakter, till exempel i folkmassor, inte nödvändigtvis för att kyssa varandra. Det kan spridas genom spotta och nysa eller hosta. Men om människor håller sig isolerade kan viruset inte hitta någon annan att reproducera sig själv, det kommer att dö när personen återhämtar sig. Därför isolation är mycket viktigt.
Ju tidigare, snabbare att isolera, desto bättre. Till exempel, om svenska avbröt resan, avbröt Eurovisionen, avbröt födelsedagsfesten och isolerats snabbare och tidigare i andra områden där du bara hittade ett par fall, har antagligen viruset utplånats nu. Men ingen vet. Kanske det skulle ta två till tre månader på något sätt.
Tack vare svenska människor som är bra på att hålla socialt avstånd och mestadels följer regeringens instruktioner offrade inte svenska folk för mycket frihet och samhället fortsätter att fungera, vilket kan ses som en positiv aspekt. Men om människor särskilt de som blev smittade, men inte så långt de går till sjukhusintensivvård, kanske om de tar Lianhuaqingwen, kan det inte bara trösta dem, utan också förhindra att de blir värre.
För att säga att det bara har mynta i sig och inte fungerar, det låter som det bara är mintgodisen. Men det är helt enkelt inte sant.
Lianhuaqingwen var faktiskt bara en av de kinesiska medicinerna som hanterade covid-19. Jag antar att det blev berömt på grund av dess massproduktion och förpackning. Qingfei Paidu Tang eller lungrengöring och gift-utrotande sirap var också mycket populärt under covid 19-kampanjen i Wuhan, centrala Kina. Men den här typen av medicin är ren ört och måste kokas varje gång för varje person. Det är svårt att få massproduktion. Kinesiska läkare betonade också att det var en person med sitt eget recept enligt hans eller hennes eget symptom.
Kinesisk medicin stressar inte att döda viruset eller bekämpa viruset, utan betonade att läkemedlet hjälper patienter att bli starkare i immunsystemet och fysisk styrka genom att bli av med eller lindra symptomet.
Covid-19 är verkligen ett starkt virus som är mycket svårt att hantera eftersom det reproducerar sig så snabbt när det kom in i människokroppen och attackerade lungan. Genom att slå tillbaka med immunsystemet kan patienter andra organ som lever eller hjärta eller njure också skadas. Det var därför många människor dog av inte bara virus utan också flera andra kombinerade sjukdomar. Viruset kan skada ditt immunsystem. Således betonade de kinesiska läkarna att människor borde äta tillräckligt med protein och sova bra. Att bli dig själv stark är nyckeln till att ta itu med Covid 19.
STOCKHOLM, May 10, (Greenpost) — Swedish Radio and Aftonbladet reported on May 6 that Swedish Customs had tested the Chinese traditional herb content medicine Lianhuaqingwen which they confiscated and gave a conclusion that the medicine doesn’t function and only contains mint in it.
This report was spread to China and Chinese medical academicians and relevant organisations immediately reacted on this news saying that the Swedish conclusion was not right.
The news said Swedish official in Drugs Administration lab said he didn’t find enough evidence to show the medicine is useful because he only found mint in it, but didn’t mention anything else and claimed that there was no any relevant articles about it.
This conclusion shows that he didn’t find the article about Lianhuaqingwen, nor read it. He neither found nothing else in the medicine or he found something but he didn’t know what they were.
News from Chinese Dr. Li said in fact there were 13 ingredients or components in the medicine. But only mint has a well recognised chemical format while the other Chinese herbs didn’t have chemical format.
The 13 ingredients are Lianqiao which is Forsythoside E, Jinyinhua which is honeysukle, similar to dandelion, luding, Rutin, mahuang, dahuang, which is Epherdra, bitter apricot core, plaster, banlangen which is a well-known antivirus medicine in China. Banlangen was invented in mid 1990s. I remember once I was asked to translate the specifications of the medicine because my radio people were often asked to do this kind of translation job. But I refused to do it because I felt I knew so little about medicine knowledge. But over the years Banlangen has become a daily medicine like Alvedon in Sweden. If people have virus type of flu, people can take Banlangen. If they have bacteria type of cold, they can just take the ganmaochonji a kind of small particle type of powder and just putting it into a cup of hot water and drink it.
Mianmaguanzhong, I do not know what it is, but Yuxingcao is also a well known name in dealing with eyes, Huoxiang is also a very common but well effective Chinese medicine. Hongjingtian is produced in Tibet and famous for its function to help you to have enough oxegon. When we were in Tibet, a colleague felt he was lack of oxegon, he went to buy oxegon bottle and Hongjingtian. Gancao was also a Chinese medicine both in tablet form or in raw herb form. One can swallow tablet, but drink water which soaks the gancao herb. The last one is exactly mint.
Mint is indeed a good component for various kinds of Chinese medicine. Mint sugar in Lidl actually helps you if you have a sore throat.
Academician Zhong Nanshan who was the key person in leading the campaign against Covid-19. President Xi Jinping was leading the campaign by sending hospitals, doctors and nurses and all the supplies through various provinces, Zhong Nanshan was a symbol of technical part, a lot of information was sent out through Zhongs name. Academician Zhang Boli who has retired at home, but he was summoned to lead in the campaign with his rich Chinese medicine knowledge. They both are authoritative while Zhong was in western medicine and Zhang was in Chinese medicine. But both recommended Lianhuaqingwen. During China’s campaign against covid-19, it was a perfect example of cooperation and combination of Chinese and western medicines.
Let’s look at the corona virus again. It is a virus, but the symptom when people catch it is coughing, fever, running or drying nose and fatigue. Thus the symptom is similar to a serious cold. Only when people are either too weak or too healthy to fight against the virus with no strength or too much strength, fiercely, then it hurt your organs especially the lung. The most typical symptom in the end is respiratory problem. But at the beginning, you just got a cold.
And all these components or ingredients of Lianhuaqingwen are useful in fighting against cold. Thus it can almost be treated as a most powerful kind of medicine in treating cold.
According to a joint study by researchers in Nanjing Chinese Medicine University and Shanghai Drug Research Institute under the Chinese Academy of Sciences, Lianhuaqingwen is so far the most functional medicine in inhibiting corona virus from reproduction and curing the fever of the patients.
Fang was in charge of a unit which had 108 patients who had light symptoms. He found that the patients had one unified symptom, that was panic and fear, worries and weak nerves. After trying western medicine for one week, he found it not only did not give positive results but give negative effects on liver. Thus he prescribed all the time Chinese medicine after a week. The Chinese medicine mainly calmed down the patients, relieved them from panic feelings and got them sleep better.
As a result, no one became more serious and no death happened in his unit. In comparison, those who were treated with western way or medicine, a lot of them could become worse and were sent to ICU.
According to the research paper, Lianhuaqingwen was used for severe patients too and by alleviating their fever or other symptoms, lives were rescued.
But so far, experts admit that there were no cure all kind of medicine against covid-19. But as a supportive medicine or healthy product, those who are confirmed to get covid-19 positive, they can take Lianhuaqingwen. Whether it works or not, one can feel it after three days. If after three days it did not function, you feel worse, then you must call the doctor again.
Sweden has tried its best to deal with the covid-19 and avoided the problem China had at the beginning of getting the hospital too crowded and almost collapse. Sweden wanted to let the covid-19 happen slowly, which is a good idea.
However, the virus loves people and can quickly spread from one person to two persons if you have close contacts, for example in crowds, not necessarily to kiss each other. It can spread through spit and sneeze or cough. But if people keep themselves isolated, the virus couldn’t find another person to reproduce itself, it will die with the recovery of the person. Thus isolation is very very important.
The earlier, quicker to isolate, the better. For example, if Swedish cancelled the travel, canceled the Eurovision, canceled the birthday party, and isolated quicker and earlier in other areas where you just found a couple of cases, then the virus probably got wiped out by now. But no one knows. Maybe it would take two to three months any way.
Thanks to Swedish people who are good at keeping social distance and mostly follow the government instructions, Swedish people did not sacrifice too much freedom and the society continues to function, which can be seen as a positive aspect. But if people especially those who got infected, but not to the extend to go to hospitals intensive care, maybe if they take the Lianhuaqingwen, it can not only comfort them, but also prevent them from getting worse.
To say it just got mint in it and does not function, it sounds like it is just the mint candy. But it is simply not true.
In fact, Lianhuaqingwen was just one of the Chinese medicines that dealt with covid-19. I guess it became famous because of its mass production and packaging. Qingfei Paidu Tang or Lung cleaning and poison eradicating syrup was also very popular during covid 19 campaign in Wuhan, Central China. But this kind of medicine is pure herb and needs to be boiled each time for each person. It is hard to get mass production. Chinese doctors also stressed that it was one person with his or her own prescription according to his or her own symptom.
Chinese medicine does not stress killing the virus or fighting the virus, instead, it stressed that the medicine helps patients to get stronger in immune system and physical strength by getting rid of or alleviating the symptom.
Covid-19 is indeed a strong virus which is very hard to deal with because it reproduces itself so quickly once it entered humans body and attacked the lung. By fighting back with the immune system, patients other organs such as liver or heart or kidney could be damaged too. That was why a lot of people died of not only virus, but also several other combined diseases. The virus can damage your immune system. Thus, the Chinese doctors emphasized that people should eat enough protein and sleep well. Getting yourself strong is the key to deal with the covid 19.
The following is the research paper about Lianhuaqingwen.
Lianhuaqingwen (LH) as traditional Chinese medicine (TCM) formula has been used to treat influenza and exerted broad-spectrum antiviral effects on a series of influenza viruses and immune regulatory effects [1]. The goal of this study is to demonstrate the antiviral activity of LH against the novel SARS-CoV-2 virus and its potential effect in regulating host immune response.
Methods
The antiviral activity of LH against SARS-CoV-2 was assessed in Vero E6 cells using CPE and plaque reduction assay. The effect of LH on virion morphology was visualized under transmission electron microscope. Pro-inflammatory cytokine expression levels upon SARS-CoV-2 infection in Huh-7 cells were measured by real-time quantitative PCR assays.
Results
LH significantly inhibited SARS-CoV-2 replication in Vero E6 cells and markedly reduced pro-inflammatory cytokines (TNF-α, IL-6, CCL-2/MCP-1 and CXCL-10/IP-10) production at the mRNA levels. Furthermore, LH treatment resulted in abnormal particle morphology of virion in cells.
Conclusions
LH significantly inhibits the SARS-COV-2 replication, affects virus morphology and exerts anti-inflammatory activity in vitro. These findings indicate that LH protects against the virus attack, making its use a novel strategy for controlling the COVID-19 disease.
Coronaviruses are a group of enveloped viruses named for their coronary appearance with positive single-stranded RNA genomes [2]. In addition to six known strains of coronaviruses that are infectious to humans, a novel coronavirus (SARS-CoV-2) was detected recently in Wuhan, China [3,4]. Like the other two highly pathogenic coronaviruses SARS-CoV and MERS-CoV, SARS-CoV-2 also caused severe respiratory illness and even death. Moreover, the population’s susceptibility to these highly pathogenic coronaviruses has contributed to large outbreaks and evolved into the public health events, highlighting the necessity to prepare for future reemergence or the novel emerging viruses [5].
Similar to SARS-CoV and MERS-CoV, SARS-CoV-2 is initiated by zoonotic transmission likely from bats and spreads rapidly among humans [6]. The basic reproduction number (R0) of person-to-person spread is about at 2.6, which means that the SARS-CoV-2 infected cases grow at an exponential rate. As of February 07, 2020, 57,620 cases of the SARS-CoV-2 have been reported in China, including 26,359 suspected cases, and a sustained increase is predictable. The initial patient cluster with confirmed SARS-CoV-2 infection was reported Wuhan pneumonia with unknown aetiology, which bore some resemblance to SARS-CoV and MERS-CoV infections and was associated with ICU admission and high mortality. Moreover, High concentrations of cytokines were recorded in plasma of patients requiring ICU admission, such as GCSF, IP10, MCP1, MIP1A, and TNFα, suggesting that the cytokine storm was associated with disease severity [7]. A retrospective clinical study indicated the risk of fatality among hospitalized cases at 4.3% in single-center case series of 138 hospitalized patients [8], and the infection fatality risk could be below 1% or even below 0.1% in a large number of undetected relatively mild infections [9]. However, It is challenging to judge the severity and predict the consequences with the information available so far. Since no specific antiviral treatment for COVID-19 is currently available, supportive cares, including symptomatic controls and prevention of complications remain the most critical therapeutic regimens, especially in preventing acute respiratory distress syndrome [10]. Although the control of SARS-CoV-2 still presents multiple challenges in the short term, more potent antiviral drugs are urgent to be developed [4].
At present, some drugs are effective in eliminating SARS-CoV-2 and improving symptoms. The most promising antiviral drug for SARS-CoV-2 is remdesivir that is currently under clinical development for the treatment of Ebola virus infection [11]. However, the efficacy and safety of remdesivir for SARS-CoV-2 pneumonia patients need to be assessed by further clinical trials. In addition, in the prevention and treatment of COVID-19, Tranditonal Chinese medicines have received broad adoption, especially in treating cases of mild symptoms [12]. Lianhuaqingwen (LH), a Chinese patent medicine composed of 13 herbs, has played a positive role in the treatment of SARS-CoV-2. A retrospective analysis of clinical records was conducted in the SARS-CoV-2 infected patients at Wuhan Ninth Hospital and CR & WISCO General Hospital. LH combination could significantly relieve cardinal symptoms and reduce the course of the COVID-19 [13], making it successively included in the Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia (On Trials, the Fourth/Fifth/Sixth/Seventh Edition) issued by National Health Commission of the People’s Republic of China and also recommended by 20 provincial health commissions including Hubei, Beijing, and Shanghai as well as National Administration of Traditional Chinese Medicine for the treatment of COVID-19. Moreover, LH exerted broad-spectrum effects on a series of influenza viruses by inhibiting viral propagation and regulating immune function and achieved similar therapeutic effectiveness with Oseltamivir in reducing the course of H1N1 virus infection [1,14,15]. Notably, the anti-influenza activity of LH in infected mice might depend on the regulation of cytokines, particularly in cytokine storm associated cytokines, such as IP-10, MCP-1, MIP1A, and TNF-α [1]. In the present study, we evaluated the antiviral and anti-inflammatory efficiency of LH against a clinical isolate of SARS-CoV-2 from Guangzhou in vitro.
2. Materials and Methods
2.1. Cell lines and virus
The African green monkey kidney epithelial (Vero E6) cells and the human hepatocellular carcinoma (Huh-7) cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS) at 37 °C. A clinical isolated SARS-CoV-2 virus (Genebank accession no. MT123290.1) was propagated in Vero E6 cells, and viral titer was determined by 50% tissue culture infective dose (TCID50) according to the cytopathic effect by use of Reed-Muench method [17]. All the infection experiments were performed in a biosafety level-3 (BLS-3) laboratory.
2.2. Reagent preparation
LH capsule (Lot No.A2001108) was obtained from Yiling Pharmaceutical Co. Ltd. (Shijiazhuang, China). UPLC fingerprints of LH consist of 32 common peaks. 9 of 32 common peaks are identified. The similarities in 10 batches of LH Capsules samples were all above 0.96 (Supplementary Fig. 1). The black powder of raw material of LH was first dissolved in dimethyl sulfoxide (DMSO) to 240 mg/mL. After shaking for 30 min at room temperature, the LH solution was diluted with serum-free DMEM to 24 mg/mL as a stock solution and stored at −20 °C before using. Remdesivir was kindly provided by Prof. Jiancun Zhang from Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences and was dissolved in DMSO to 100 mM and stored at −20 °C before using. DMEM with 2% FBS was used as the dilution buffer in the follow-up experiments.
2.3. Cytotoxicity assay
The cytotoxic effects of the LH on Vero E6 and Huh-7 cells were evaluated by Methyl Thiazolyl Tetrazolium (MTT) assay. Briefly, monolayers of Vero E6 cells and Huh-7 cells in 96-well plates were rinsed with phosphate-buffered saline (PBS) followed by incubation with indicated concentrations of LH. After 72 h, the cells were stained with MTT solution at 0.5 mg/mL for 4 h. The supernatants were then removed, and the formed formazan crystals were dissolved in 200 μL DMSO. The absorbance was measured at 490 nm using Multiskan Spectrum reader (Thermo Fisher, USA). The 50% cytotoxic concentration (CC50) was calculated by the GraphPad Prism 7.0 software.
2.4. Cytopathic effect (CPE) inhibition assay
The Vero E6 cell monolayers were grown in 96-well plates and inoculated with 100 TCID50 of coronavirus strains at 37 ̊C for 2 h. The inoculum was removed, and the cells were subsequently incubated with indicated concentrations of LH or the positive control remdesivir. Following the 72 h of incubation, the infected cells shown 100% CPE under the microscope. The percentage of CPE in LH-treated cells were recorded. The 50% inhibition concentration (IC50) of the virus-induced CPE by LH was calculated by the Reed-Muench method [17].
2.5. Plaque reduction assay
The Vero E6 cell monolayers in 6-well plates were infected with 50 plaque-forming units (PFU) of SARS-CoV-2 for 2 h at 37 °C. After incubation, the cell monolayers were covered with agar overlay (final concentration: 0.6% agar, 2% FBS, indicated concentrations of LH or remdesivir). The plates were then incubated for 48 h at 37 °C with 5% CO2. Subsequently, the agar overlays were removed, and the cell monolayer was fixed with 10% formalin, stained with 1% crystal violet, and then the plaques were counted and photographed.
2.6. RNA isolation and reverse transcriptase-quantitative PCR analysis (RT-qPCR)
The Huh-7 cell monolayers in 12-well plate were rinsed with PBS and then exposed to coronavirus at a multiplicity of infection (MOI) of 1 for 2 h at 37 °C. The inoculum was removed and replaced with the indicated concentrations of LH or mock-treated with DMEM supplemented with 2% FBS for subsequent 48 h incubation at 37 °C with 5% CO2. The cells were then harvested for RNA isolation and qPCR as described previously [16]. The primer and probe sequences used for analysis are listed in Supplementary Table 1. The relative mRNA expression was calculated using the 2-△△Ct method with GAPDH as an internal reference gene.
2.7. Electron microscope
Monolayers of Vero E6 cells in 6-well plates were incubated with SARS-CoV-2 at a MOI of 0.001 for 2 h at 37 °C. The virus inoculum was then removed and replaced with DMEM medium supplemented with 2% FBS containing LH (600 μg/mL) or remdesivir (5 μM). At 48 h p.i., the cells were fixed, dehydrated and embedded as described previously [18]. Ultrathin sections (70 nm) of embedded cells were prepared, deposited onto Formvar-coated copper grids (200 mesh), stained with uranyl acetate and lead citrate, and then observed under JEM-1400 PLUS transmission electron microscopy (Japan Electron Optics Laboratory Co., Ltd., JEM-1400 PLUS).
2.8. Statistical Analyses
Statistical analysis was performed using GraphPad Prism 7.0 software. The differences in mRNA expression levels of cytokines were compared using a one-way analysis of variance (ANOVA). Values of p < 0.05 was considered to be statistically significant.
3. Results
3.1. Antiviral activity of LH on SARS-CoV-2 in vitro
The cell viability after LH or remdesivir treatment was determined by MTT assay in both Vero E6 and Huh-7 cells. LH showed unapparent cytotoxicity for both cell lines at concentrations up to 600 μg/mL (Fig. 1A, C). The positive control remdesivir showed no cytotoxicity to cells at a concentration of 50µM (Fig. 1B, D).
To investigate the antiviral effect of LH against SARS-CoV-2 virus, the Vero E6 cells were infected with 100 TCID50 of virus and incubated with LH at various concentrations for 72 h. As shown in Fig. 2A, LH inhibited the replication of SARS-CoV-2 virus with an IC50 value of 411.2 µg/mL by CPE assay (Fig. 2A). Meanwhile, treatment with LH following infection also had a dose-dependent inhibitory effect on plaque formation of the SARS-CoV-2 virus (Fig. 2C). We selected remdesivir as the positive control in our study and the results showed that remdesivir potently inhibited virus-induced CPE with an IC50 of 0.651 µM and a total plaque formation inhibition at 5 μM (Fig. 2B, C).
To further confirm the efficacy of LH in inhibiting SARS-CoV-2 virus replication in cells, we detected the viral particles in ultrathin sections of infected cells under electron microscopy. At 48 h p.i., viral particles were found in cytoplasm, intracellular vesicles, endoplasmic reticulum, and cell membrane and presented spherical crown-like appearance, which was typical coronavirus morphology (Fig. 3B, G). LH (600 μg/mL) and positive control remdesivir (5 μM) treatment resulted in a reduction of the number of virions compared with mock-treated infected cells (Fig. 3G–J). It was interesting to note that some virions in the surface of LH-treated cells presented spindle sharp which was in contrast to the typical spherical particles in the mock-treated cells (Fig. 3I).
3.2. Inhibition of SARS-CoV-2-induced cytokine and chemokine expression by LH in vitro
To determine the effect of LH on the expression of cytokines and chemokines induced by SAR2-CoV-2, the mRNA expression levels of TNF-α, IL-6, CCL-2/MCP-1, and CXCL-10/IP-10 were detected and compared between the LH-treated and mock-treated Huh-7 cells. The results showed that the elevated expressions of these four cytokines were significantly inhibited by LH treatment in a concentration-dependent manner (Fig. 4).
4. Discussion
Starting from December 2019, a pandemic of respiratory illness caused by a novel coronavirus named SARS-CoV-2 is sweeping the mainland of China. This virus has spread to several foreign countries, threatening to trigger a global outbreak. Several antiviral agents can be envisaged to control or prevent viral infections by antiviral assay in vitro [14,17]. However, the efficacy and safety of novel candidates need validations in vivo, even for those clinically approved medicines, which means that it will take months to years for clinical practices. At present, symptomatic and supportive treatments remain key to clinical practices. Thus, Traditional Chinese Medicines (TCM) carried both the antiviral effect and the symptomatic relief might bring more clinical benefits [12]. As a classical TCM prescription for respiratory diseases, LH is the only approved medicine in the treatment of SARS and influenza. After the outbreak of SARS-CoV-2, LH as a representative TCM prescription was recommended again in the latest Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia issued by National Health Commission of the People’s Republic of China The purpose of this study was to demonstrate whether the therapeutic effects of LH on the COVID-19 targeting virus replication and immunological regulation as it did on the infection caused by influenza viruses.
Our previous study showed that LH exhibited in vitro anti-influenza activity with IC50 ranging from 200-2000 μg/mL [1]. Here we demonstrated that LH also has a comparable antiviral potency against the SARS-CoV-2 virus with an IC50 value of 411.2 μg/mL (Fig. 2). Transmission electron microscopy (TEM) has been a potent tool to observe virus entry, virus particle assembly, viral ultrastructure, and budding from the plasma membrane [17]. To understand the antiviral details of LH, EM pictures were taken from each group. Abundant virus particles assembled at the surface of membrane, cytoplasm, and plasma vesicles in the SARS-CoV-2 infected cells, decreased in the treatment of LH at 600ug/mL. Notably, slight deformation of virus particles was seen in the LH treatment, which required us to make further studies.
Highly pathogenic coronaviruses such as SARS-CoV and MERS-CoV cause fatal pneumonia, which is mainly associated with rapid virus replication, massive inflammatory cell infiltration and elevated proinflammatory cytokine/chemokine responses. Although the pathophysiology of fatal pneumonia caused by highly pathogenic coronaviruses has not been completely understood, accumulating evidence suggests that the cytokine storm plays a crucial role in causing fatal pneumonia [18]. Excessive amounts of proinflammatory cytokines were reported (e.g., IL-1β, IL-6, IL-12, IFN-γ, IP-10, and MCP-1) in the serum of SARS patients [18], similar in the serum of MERS patients [19]. Chaolin Huang et al. confirmed the occurrence of the cytokine storm in the COVID-19 patients in ICU rather than those in non-ICU patients [7]. Based on the excessive cytokines responses, Suxin Wan et al. claimed that IL-6 and IL-10 levels could be used as one of the bases for predicting the outcome and prognosis of the COVID-2019 [20]. In this study, host cells infected with HCoV-229E and SARS-COV-2 increased the cytokine release such as TNF-α, IL-6, CCL-2/MCP-1, and CXCL-10/IP-10, which was suppressed by LH in a dose-dependent manner. The change of cytokine profiles suggested that LH might have a potential effect on the inhibition of cytokine storm induced by SARS-COV-2, which also needed to be validated in vivo.
5. Conclusion
Since the launch of LH, it has been widely used as a broad spectrum of antiviral agent in the clinical practice, especially for various respiratory virus infections. Previous studies have shown that LH a broad spectrum of effects on a series of influenza viruses by interfering with both viral and host reactions. Although LH significantly relieved the clinical symptoms of the COVID-19, the underlying mechanism of antiviral effects on coronavirus, especially in the SARS-COV-2, was still elusive. In this study, we demonstrated that LH exerted its anti-coronavirus activity by inhibiting virus replication and reducing the cytokine release from host cells, which supported the clinical application of LH in combination with existing therapies to treat COVID-2019.
Acknowledgements
The study was funded by Beijing Municipal Science and Technology Commission NCP Emergency Project; Hebei Provincial Department of Science and Technology NCP prevention and control emergency scientific research project (Grant no. 20277708D); The Science research project of the Guangdong Province (Grant no. 2020B111110001); Daxing District Science and technology development projects (Grant no. KT202008013).
Appendix A. Supplementary data
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