Objective

2

To explore the clinical efficacy of compound Wufengcao liquid （CWL） on tuberculous ulcer and the influence on macrophage polarization.

Method

2

① Clinical experiment： A total of 145 patients with tuberculous ulcer who were treated in Nanjing Integrated Traditional Chinese and Western Medicine Hospital were randomized into observation group， control group Ⅰ， and control group Ⅱ according to the random number table method. In addition to the basic anti-tuberculosis chemotherapy， CWL， Kangfuxin liquid， and isoniazid solution （local external application） were respectively used in the observation group， control group Ⅰ， and control group Ⅱ. The treatment lasted 4 weeks for each group. The total effective rate in wound healing， traditional Chinese medicine（TCM） syndrome score， and histopathological morphology of wound were observed and the expression of inducible nitric oxide synthase （iNOS） and arginase-1 （Arg-1） in wound tissue was measured. ② Cell experiment： RAW264.7 cells were cultured in DMEM （10% fetal bovine serum， 1% double-antibody solution） in a cell incubator （37 °C， 5% CO

2

）. Phorbol 12-myristate 13-acetate （PMA） was used to induce the differentiation of RAW264.7 cells into macrophages. Lipopolysaccharide （LPS） was employed to stimulate polarization of macrophages into M1 type and interleukin-4 （IL-4） to induce the polarization into M2 type. Kangfuxin solution， isoniazid solution， and CWL were respectively applied to the above cell model for 36 h. The cell supernatant was collected and centrifuged. Western blot was used to detect the protein expression of tumor necrosis factor-

α

（TNF-

α

）， transforming growth factor-

β

（TGF-

β

）， iNOS， and Arg-1， and flow cytometry （FCM） to detect the expression of CD86 and CD206.

Result

2

①Clinical experiment： The total effective rate in the CWL group ［98.0% （48/49）］ was higher than that in the control group Ⅰ ［87.5% （42/48），

χ

2

=3.962，

P

<

0.05］ and control group Ⅱ ［83.3% （40/48），

χ

2

=6.162，

P

<

0.05］. After 28 days of treatment， compared with control group Ⅰ and control group Ⅱ， CWL decreased the TCM syndrome score （

P

<

0.05） and obviously improved the histopathological morphology of the wound. Immunohistochemistry results showed that the iNOS expression in local focus tissue was lower （

P

<

0.05） and the expression of Arg-1 was higher （

P

<

0.05，

P

<

0.01） in the CWL group than in the control group Ⅰ and control group Ⅱ after 28 days of treatment. ② Cell experiment： Western blot assay showed that the expression of iNOS and TNF-

α

in LPS group increased compared with that in the M0 group （

P

<

0.01） and the expression in the LPS+ isoniazid group， LPS+ Kangfuxin group， and LPS+CWL group was lower than that in the LPS group （

P

<

0.05）. The expression of iNOS in LPS+Kangfuxin group and LPS+ CWL group was lower than that in the LPS+isoniazid group （

P

<

0.05，

P

<

0.01）， and the expression of TNF-

α

in LPS+ CWL group was lower than that in LPS+isoniazid group （

P

<

0.01）. The expression of TNF-

α

in LPS+ CWL group decreased compared with that in the LPS+ Kangfuxin group （

P

<

0.05）. The expression of Arg-1 and TGF-

β

in IL-4 group was higher than that in the M0 group （

P

<

0.01）， and the expression in the IL-4+isoniazid group， IL-4+Kangfuxin group， and IL-4+ CWL group was higher than that in the IL-4 group （

P

<

0.05）. The expression of Arg-1 and TGF-

β

in the IL-4+ Kangfuxin group and IL-4+CWL group was higher than that in the IL-4+isoniazid group （

P

<

0.05，

P

<

0.01）， and the expression was higher in the IL-4+CWL group than in the IL-4+Kangfuxin group （

P

<

0.05，

P

<

0.01）. The FCM result showed that the expression of CD86 and CD206 in LPS group and IL-4 group was higher than that in M0 group （

P

<

0.01）. CD86 expression in LPS+isoniazid group， LPS+ Kangfuxin group， and LPS+CWL group was lower than that in the LPS group （

P

<

0.01）. The expression of CD86 in LPS+Kangfuxin group and LPS+ CWL group increased compared with that in the LPS+isoniazid group （

P

<

0.01）， and the expression was higher in the LPS+ CWL group than in the LPS+Kangfuxin group （

P

<

0.01）. CD206 expression in IL-4+ isoniazid group， IL-4+Kangfuxin liquor group， and IL-4+ CWL group was increased compared with that in the IL-4 group （

P

<

0.01）. CD206 expression in IL-4+Kangfuxin liquid group and IL-4+ CWL group was decreased compared with that in the IL-4+isoniazid group （

P

<

0.01）. CD206 expression in IL-4+CWL group was lower than that in the IL-4+ Kangfuxin group （

P

<

0.05）.

Conclusion

2

CWL can promote the healing of tuberculous ulcers， and the mechanism is that it inhibits the expression of iNOS， TNF-

α

， and CD86 and promotes the expression of Arg-1， TGF-

β

， and CD206， thereby regulating M1/M2 polarization balance.