The Effect Of TXL On Macrophage Proliferation Induced By Hypoxia And Its Mechanism

Objective: The macrophage is the key initial player in an innateresponse, functioning both to eliminate the pathogen and to recruit otherinnate inflammatory cells. Macrophages secrete a number of factors such asgrowth factors and other cytokines to mediate inflammatory reaction. It wasreported that hypoxic exposure induced an inflammatory response within thevessel wall, and involved in many development process of diseases such asinfection, chronic inflammation and ischemia.Tongxinluo (TXL) is a compound traditional Chinese medicine, hasbeen widely used in China to treat patients with diseases such asarteriosclerosis and apoplexy. Recently research found that TXL could inhibitthe inflammatory response.In this study, we observed the effect of TXL on the proliferation ofhypoxia-induced macrophages in vivo and in vitro and found that hypoxiacould significantly induce the expression of hypoxia inducible factor andinflammation related genes in macrophages. TXL inhibited the proliferation ofmacrophages by down-regulating the expression of inflammation relatedgenes induced by hypoxia.Methods: Hypoxia model of RAW264.7cells were established bytreatment with CoCl2. The expression of HIF-1α, TLR-4, iNOS, NF-κB inthe RAW264.7cells was measured by RT-PCR and Western blotting. Thecell viability was evaluated using MTS assay. The chronic and acute hypoxiaanimal model was established. The protein expression of HIF-1α, TLR-4,iNOS, and NF-κB were detected by immunohistochemistry.Results:1CoCl2promoted RAW264.7cells proliferation.RAW264.7cells were used to study the effect of hypoxia on the proliferation of macrophages. MTS assays showed that the proliferation ofRAW264.7cells was induced by CoCl2stimulation, especially in theconcentration of100μM.2CoCl2increased expression of HIF-1α, TLR-4, iNOS and NF-κB.In order to further study the molecular mechanism of macrophageproliferation induced by hypoxia, we observed the expression of HIF-1α andinflammation related genes. Western blotting and RT-PCR results showed thatCoCl2could significantly induced the expression of HIF-1α, TLR-4, iNOS,and NF-κ B compared with the control group.3TXL inhibited RAW264.7cells proliferation induced by CoCl2The effect of TXL on proliferation of RAW264.7cells was furtherexamined using MTS assay. The results showed that TXL inhibited theproliferation of RAW264.7cells with dose dependent manner.4TXL inhibited expression of HIF-1α, TLR-4, iNOS and NF-κB inRAW264.7cells induced by CoCl2.To further investigate the molecular mechanism of TXL on macrophageproliferation, the expression of HIF-1α, TLR-4, iNOS and NF-κB wasdetected by RT-PCR and Western blotting. The results showed that hypoxiacould induce the expression of the above genes, while TXL significantlyinhibited the expression of hypoxia-induced genes.5TXL inhibited expression of HIF-1α, TLR-4, iNOS and NF-κB inchronic hypoxia mice.To further verify TXL effect on macrophage proliferation in vivo, chronichypoxia and acute hypoxia mice model was established. The expression ofHIF-1α, TLR-4, iNOS, and NF-κB was observed by immunohistochemistry inlung tissue. The results showed that the expression of HIF-1α, TLR-4, iNOS,and NF-κB increased in chronic hypoxia mice, while TXL inhibited theseprotein expression in lung tissue. However, TXL had no effect on survivaltime and protein expression in acute hypoxia mice.Conclusions:1CoCl2promoted RAW264.7cells proliferation. 2CoCl2increased the expression of HIF-1α, TLR-4, iNOS and NF-κB inRAW264.7cells.3TXL inhibited RAW264.7cells proliferation induced by CoCl2.4TXL inhibited the expression of HIF-1α, TLR-4, iNOS and NF-κB inRAW264.7cells induced by CoCl2.5TXL inhibited expression of HIF-1α, TLR-4, iNOS, and NF-κB inchronic hypoxia mice.