Treating rosacea with botulism toxin: Protocol for a systematic review and meta‐analysis

Rosacea is a chronic inflammatory disease usually associated with persistent erythema and periodic flushing. This disease is difficult to treat, and the outcomes are often unsatisfactory and prone to recurrence. In recent years, botulinum toxin has been used as a new treatment for rosacea; however, its efficacy and safety remain under discussion. Although a systematic review of the effectiveness and safety of botulinum toxin has been previously conducted by other researchers, our systematic review and meta‐analysis evaluate the efficacy of botulinum toxin from a more comprehensive and detailed perspective to provide evidence for clinicians.

including rosacea.Significant improvements in clinical signs and symptoms have attracted the attention of both doctors and patients.Yet, no consensus has been reached regarding this treatment method, and its safety, efficacy, and long-term side effects remain to be explored.We hope that our systematic review will provide a reference for clinicians when making decisions for the treatment of rosacea.

| Data extraction and evaluation of literature quality
Two reviewers independently screened the literature and extracted and cross-checked the data; if there were differences in the F I G U R E 1 Flowchart of included studies.

Identification
Records identified from:   The Cochrane risk of bias tool was used to evaluate RCTs, and the Newcastle-Ottawa Scale was used to evaluate cohort and casecontrol studies.The methodological index for non-randomized studies (MINORS) was used for non-randomized controlled trials, 7 while case reports and case series were evaluated using the JBI critical appraisal tools. 8atistical analysis of the included observational outcome data was performed using the Review Manager 5.4 software from Cochrane.Relative risk (RR), mean difference (MD), and 95% confidence intervals (CI) were used as statistical indicators for effect analysis.Heterogeneity among the included studies was quantitatively determined using I 2 .If statistical heterogeneity among the studies was small (I 2 < 50%) or not present, a fixed effects (FE) model was used for the meta-analysis; if statistical heterogeneity was large (I 2 ≥ 50%), a random effects (RE) model was used.Statistical significance was set at p = 0.05.Significant clinical heterogeneity (I 2 ≥ 50%) indicated the presence of heterogeneity between studies and required a subgroup analysis to investigate its source.The stability of the meta-analysis results was evaluated through sensitivity analysis.

| RE SULTS
In total, 201 relevant records were obtained during the initial screening of the database.After removing 92 duplicate records, 35 publications were excluded on analysis of the titles and abstracts, records of animal experiments, guidelines, and other noncompliant literature.
After a layer-by-layer screening, 22 studies were finally included, of which one retrospective and one prospective self-controlled trials were included in the quantitative meta-analysis.Other studies were qualitatively analyzed.The literature screening process is illustrated in Figure 1.

| Basic characteristics of the included studies
The basic characteristics of the included studies are presented in Table 1.Twenty-two studies were published between 2012 and 2022 and included 720 participants, with a minimum sample size of one and a maximum of 120.  Theatients included in these studies were adults, with a predominance of female patients.The age range of the participants was large, ranging 18-75 years.The disease duration ranged from 49 days to 15 years.Nine of the 22 studies were clinical randomized controlled trails, whereas the remaining studies were of different types.
Ten studies described the erythematotelangiectatic rosacea subtype.Three studies reported interventions in patients with erythematotelangiectatic and/or papulopustular rosacea.The remaining nine studies did not specify any subtypes.Botulinum toxin A was used in all studies.However, the brands and manufacturers of botulinum toxin A differed.Studies by Bloom et al., 24 Dayan et al., 14 Park et al., 13 and Kim et al. 19 were supported by grants from pharmaceutical companies that produce botulinum toxin.
The method of botulinum toxin dilution and the mode of administration of the total dose used for each patient varied among the included studies.To enhance confidence in treatment, some researchers chose to combine a pulsed dye laser (PDL), intense pulsed light (IPL), a novel thermomechano-ablative device, and ultrasound impact system, 30% supramolecular salicylic acid and hyaluronic acid gel, as well as other treatments to treat rosacea.
Objective and subjective indicators were used to evaluate efficacy.The influence on the life and work of patients with rosacea and their satisfaction with treatment was evaluated using the DLQI

| CEA scores
The higher the CEA score, the more severe the rosacea.The CEA scores were used to assess efficacy in four studies, [18][19][20]28 but only two studies that assessed CEA scores simultaneously could be combined and analyzed. Thee two studies compared the CEA scores at baseline and 1 month after accepting botulinum toxin treatment.20,28 There was no significant heterogeneity in CEA scores between the two clinical trials (p = 0.74; I 2 = 0%).Statistical analysis showed that the CEA scores 1 month after botulinum toxin treatment were significantly lower than baseline in both studies, and patients showed a statistically significant improvement in clinical symptoms (95% CI = [−2.22,−1.60], p < 0.00001) (Figure 3).

| Subgroup analysis and sensitivity analysis
Only two studies reported CEA scores that could be combined; therefore, there were no conditions for subgroup or sensitivity analyses.

| DISCUSS ION
The erythema, and telangiectasia in patients with rosacea.Therefore, the indications for treatment of rosacea with botulinum toxin are refractory paroxysmal flushing, moderate-to-severe persistent facial erythema, and rosacea that is unresponsive to conventional treatment. 32The included studies showed that botulinum toxin was committed to resolving the flushing and erythema caused by rosacea, and some studies have focused on the treatment of flushing and erythema related to papulopustular rosacea.No hypertrophic skin lesions or ocular manifestations were observed.According to our experience, the effect of botulinum toxin injection is generally gradual after 3 days, and the onset time varies among individuals.
The efficacy was maximized 2 weeks after injection.If the curative effect was not satisfactory at the 2-week follow-up after the operation, supplementary therapy could be administered according to the situation.The IPL and PDL can both be used to improve erythema and telangiectasia caused by rosacea, and botulinum toxin can be used in combination to improve efficacy.To avoid the thermal effect that accelerates the diffusion of local botulinum toxin, the skin   appear to be reliable, with no serious or unpredictable side effects.
However, the introduction of botulinum toxin as a new treatment for rosacea is an off-label, novel attempt.Therefore, there are no recommendations regarding the best dilution formula, administration of botulinum toxin, therapeutic dose, or treatment frequency.
The botulinum toxin formulations used in the various studies included in this meta-analysis differed, which may confuse clinicians when using it to treat rosacea.In addition, the duration of response varies from person to person and there is a lack of guidance on subsequent botulinum toxin therapy when the response wears off.In the next step, we aim to systematically sort and summarize the detailed procedures and specific dilution ratios of botulinum toxin in the treatment of rosacea as a reference for clinicians.
Previously, Zhang et al. conducted a review and analysis of this new treatment method. 33However, we included more studies and performed a meta-analysis of the baseline and 1-month posttreatment CEA scores from two non-randomized self-controlled clinical trials.The comprehensive results showed that the CEA score significantly decreased after treatment, and erythema and flushing were notably relieved.Both studies followed the patients for 6 months and found that erythema and flushing gradually returned at 6 months.However, neither symptom returned to its original severity, which indicates that Botox treatment of rosacea is not a oneoff treatment and that periodic treatments are required to achieve sustained relief.
Worldwide, the reported prevalence varies widely according to skin color, ranging from less than 1% to more than 20%. 34However, the actual situation may be more worrisome.This is because darker skin makes early recognition of erythema and telangiectasia challenging.At the same time, inconsistent diagnostic criteria may lead to the underdiagnosis of patients with rosacea. 35In addition, because there are few epidemiological studies on rosacea, these data may be biased against the true prevalence of the condition.
The presentation of rosacea may vary according to the Fitzpatrick skin type.The common symptom of skin types I-IV is persistent erythema; however, for patients with types V or VI, identifying erythema early is challenging and the first obvious symptoms may be papules and pustules.In this systematic review, the selected literature comes from the United States, China, India, and other countries.
Although some studies do not describe the Fitzpatrick skin types, pale white skin types are usually type I or type II.The skin types of Asians range from Chinese type III and IV to Indian type IV and V. 36 Therefore, the literature included in this review contains more comprehensive skin types, providing a reference for clinicians when dealing with patients of different races and skin types.
The incidence of rosacea is believed to result from a combination of genetic and environmental factors.These factors, involved in the pathogenesis and development of the disease include ultraviolet radiation, alcohol consumption, smoking, and age.Rosacea is a chronic inflammatory disease that results from a combination of multiple interacting pathways and factors.Abnormal functioning of the immune system, mast cells, and the nervous and vascular systems play an important role in this process. 1,37The pathophysiology of this disease involves multiple mediators and pathways.The roles of vascular abnormalities and inflammation have been demonstrated in previous studies.
F I G U R E 3 Forest plot of CEA scores at baseline and 1 month after botulinum toxin treatment.IV, interval variable; CI, confidence interval.
Release of acetylcholine can mediate vasodilation and cause skin flushing. 38Vasodilation is also mediated by the release of neuropeptides, along with the release of inflammatory transmitters. 39Testing revealed that toll-like receptor 2 (TLR2) expression increases in rosacea, and TLR2 can induce keratinocytes to release kallikrein 5, a key protease participating in disease pathogenesis. 40The antimicrobial peptide levels in the affected individuals were also much higher than normal. 41The activated form of antimicrobial peptide LL-37 activated the inflammatory signaling pathway by binding to TLR2, 42 increased expression of inflammation-related biomarkers was induced, 43 which aggravated skin inflammation.The NLRP3 inflammasome, which causes skin inflammation, is activated by LL-37, a pathway that plays a critical role in disease development. 44Activation of TLR2 is crucial for the NLRP3 signaling pathway. 45Therefore, TLR2 and LL-37 are considered vital mediators that initiate signal transduction pathways that allow an organism to develop an inflammatory response.Mast cells are capable of releasing a diverse range of substances that lead to vasodilation, angiogenesis, and tissue fibrosis, which play a prominent role in the pathogenesis of rosacea. 46There is a correlation between angiogenesis and chronic inflammatory skin diseases.LL-37 induces pathological angiogenesis in rosacea through the signaling cascade of the proangiogenic molecule VEGF produced by microorganisms and mast cells. 47In animal experiments, studies have shown that botulinum toxin can inhibit TLR2-mediated inflammation, 48 and reduce erythema induced by LL-37-induced inflammation.Moreover, it inhibits mast cell degranulation, thereby reducing skin inflammation. 49Therefore, the possible mechanism by which botulinum toxin functions in rosacea is to prevent the release of acetylcholine and neuropeptides, and inhibit the function of TLR2, LL-37, and mast cells, thereby blocking the release of relevant mediators and inhibiting inflammation and pathological angiogenesis.Rosacea pathogenesis is closely associated with TLR2, LL-37, kallikrein 5, and mast cells, in addition to lymphocytes, and vasoactive peptides.The underlying mechanisms by which botulinum toxin works in rosacea are complicated, and the current research is not sufficiently comprehensive.
The precise mechanisms need to be further explored.

| LI M ITATI O N S
The evidence provided in this systematic review for clinicians has some limitations.First, because there are few high-quality randomized controlled clinical trials on the potential use of botulinum toxin for rosacea, we included non-randomized controlled clinical studies, case reports, and case series.However, this may increase the risk of bias.Second, the duration of follow-up in the included studies was inconsistent, with some studies being followed up for only a month.Rosacea is characterized by its tendency to recur; Additionally, there is an incomplete understanding of the prolonged adverse effects of botulinum toxin in patients requiring regular treatment.High costs can also discourage physicians and patients from using it as the preferred treatment modality.
the publication presented duplicate data.The search strategy was determined according to the PICO framework: P-patients diagnosed with rosacea according to the diagnostic criteria; I-interventions, including botulinum toxin alone or in combination with other treatments; C-comparison with placebo or other treatments; O-outcomes, such as the Clinician's Erythema Assessment (CEA), Rosacea Clinical Scorecard (RCS) assessment, and Dermatology Life Quality Index (DLQI) scores.To screen suitable studies, two independent researchers conducted a comprehensive literature search without language restrictions.
photographs and erythema quantification measurement.The severity scores of erythema and flushing were evaluated based on the Clinician's Erythema Assessment Grading Scale
clinical manifestations of rosacea are varied.Paroxysmal flushing of the skin, persistent erythema, papules, pustules, and telangiectasia may appear either singly or together on a patient's face; a few patients may even have hypertrophy and ocular manifestations.In daily clinical practice, any one clinical feature of rosacea may dominate and other clinical features may coexist.31Botulinum toxin regulates neurovascular function by inhibiting the release of acetylcholine and neuropeptides, thereby improving flushing,

13 F
Cochrane risk of bias assessment graph of RCTs.TA B L E 3 List of nine items of the definitive minors. .Al-Niaimi et al., cooled after photoelectric treatment and then injected with botulinum toxin.Alternatively, photoelectric treatment can be administered at least 2 weeks after injection.Patients with severe lesions, such as papular pustules or inflammatory reactions, should consider systemic medication to quickly control inflammation, and use Botox to improve erythema and flushing after inflammation control.Additionally, metronidazole can be combined with other topical drugs to increase efficacy.The efficacy of botulinum toxin treatment for rosacea has been maintained for approximately 3-6 months.Doctors should follow-up at 2 weeks, 1 month, 3 months, and 6 months after the injection.If recurrence is observedduring the follow-up process, botulinum toxin treatment can be carried out again.Generally, a 1-3 time treatment can achieve a relatively stable effect.32Both the experience of the authors and the results of the collected studies show that botulinum toxin has a significant therapeutic effect.The retained imaging data of patients before and after treatment and related scales, such as CEA, PSA, and DLQI, showed varying degrees of improvement in signs and symptoms.In our systematic review, only one study made attempted to use a new nonlaser thermodynamic system to promote the transdermal absorption of drugs for the treatment of rosacea-associated flushing and erythema, whereas the remaining studies administered the drug through intradermal injection.Both regimens showed satisfactory efficacy and safety profiles.Analyses have shown that the treatments used for intradermal injections or topical application of botulinum toxin

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therefore, an insufficiently short follow-up period may misrepresent the true efficacy of the botulinum toxin treatment by making it difficult to record the maintenance time of treatment efficacy and over-enhancing the confidence of clinicians in it.Although skin color types were more comprehensive in the studies we included, no studies directly compared the efficacy in patients with different skin color types.It is uncertain whether there are any differences in the effective treatment dose and maintenance of efficacy among patients with different skin color types.CON CLUS IONS In conclusion, botulinum toxin is a novel option worth trying in terms of reduction in CEA score, improvement in signs and symptoms, and patient satisfaction.However, this conclusion is limited by the number and quality of research studies available.Larger samples and high-quality RCTs are needed to validate its long-term efficacy and recurrence rates to provide more credible evidence to clinicians.

TA B L E 2 Clinical characteristics of the included studies. Author, year Botulinum toxin forms Dilution, dosing Administration route Combined treatment
JBI critical appraisal checklist for case reports.
TA B L E 4 JBI critical appraisal checklist for case series.