| In previous works, to study the influence of DNA damage of host cells on innate immune response, we have designed a series of oligonucleotides(CCT ODN) based on the sequences of human microsatellite DNA. Among them, an ODN composed of CCT repeats(named as SAT05f) was found to down-regulate TLR7/9-dependent IFN-α production in cultured human peripheral blood mononuclear cells(PBMCs).Based on the results we speculate that CCT ODN may have the potential to cure some autoimmune diseases. To prove this speculation, we designed the following experiment: First we established a chronic graft versus host(c GVHD) induced lupus nephritis mouse model and then testified the therapeutic effect of CCT ODN on experimental lupus nephritis. In the process, we unexpectedly found that CCT ODN could relieve the symptoms of experimental lupus nephritis, at the same time it could induce patchy hair loss around mouth of male mice. The finding hinted that the CCT ODN could impact HFs. Further, since our previous work revealed that the CCT ODN,when subcutaneously injected, caused massive infiltration of NK cells in drainage lymph nodes,20 we suspected that NK cells or possibly other NKG2D+ cells could be mobilized or activated by the CCT ODN and therefore were involved with the development of hair loss in the male mice.Human Hair loss is a common clinical disease, both men and women, regardless of age, all suffered by hair loss in different degree. Hair loss seriously affect the patient’s appearance, thereby affecting the patient’s social, personal charm and self-confidence, therefore bring great harm to people’s quality of life and physical and mental health. Due to the reasons of hair loss causes, there is a big difference in terms of duration, alopecia size, whether recurrence, there is no effective treatment for hair loss at present. Therefore, revealing the reasons and mechanisms of human hair loss,looking for effective treatment for hair loss is still an urgent problem to be solved. ForODN induced hair loss found in preliminary experiment was not an accidental phenomenon, we tried to reveal the law of hair growth inhibited induced by ODN in male mice and its possible mechanisms, and therefore to reveal the reasons and mechanisms of human hair loss, so as to provide theoretical basis for the research and development of drugs for hair loss treatment.1. Effect of ODN on hair and hair follicle growth in F1 mice with experimental lupus nephritisTo induce c GVHD induced lupus nephritis mouse model, 8-week old F1 mice were intraperitoneally(i.p.) injected with 5×107 single-cell suspension containing a mixture of thymus, spleen, and lymph node cells from Balb/c mice at a ratio of 3:2:1for 4 times at 3-day intervals. To observe the effect of CCT ODN on the hair growth,after induction, F1 mice were injected i.p. with PBS, CCT ODN or Cp G ODN. First injection began at 2h later of the first lymphocyte injection and the injection lasted for9 weeks(twice a week). Photos were taken every week to record the hair growth of the mice all over the body. The results showed that the hairs began to lose at Week 5,until Week 9 all the hairs around the mouth lost.Collected the serum of the mice in each group after the 8th injection, the 12 th injection and the 18 th injection, respectively. To test whether ODN induced hair loss was associated with the serum anti-ds DNA antibody level in mice, we detected the serum anti-ds DNA antibody level with ELISA. The results showed that the serum anti-ds DNA antibody level increased after the c GVHD induction, but the the serum anti-ds DNA antibody level in the mice with hair loss wasn’t higher than the mice without hair loss, showing that there was less association of ODN induced hair loss with the increased serum anti-ds DNA antibody level. To test whether ODN induced hair loss was associated with the catagen-like change of the vibrissa HFs, 3 days after the last injection, half of the mice were randomly sacrificed and their whisker pads were isolated for H&E staining. The results showed that there was no significant differences in the numbers of the regressive vibrissa HFs in the mice with hair loss compared to the mice without hair loss, showing that there was less association ofODN induced hair loss with the catagen-like change of the vibrissa HFs.2. Effect of ODN on hair and hair follicle growth of Balb/c miceTo observe the effect of ODN on the hair growth, 6-week old Balb/c mice were injected i.p. with PBS, CCT ODN, Cp G ODN or MS ODN twice a week for 4 weeks.Photos were taken every week to record the hair growth of the mice all over the body.The results showed that in the CCT ODN group, the hairs of the male mice began to lose at Week 3(the shortening of the long vibrissa), until Week 4 the long vibrissa shortened more obviously, but they didn’t lose. Cp G ODN and MS ODN showed no effect on hair growth.To test whether ODN induced hair loss was associated with the catagen-like change of the vibrissa HFs, 3 days after the last injection, half of the mice were randomly sacrificed and their whisker pads were isolated for H&E staining. The results showed that there was no significant differences in the numbers of the regressive vibrissa HFs in the mice with hair loss compared to the mice without hair loss, showing that there was less association of ODN induced hair loss with the catagen-like change of the vibrissa follicles.3. Effect of ODN on serum anti-ds DNA level of Balb/c miceCollected the serum of the mice in each group after the 6th injection and the 8th injection, respectively. To test whether ODN induced hair loss was associated with the serum anti-ds DNA antibody level in mice, we detected the serum anti-ds DNA antibody level with ELISA. The results showed that the serum anti-ds DNA antibody level in the mice with hair loss wasn’t higher than the mice without hair loss, showing that there was less association of ODN induced hair loss with the auto antibody response.4. Effect of ODN on growth of hair and hair follicle in depilated Balb/c mice6-8 week old Balb/c mice, first depilated with depilatory cream at the back, then injected i.p. with PBS, CCT ODN or Cp G ODN, twice a week for 4 weeks. Photos were taken every week to record the hair growth of the mice at the back. The resultsshowed that the hairs grew more slowly in the CCT ODN group compared with the control, showing that CCT ODN might slow down the speed of hair re-growth after depilation, but the results needed further proved.5. Effect of ODN on NKG2D+ and the expression of NKG2 DL H60 in vibrissaHFs of Balb/c miceTo test whether CCT ODN induced hair loss was associated with the NKG2D+cells infiltration in the vibrissa HFs, on day 3 of the last injection, the whisker pads on the one side of the mice were isolated and then stained with anti-NKG2 D antibody; at the same time the whisker pads on the other side were isolated ant then extracted protein, the expression of H60 protein was detected by Western Blotting. The results showed that there were more NKG2 D positive cells in and around the vibrissa HFs of the mice with hair loss, showing that the increased NKG2 D positive cells in and around the vibrissa HFs was involved with the ODN induced hair loss.6. Effect of ODN on the ratio of NKG2D+ cells in splenocytes and PBMCs ofBalb/c mice6-8 week old Balb/c mice, injected i.p. with PBS, CCT ODN or Cp G ODN. 4 h or 24 h after the last injection, the mice were sacrificed and the splenocytes and PBMCs were isolated for flow cytometry. The results showed that after the injection of ODN, the percentage of NKG2D+ cells tended to be decreased in the splenocytes or PBMCs(no statistic difference). These results implied that the NKG2D+ cells might be mobilized to HFs to result in hair loss in the male mice.7. Effect of ODN on growth of cultured vibrissa HFs of Balb/c miceWhisker pads from the 1-week old normal male Balb/c mice were isolated and whole vibrissa HFs were dissected out of the specimens under a dissecting microscope. Early- or mid- anagen growth phase follicles were selected for culturing.They were maintained in the serum-free Williams’ E medium supplemented with or without CCT ODN or Cp G ODN(4, 8, 16 μg/ml) or the supernatants of the splenocytes stimulated with CCT ODN or Cp G ODN. HFs were incubated for a totalperiod of 2 weeks, and half of the medium was changed every 3 days. Photos of each HF were taken every week to record the morphologic changes. The results showed that with the prolongation of the incubation time, all the vibrissa HFs in anagen began to change into catagen, the length of the hair shaft increased, the total length and width of the hair follicle decreased, the HFs cultured with the CCT ODN conditioned supernatants for 14 days were obviously smaller than those cultured with the supernatants without CCT ODN treatment(P<0.05). These results showed that CCT ODN could speed up the catagen-like change of the vibrissa HFs, promote the cultured HFs in anagen to change into catagen in advance.8. Effect of ODN on the expression of NKG2 DLs m RNA in cultured vibrissa HFs of Balb/c miceThe vibrissa HFs isolated from the whisker pad of the 8-week old normal Balb/c mice were cultured with CCT ODN or Cp G ODN at the concentration of 4, 8, 16μg/ml for 24 h. Then total RNA of the vibrissa HFs was extracted and the NKG2 DLs expression was analyzed by RT-PCR. The results showed that there was no NKG2 DLs expression in the vibrissa HFs of both the male and female mice cultured with the basal medium compared with the positive control YAC-1 cells(the standard NK target cell line). And after stimulation with CCT ODN or Cp G ODN, there was still no NKG2 DLs expression. The results showed that CCT ODN might not directly influence the NKG2 DLs expression of the vibrissa HFs cultured in vitro.9. Effect of ODN on the expression of IFN-g and TGF-b m RNA in splenocytes ofBalb/c mice6-8 week old Balb/c mice, injected i.p. with PBS, CCT ODN or Cp G ODN. 24 h after the injection, the mice were sacrificed and the splenocytes were isolated from the mice. Then total RNA of the splenocytes was extracted and the expression of IFN-γand TGF-β was analyzed by RT-PCR. The results showed that CCT ODN could up-regulate the m RNA expression of IFN-γ(P<0.05), not TGF-β, in the splenocytes of the male mice, implying that CCT ODN might cause hair loss in the male mice byup-regulating the expression of IFN-γ.Overall, ODN might induce the suppression of the hair growth and the catagen-like change of the HFs; the increased NKG2 D positive cells in and around the vibrissa HFs was involved with the ODN induced hair loss; the suppression of the hair and HFs might be related with the systemic immune regulation activity of the ODN,for the ODN conditioned supernatants promoted the cultured HFs in anagen to change into catagen in advance and induced the up-regulation of the IFN-γ m RNA in the splenocytes.Although our research seems to be of little clinical significance, the general application of the immune modulators and the common phenomenon of the collapse of the immune privilege during treatment, causing undetectable pathological changes,the accumulation of pathological changes might be responsible for the susceptibility to certain diseases. The possible mechanisms of the ODN induced hair loss are not well understood, but this induction leading to the collapse of the immune privilege of the HFs, and it might be a potential model of some diseases. Our research is still in the early stages of exploration, although tried to explore the mechanisms, the results are mainly about some phenomenons. This might be the biggest limitation of our research, however, it might also be an important clue and breakthrough point of our research after deeply thinking in the future. |
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