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Individual HFs were photographed 72 h after the start of incubation Edmund Optics Ltd, UK. After washing with PBS, the samples were next incubated with the in situ cell death detection kit reagents ab, Abcam, Cambridge, MA, USA according to the manufacturer's instructions.
The TUNEL-positive cells and the number of TUNEL-positive cells that expressed or did not express cleaved caspase-3 were detected using fluorescence microscopy Eclipse E RIPK1 and RIPK3 were silenced in C 3 H mice in vivo by transfecting with 6 μg of RIPK1 vs.
Luminescence was measured using a Glomax Discover microplate Reader Promega. We first evaluated the hair growth-promoting effect of the three Nec analogs in the mouse vibrissa organ culture.
Only Nec-1s increased the length of the mouse vibrissae HF at day 3 Fig. Also, only Nec-1s increased the hair length in the pig hair organ culture Fig.
The percentage of pig skin and mouse vibrissae HF in the anagen phase was higher in Nec-1s than other Nec analogs Fig. In addition to that, Nec-1s significantly enhanced the proliferation of ORS cells Fig. Hair growth-promoting effects of necrostatin derivatives. Among the necrostatin analogs Necs , Nec-1s treatment significantly promoted mouse vibrissa follicle A and pig HFs B growth ex vivo.
C The number of pig HFs and mouse vibrissae follicles in the anagen and non-anagen phase was counted. Anagen and non-anagen pig HF or mouse vibrissae follicle was observed C, right panel.
D The proliferation of outer-root sheath ORS cells was measured. White bars: control, blue bars: Nec-1i, green bars: Nec-1, red bars: Nec-1s. We used telogen phase 7-week-old male C 3 H mice to investigate the telogen-to-anagen transition Fig.
Compared with the negative control, topical application of Nec-1s 0. Hair matrix cell and ORS cell proliferation in the HFs BrdU-immunopositive cells were also increased in the skin of Nec-1s-treated mice Fig.
The hair weight and anagen HF number were significantly increased by Nec-1s in a dose-dependent manner Fig. We next investigated whether Nec-1s can prolong the anagen phase of the hair cycle in C 3 H mice Fig.
More catagen HFs was found in control-treated mouse, and the anagen phase was prolonged by topical treatment of Nec-1s 0.
Hair growth-promoting effects of necrostatin-1s. A The experiment design. The back skin of 7-week-old C 3 H mice was shaved, and 0.
B Compared with the negative control, topical application of Nec-1s 0. E Anagen elongation experiment design. F Photographs were taken after topical application of Nec-1s for 5 days. Nec-1s had been developed to inhibit RIPK1-dependent necroptosis 7 , 19 , 20 , 21 , Therefore, we examined where RIPKs are expressed in the HF and analyzed RIPK1 and RIPK3 proteins by immunofluorescence in human and pig HFs.
Both RIPK1 and RIPK3 were highly expressed in the ORS region of human scalp HFs and ORS region of pig HFs which were indicated by co-staining with keratin 17 k17, an ORS marker in pig HF Figs.
These results suggested that Nec-1 s may target RIPK1 and RIPK3 in the ORS region. RIPK1 expression in the hair follicle. A Human HFs were kindly received from Prof. Sang Ho Oh Severance Hospital, Yonsei, University College of Medicine and B pig HFs were isolated from 1-week-old pig back skin.
Immunostaining showed that RIPK1 green localized in the outer-root sheath ORS region of HFs and co-localized with keratin 17 k17, blue, an ORS marker in the HF. DAPI staining blue indicates nuclei. In our study, RIPK1 was expressed in ORS region of HFs at anagen stage of human, pig and mice Fig.
In addition, the expression level of RIPK1 in ORS region was highest at catagen Fig. To investigate which cell death occurred during hair cycling, we performed double labelling with TUNEL and cleaved caspase-3 c-cas3 during hair cycling. According to our result on Fig.
These results suggested that both apoptosis and necroptosis occurred in mainly ORS cells at catagen stage during normal hair cycling, and necroptosis still occurred at telogen stage. Necroptosis occurred during mouse hair cycle. The paraffin sections were isolated from 4-, 5-, and 7-week-old C 3 H mice.
A Immunofluorescent detection of RIPK1 green in mouse dorsal skin. DP, dermal papillae; HG, hair germ. B TUNEL and cleaved caspase-3 dual immunofluorescent labelling in the skin tissue. TUNEL Red and cleaved caspase-3 Green staining were performed.
We further studied whether RIPK1 or RIPK3 inhibition would enhance the hair growth. In the vibrissa organ culture, both treatments of RIPK1 siRNA and RIPK3 siRNA promoted mouse vibrissa length for 1. Furthermore, siRNA injection for RIPK1 and RIPK3 accelerated telogen-to-anagen transition in C 3 H mice Figs.
In addition, RIPK1 pharmacological inhibition was tested. Sibiriline, GSK᾽, and GSK treatment significantly elongated hair length in the mouse vibrissa organ culture Fig. These results indicated that RIPK1 inhibitors promote hair growth by inhibiting necroptosis.
Hair growth-promoting effects of RIPK inhibition. A Mouse vibrissa follicles were isolated and cultured with 1 μg of RIPK1 siRNA treatment for 3 days. RIPK1 knockdown increased hair fiber length in the organ culture.
B The experiment design. After depilation of the dorsal skin, 6 μg of siRNA was injected for every 2 days. Injection of siRNA for RIPK1 accelerated the telogen-to-anagen transition in C 3 H mice C.
D—F Hair growth-promoting effect of several RIPK1 inhibitors. Sibiriline D , GSK' E , or GSK F treatment significantly promoted mouse vibrissa follicle growth ex vivo.
They also increased hORS cell proliferation G. ORS cells were treated with Nec-1s 1 nM— μM for 48 h, and both ORS proliferation and migration were enhanced by Nec-1s in a dose-dependent manner Fig.
Therefore, we explored the underlying mechanism of hair growth promotion by Nec-1s in hORS cells. In a previous study, Nec-1s led to an increased Wnt3a We measured the upregulated mRNA levels for the Wnt signaling pathway using PCR array. Furthermore, Nec-1s increased active β-catenin ser expression, which protects its degradation from GSK3β and promotes its translocation into the nucleus Fig.
Therefore, Nec-1s treatment increased β-catenin accumulation in nucleus of ORS cells Fig. The direct effect of the treatment with Nec-1s on β-catenin activity was confirmed by a β-catenin promoter-Luciferase activity assay in human HaCaT and T cells Fig.
A,B Nec-1s treatment increased hORS cell proliferation A and migration B in a dose-dependent manner. C Wnt signaling pathway was observed using a RT 2 profiler PCR assay.
Among the 84 Wnt-related genes, five genes were increased Wnt3a, Wnt5b, Wnt6, CCND1, and PPARD and three CXXC4, SFRP1, and DKK1 were decreased. D qRT-PCR was used to confirm upregulation and downregulation of mRNA expression. E Nec-1s μM increased the total and active β-catenin ser protein expression in ORS cells, the blot density was quantified by Image J.
F β-catenin immunostaining was carried out in hORS cells. β-catenin green translocation from the cytosol to the nucleus was detected in Nec-1s-treated cells arrowhead. G Nec-1s enhance b-catenin promoter activity. A luciferase assay was performed by Human HaCaT and HEKT cell, which stably expressed β-catenin-Luc.
Full-length original blots are shown in Fig. We first investigated whether or not Nec analogs and RIPK1 inhibitors promote hair growth and further examined the underlying molecular mechanisms in the present study.
Nec-1s was the most effective hair growth promoter among the Nec derivatives. Nec-1s induced ORS cell proliferation and increased the HF length in the mouse and pig organ cultures. In addition, it accelerated the telogen-to-anagen transition and anagen elongation in C 3 H mice.
We measured the expression of RIPK1 during the anagen to telogen phase in C3H mice, and RIPK1 was highly expressed in ORS cells during the hair regression period catagen and telogen phases. The transection of RIPK1- or RIPK3-siRNA increased HF length in hair organ cultures, and accelerated telogen-to-anagen transition in the animal model.
Nec-1s upregulated Wnt3a and Wnt5b mRNA expression and increased the translocalization of β-catenin into the nucleus by stimulating β-catenin promoter binding activity. Collectively, these results indicated that Nec-1s has hair growth promoting effects, and necroptosis is involved in normal hair cycle regulation.
Jang et al. examined whether or not necroptosis is associated with the pathogenesis of AA, and found that RIPK1 and RIPK3 mRNA and protein expressions were not upregulated in the skin lesions of AA patients They also reported that keratinocyte necroptosis inhibition prevented psoriatic inflammation, and both the inhibitors of RIPK1 Nec-1s and MLKL e.
RIPK1 was highly expressed in the epidermal basal layer, although its expression decreased significantly in the skin lesions of psoriasis vulgaris. We also found that RIPK1 was highly expressed in epidermal layers and in the outer layer of HFs ORS cells, bulge, and hair germ cells.
Therefore, it is reasonable to assume that RIPK1 may regulate hair cycle regression by inducing epidermal cell death in HFs.
RIPK1 mediates apoptosis and necroptosis, and both are important for late embryonic development and the prevention of inflammation in epithelial barriers 27 , 28 , RIPK1 mediates inflammatory and cell death signaling, and drives RIPK3 and MLKL-mediated necroptosis.
For example, Dannappel et al. Lin et al. Duan et al. Of interest, RIPK3 kinase activity is essential for necroptosis and governs whether a cell activates caspase-8 and dies by apoptosis We found in the present study that Nec-1s inhibited TNF-α- and LPS-induced RIPK1 and RIPK3 phosphorylation; however, it did not attenuate cleaved caspase-3 formation Fig.
These results indicated that both apoptosis and necroptosis occurred in mainly ORS cells at catagen stage during normal hair cycling, and necroptosis still occurred at telogen stage. These results correspond to the result that expression level of RIPK1 was highest at catagen stage and decreased at telogen stage Fig.
These results suggested that apoptosis by RIPK1 may occur mainly at catagen stage, but necroptosis by RIPK1 may occur at both catagen and telogen stages.
The reason why RIPK1 still being expressed at telogen in bulge cell and hair germ, is probably because necroptosis was occurring to some extent until telogen stage though further study may be needed.
Wnt signaling inhibition after re-epithelialization completely abrogated the wounding-induced folliculogenesis, whereas Wnt ligand overexpression increased the number of regenerated HFs Wnt ligand expression is increased within the regenerating epithelium during the anagen phase induction and plays a key role in epidermal stem cell activation Of note, Nec-1s led to increased neurite number in the retinal ganglion cell via Wnt3a-dependent regulation Therefore, we hypothesized that Nec-1s promotes hair growth via the Wnt signaling pathway.
As expected, Nec-1s upregulated the WNT3A, WNT 5A, WNT6, CCND1, and PPARD, whereas it downregulated CXXC4, SFRP1, and DKK1, and thereby translocated β-catenin into the nucleus. Necroptosis has an important role in the programed cell death in embryonic development, tissue homeostasis, immunity, and inflammation Therefore, many pharmaceutical companies have pursued targeting RIPK1 and, to a lesser extent, RIPK3 for drug development.
Novel small-molecule drugs were classified and existed based on their pharmacodynamic type1—3 binding mode 22 , Nec analogs are the first RIPK1 inhibitors to be discovered with an ATP-competitive type 3 binding mode, and we further tested three type 3 inhibitors for hair-loss treatment Figs.
Most RIPK1 inhibitors were effective, although Nec-1s exhibited the strongest hair growth-promoting effect in vitro and in animal experiments.
Therefore, we can conclude that RIPK inhibitors, such as Nec-1s, can be a new target for hair-loss treatment Fig. An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Degterev, A. Targeting RIPK1 for the treatment of human diseases. USA , — Article CAS PubMed PubMed Central Google Scholar.
Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models. Cell Death Dis. Berger, S. Cell Death Discov. Le Cann, F. Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.
FEBS J. Article CAS PubMed Google Scholar. Yang, S. EMBO Mol. Article ADS CAS PubMed PubMed Central Google Scholar. Researchers do not know whether supplements will prevent hair loss in people that do not have nutrient deficiencies.
In fact, the over supplementation of certain nutrients, such as vitamin A, vitamin E, and selenium , can lead to hair loss. There appears to be a direct link between stress and hair loss. Likewise, a shock to the system, be that through physical or emotional trauma, can also act as a shock to the hair follicles and they can stop growing.
In some cases , the use of red ginseng , which can be taken as a supplement, has been shown to promote hair growth. Exactly why this happens is unclear, although researchers think it could be because of indirect stimulation of the hair follicles.
The omega-6 fatty acid arachidonic acid has been shown to promote hair growth by helping to speed up the production of the follicles.
This natural hair growth supplement was shown to promote hair growth in women experiencing temporary thinning. However, a further study also showed its ability to decrease hair loss.
The use of geranium oil as a treatment significantly promotes hair growth and can prevent hair loss. People can apply it by mixing a few drops into a shampoo or conditioner. Coconut oil is widely used to treat damaged hair of various types.
It significantly reduces the protein loss in both damaged and undamaged hair as it can penetrate the hair shaft. The use of aloe vera as a treatment for a variety of different things has been traced back as far as 6, years ago in Ancient Egypt. One such treatment is for hair loss, and evidence has shown it has a soothing effect on the skin that can help alleviate certain conditions.
Using rosemary oil for at least 6 months has been shown in a study to increase hair count. However, many of the group experienced scalp itching as a side effect. Here are some commonly told mistruths and also some genuine reasons behind hair loss. Some women experience hair loss after pregnancy, as a symptom of polycystic ovary syndrome, or if they have problems with their thyroid gland.
TRUE : Although it is not limited to either side of the family. The hair loss gene can be passed from either the father or the mother. FALSE : There is no known link between masturbation or having sex that can cause hair loss.
TRUE : As both men and women age, the number of follicles on the scalp reduces, which means the hair will gradually become thinner and sparser. FALSE : Unless the hat is too tight and causes tension on the hair, wearing a hat has no link to hair loss whatsoever. FALSE : Hair loss occurs at the follicle, meaning at the base of the hair.
The sun cannot reach the follicle because the hair acts as a shield against it. While most women lose between 50 and strands of hair per day, this hair is usually quickly replaced by new growth.
When bald patches or thinning….
Thank you promotiln Oral health tips nature. You Small-batch coffee beans using Oral health tips grosth version with limited support grotwh CSS. Promotiob obtain Hair growth promotion best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. An Author Correction to this article was published on 21 April Necrostatins Necs have been developed as a receptor-interacting protein kinase 1 RIPK1 inhibitor, thus inhibiting necroptosis. Performance-enhancing drug testing Updated: September 6, References. This article was co-authored Oral health tips Courtney Foster. Courtney Foster is a Promotoon Cosmetologist, Certified Hair Loss Practitioner, proomotion Cosmetology Educator based out of New York City. She received her Cosmetology License from the State of New York after training at the Empire Beauty School - Manhattan. There are 11 references cited in this article, which can be found at the bottom of the page. This article has been viewedtimes.
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