Study On The Mechanism Of Antiviral And Anti-inflammatory Based On Traditional Chinese Medicine Small Molecule Shikonin

Coronavirus disease 2019（COVID-19）that broke out in December 2019 has been a global pandemic that severely threatens global health with concordant economic damage.More than 91.5%of the confirmed cases in China are treated with traditional Chinese medicine,and clinical observation shows that the treatment rate of traditional Chinese medicine reaches 90%.The safety of traditional Chinese medicine and the overall treatment characteristics of"multi-component,multi-target,multi-effect"make it play a crucial role in the prevention and treatment of COVID-19.At present,traditional Chinese medicine small molecule shikonin has successfully targeted the new coronavirus main protease(SARS-Co V-2 M^pro),and its complex spatial structure has been successfully analyzed.A study has demonstrated that shikonin exerts anti-coronavirus effect by inhibiting SARS-Co V-2 M^pro activity.Shikonin is expected to enter the clinic through subsequent drug optimization.Based on this,we deeply have studied the pharmacological mechanism of traditional Chinese medicine small molecule shikonin.This paper mainly studies the antiviral and anti-inflammatory mechanisms of shikonin.（1）First,we studied the more common human coronavirus NL63（HCo V-NL63）,which has caused worldwide spread.HCo V-NL63 was first isolated from a seven-month-old child in 2004 and accounts for 10%of all respiratory illnesses caused by etiological agents.HCo V-NL63 mainly infects children and the immunocompromised and is responsible for a series of clinical manifestations,including cough,fever,rhinorrhoea,bronchiolitis,and croup.However,effective antivirals against HCo V-NL63 infection are currently unavailable.The HCo V-NL63main protease(M^pro),also called 3C-like protease(3CL^pro),plays a vital role in mediating viral replication and transcription by catalyzing the cleavage of replicase polyproteins（pp1a and pp1ab）into functional subunits.Moreover,M^pro is highly conserved among all coronaviruses,thus making it a prominent drug target for antiviral therapy.Here,four crystal structures of HCo V-NL63 M^pro in the apo form at different p H values are reported at resolutions of up to 1.78（?）.Comparison with M^profrom other human betacoronaviruses such as SARS-Co V-2 and SARS-Co V reveals common and distinct structural features in different genera and extends knowledge of the diversity,function and evolution of coronaviruses.Meanwhile,we screened and optimized the crystallization conditions of the complex of HCo V-NL63 main protease and non-covalent inhibitor shikonin,and finally obtained co-crystals of the complex.The FRET method was used to determine the effect of shikonin on the HCo V-NL63main protease.The results showed that shikonin has an inhibitory effect on the main protease of HCo V-NL63,and the half-maximal inhibitory concentration（IC50）reached 16.91μM.The crystallographic study of the main protease of HCo V-NL63and its complex will help people understand the mechanism of action of the main protease of coronavirus.Combined with the existing research results,it is conducive to the development of broad-spectrum inhibitors against coronavirus.（2）In addition,we also investigated the mechanism of shikonin in preventing postoperative intraperitoneal adhesions in rats.Objective:To observe the preventive effect of shikonin on postoperative intraperitoneal adhesion in rats and explore its mechanism of action.Methods:Twenty-four SPF grade SD male rats（7 weeks old,200-210 g）were randomly divided into blank control group（Group C,n=6）,model group（Group M,n=9）,and preventive treatment group with shikonin（Group P,n=9）,in which the group C did not receive any treatment while the other two groups were given surgical treatment to create a model of abdominal adhesion.Starting 2 cm away from the ileocecal part,the intestinal tract was clamped with toothed tweezers at 1 cm intervals until subserosal hemorrhage and spotting hemorrhage occurred,a total of 10places.Meanwhile,the cecum was scraped until bleeding oozed,thus forming a model of abdominal adhesion.After the operation,group P was given shikonin by gavage（10 mg/kg/d）,group M was given the same volume of normal saline,and group C was given a normal diet for 14 days respectively.14 days later all the rats were sacrificed,adhesion scores and incidence were assessed according to the Nair scoring scale.The adhesive tissue or the tissue at the modeling site were collected for HE staining,Masson staining,and immunohistochemistry staining respectively.Arterial blood was obtained from the heart of rats,centrifuged at 1,000 x g for 15 min at 4℃,and then the supernatant was carefully collected and stored at-80℃.The concentrations of inflammatory factors（IL-1β,IL-6,TNF-α,IFN-γ）and fibrinolytic system activity indicators（t PA,PAI-1）in rat serum were detected by ELISA.Western Blot assay was used to detect the expression of fibrotic proteins（TGF-β1,α-SMA）in the adhesion tissue and intestinal tissue at the modeling site.The data were analyzed by Graph Pad Prism 7.00.If P<0.05,the difference was statistically significant;if P>0.05,the difference was not statistically significant.Results:（1）The model group had the heaviest intra-abdominal adhesions and the highest score.The adhesions mainly involved the greater omentum,stomach,liver,spleen,and abdominal wall.Groups treated with shikonin have less adhesion scores than the model group（P<0.05）.（2）The adhesion tissue in group P had less inflammation cell,less fibroblast,and less type I collagen expression than group M.（3）Compared with group C,surgery significantly enhanced the expression levels of inflammatory factors IL-1β,IL-6,TNF-αand IFN-γin group M.Group P significantly decreased the expression levels of IL-1β,IL-6,TNF-αand IFN-γthan group M.Compared with group M,the fibrinolytic system activity index t PA was increased and PAI-1 was decreased in group P（P<0.05）.（4）In the WB experiment,the expression levels of TGF-β1 andα-SMA increased in group M compared with the group C,but the trend was reversed in group P（P<0.05）.Conclusion:Shikonin can down-regulate the expression of TGF-β1,α-SMA,and type I collagen,inhibit the release of inflammatory factors,and effectively prevent the formation of postoperative abdominal adhesions.It provides a new drug direction for preventing the formation of intraperitoneal adhesions.