Effects Of Lovastatin On Neutrophil Behavior

Lovastatin is a lipid-lowering drug,which also has the effects of improving endothelial cell function,anti-inflammatory,and cancer treatment,and can be produced during the fermentation of Pu'er tea.Some studies have shown that the infiltration of neutrophils around the tumor not only reduces the therapeutic effect,but also promots the tumor metastasize.Although it has been reported that lovastatin not only has anti-inflammatory functions but also anti-tumor,its mechanism is unknown.Therefore,this experiment starts from the perspective of the lovastatin's anti-inflammatory effect and explores whether the effect of lovastatin on neutrophils can help the treatment of tumors,providing a theoretical basis for the application of lovastatin in tumor treatment.Using lovastatin,transgenic zebrafish(mpx: e GFP)zebrafish,Bal B / c mice as the main experimental materials,the following contents were studied:1.Effect of lovastatin on the number of neutrophils in zebrafish inflammation modelA disposable surgical blade was used to cut off the 0.4mm tail tip of 3dpf(days post fertilization)transgenic zebrafish larvae to establish the inflammatory model and divide them into a control group,a 30 n M lovastatin group and a 60 n M lovastatin group,and then treat them for 4h and 22 h respectively.Then the effects of lovastatin on the number of neutrophils accumulated in the inflammatory site were observed.The results showed lovastatin did not significantly reduce neutrophils number in the whole body of zebrafish,but it significantly reduced neutrophils number in the tail inflammation site at 4hpi(hours post injury)and the neutrophils number in the body and tail of the zebrafish both decreased significantly at 22 hpi,indicating that lovastatin has the effect of inhibiting the recruitment of neutrophils in the zebrafish inflammation site.Overall,lovastatin significantly reduced the number of neutrophils and inhibited the recruitment of neutrophils at the site of inflammation.It can be speculated that the decrease in the number of neutrophils may be caused by neutrophil apoptosis or migration.2.Effect of lovastatin on neutrophils apoptosis in zebrafish inflammation siteThe effect of lovastatin on the cell proliferation and apoptosis-related proteins ERK1/2and AKT1/2/3 phosphorylation was tested,and the zebrafish embryos were treated with corresponding protein inhibitors to confirm the role of these proteins in the process of neutrophil reduction.The results showed lovastatin significantly inhibited the expression of pro-proliferation anti-apoptotic proteins p-ERK and p-Akt,and the number of neutrophils in the inflammation site was reduced after using the corresponding protein inhibitors,so it can be speculated that lovastatin promotes apoptosis.Then zebrafish were respectively treated with lovastatin(30n M)for 2h,9h and 12 h,and then used zebrafish embryo immunostaining and TUNEL apoptosis labeling method to further confirm the use of lovastatin on the neutrophils apoptosis of zebrafish inflammation site.It can be seen from the results lovastatin slightly increased the number of neutrophil apoptosis at 2h,but there was no difference,and lovastatin significantly increased the level of neutrophil apoptosis at 9h and 12 h.Therefore,it can be concluded that lovastatin significantly increases the level of apoptosis in the zebrafish inflammation site.3.Effect of lovastatin on neutrophil migrationqRT-PCR was used to detect the expression of neutrophil positive migration and reverse migration related genes at different times after lovastatin treatment.The results showed the expression of all genes related migration were inhibited after treatment with lovastatin,which was contrary to the results of neutrophils reduction in the inflammation site.Therefore,it can be speculated that the reduction of neutrophils number at the site of inflammation caused by lovastatin is not related to the neutrophils migration.4.Study of lovastatin combined with bacterial treatment to inhibit tumors in colon cancer mouse modelIt is known that bacterial therapy has a good effect in suppressing tumors,but there are some deficiencies such as tumor recurrence,which speculated are related to the accumulation of a large number of neutrophils around the tumor.So we combined lovastatin with bacterial therapy to treat colon cancer tumors in mice.Five-to six-week-old male Bal B/c mice were inoculated with CT26 cells and randomly divided into PBS,S.t?pp Gpp,lovastatin,and S.t?pp Gpp+lovastatin groups.When the tumor volume reached 120-160mm3,3×107CFU bacteria were injected through the tail vein.After that,the tumor volume was measured every 2 days until the tumor volume reached ?1500mm3.The results showed that the lovastatin group significantly inhibited the growth of the tumor compared with the PBS group,indicating that the lovastatin itself has a certain tumor suppression effect;in the s.? pp Gpp group,the tumor volume gradually decreased and even fell off within 10 days after the bacterial injection,but gradually recrudesce after 10 days.However,the combination of bacterial therapy and lovastatin significantly slowed down the rate of tumor recurrence,and even made the tumor completely fall off,indicating that lovastatin has a certain anti-tumor effect,and has played a positive role in promoting tumor suppression by bacterial treatment.Two groups of S.t?pp Gpp and S.t?pp Gpp+lovastatin combined treatment was designed,and tumor,liver,lung,and spleen of mice were collected for bacterial culture and counting on the 2nd and 6th days after the treatment,in order to study the effect of lovastatin on the ability of S.t?pp Gpp bacteria to target tumors as well as their remaining time in tumors.The results showed that after 6 days treatment,the number of bacteria in the tumor of both lovastatin group and combination group were significantly higher than that of the simple bacteria treatment group;and 2nd and 6th day after treatment,the number of bacteria in the tumors of both groups was much higher than that of other tissues.Thus,it can be seen that lovastatin can effectively prolong the existencel time of S.t ?pp Gpp engineering bacteria in tumors.In summary,lovastatin reduces neutrophils number by promoting the apoptosis of neutrophils,and whether it is used alone or in combination with bacterial therapy has a significant tumor suppressing effect.